Calcium And Calmodulun Regulatiov Cell Cycle.pdf

0163-769X/03/$20.00/0 Printed in U.S.A.

Endocrine Reviews 24(6):719 –736 Copyright © 2003 by The Endocrine Society doi: 10.1210/er.2003-0008

Regulation of Cell Cycle Progression by Calcium/ Calmodulin-Dependent Pathways CHRISTINA R. KAHL

AND

ANTHONY R. MEANS

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 Many hormones, growth factors, and cytokines regulate proliferation of their target cells. Perhaps the most universal signaling cascades required for proliferative responses are those initiated by transient rises in intracellular calcium (Ca2ⴙ). The major intracellular receptor for Ca2ⴙ is calmodulin (CaM). CaM is a small protein that contains four EF-hand Ca2ⴙ binding sites and is highly conserved among eukaryotes. In all organisms in which the CaM gene has been deleted, it is essential. Although Ca2ⴙ/CaM is required for proliferation in both unicellular and multicellular eukaryotes, the essential targets of Ca2ⴙ/CaM-dependent pathways required for cell proliferation remain elusive. Potential Ca2ⴙ/CaM-dependent targets include the serine/threonine phosphatase calcineurin and the family of multifunctional Ca2ⴙ/CaM-dependent pro-

tein kinases. Whereas these enzymes are essential in Aspergillus nidulans, they are not required under normal growth conditions in yeast. However, in mammalian cells, studies demonstrate that both types of enzymes contribute to the regulation of cell cycle progression. Unfortunately, the mechanism by which Ca2ⴙ/CaM and its downstream targets, particularly calcineurin and the Ca2ⴙ/CaM-dependent protein kinases, regulate key cell cycle-regulatory proteins, remains enigmatic. By understanding how Ca2ⴙ/CaM regulates cell cycle progression in normal mammalian cells, we may gain insight into how hormones control cell division and how cancer cells subvert the need for Ca2ⴙ and its downstream targets to proliferate. (Endocrine Reviews 24: 719 –736, 2003)

I. Introduction II. Role of Calcium (Ca2⫹) in Cell Proliferation A. Requirement of Ca2⫹ for cell proliferation B. Ca2⫹ signals and the cell cycle C. Calmodulin (CaM), an intracellular Ca2⫹ receptor III. Regulation of Cell Proliferation by CaM A. CaM expression and the cell cycle B. Genetic analysis of CaM function C. Requirement of CaM for cell growth in culture D. In vivo studies of CaM function during cell growth and proliferation E. Targets of CaM and cell proliferation IV. Role of Calcineurin in Cell Proliferation A. Calcineurin structure and biochemistry B. Genetic analysis of calcineurin function C. Calcineurin function in mammalian systems V. Role of Ca2⫹/CaM-Dependent Kinases (CaMKs) in Cell Proliferation A. Structure and biochemistry of CaMKs B. Genetic analysis of CaMK function C. CaMK function in mammalian systems VI. Conclusions and Perspectives

and learning and memory (1, 2). How can a single ion carry out such a vast array of complex cellular processes? Hormones, growth factors, cytokines, and neurotransmitters all elicit increases in intracellular calcium, but differences in the temporal and spatial nature of the intracellular Ca2⫹ transients enable a cell to tailor its response to a given hormone (3, 4). Ca2⫹ can act directly on target proteins or its effects can be mediated via intracellular Ca2⫹ binding proteins. The complex nature of Ca2⫹ signals and the myriad of Ca2⫹ binding proteins in cells allow a single cell to use Ca2⫹ signals for its own unique functions. For example, a pancreatic acinar cell uses Ca2⫹ signals at its apex to control the release of secretory granules, whereas a neuron uses the frequency of Ca2⫹ signals to regulate learning and memory. However, all cells must grow and divide, and Ca2⫹ is universally required for cell proliferation. Although much is known about the many diverse Ca2⫹-dependent pathways regulating muscle contraction, secretion, and learning and memory, the nature of Ca2⫹-dependent pathways regulating cell growth and differentiation remains poorly characterized. Tremendous progress has been made in the last few decades in understanding key pathways that regulate cell growth and division. The cell cycle consists of four primary phases: G1, the first gap phase; S phase, in which DNA synthesis occurs; G2, the second gap phase; and M phase, or mitosis, in which the chromosomes and cytoplasmic components are divided between two daughter cells (Fig. 1). The transitions between these cell cycle phases are tightly regulated, and checkpoints during the cell cycle allow the cell to determine whether all is well before proceeding to the next cell cycle phase (5). For example, if DNA damage occurs during G2, the cell will pause and repair its DNA before entry into mitosis (6, 7). Understanding the pathways that regulate these cell cycle transitions has been facilitated by studies in

I. Introduction

C

2⫹

ALCIUM (Ca ) IS a universal second messenger that regulates a number of diverse cellular processes including cell proliferation, development, motility, secretion,

Abbreviations: Ca2⫹, Calcium; CaM, calmodulin; CaMK, Ca2⫹/CaMdependent kinase; CAMKK, Ca2⫹/CaM-dependent protein kinase kinase; cdk, cyclin-dependent kinase; CKI, cdk inhibitor; FKBP, FK506binding protein; hsp, heat shock protein; MEF, mouse embryonic fibroblast; NFAT, nuclear factor of activated T cells; NLS, nuclear localization sequence; NRK, normal rat kidney.

719 Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

720

Endocrine Reviews, December 2003, 24(6):719 –736

FIG. 1. Schematic diagram of the mammalian cell cycle transitions. Cell cycle transitions are regulated by a series of cdks and their cyclin partners. Upon reentry from quiescence (G0), cyclin D accumulates and associates with cdk4. Cyclin D/cdk4 complexes preferentially phosphorylate pRb in mid-late G1. Then, in a sequential manner, pRb is phosphorylated by cyclin E/cdk2 complexes. As cells enter S phase, cyclin A/cdk2 complexes become activated. The transition from G2 to mitosis is primarily regulated by the activity of cyclin B/cdc2. Ca2⫹/ CaM is required at two points during the reentry from quiescence, early after mitogenic stimulation and later near the G1/S boundary. Additionally, Ca2⫹/CaM is implicated in the G2/M transition, M phase progression, and exit from mitosis.

unicellular eukaryotes, such as yeast. Importantly, homologous pathways have been identified in multicellular eukaryotes that also serve to control cell cycle progression. One group of proteins that acts as a fundamental regulator of cell cycle transitions is the cyclin-dependent kinases (cdks). The cdks are a family of serine/threonine protein kinases that are dependent upon cyclin binding for activity (8 –10). Both the cdks and cyclins as well as their functions in regulating the transitions between cell cycle phases are highly conserved among eukaryotes. cdk Proteins were initially identified in a yeast mutant screen to identify temperature-sensitive mutants that displayed dramatic cell division defects at the restrictive temperature. The major cdk implicated in cell cycle control in Saccharomyces cerevisiae is cdc28p (11). This single cdk associates with different cyclin partners during each stage of the yeast cell cycle. Importantly, the regulation of the cell cycle by cyclins and cdks is conserved throughout eukaryotes. Mammalian cells contain multiple cdks and cyclins, which act at different phases of the cell cycle (Fig. 1) (8 –10). As cells progress through G1, cyclin D/cdk4 complexes are first activated and phosphorylate the tumor suppressor protein, retinoblastoma (pRb) (12–14). Next, cyclin E/cdk2 complexes phosphorylate pRb in a sequential manner after cyclin D/cdk4 phosphorylation (15). The hyperphosphorylation of pRb enables the activation of the family of E2F transcription factors, which, in turn, regulate the expression of numerous genes required for S phase progression (16 –18). As cells enter S phase, cyclin A/cdk2 becomes activated and remains activated into G2 phase (19, 20). In late G2, cyclin B/cdc2 is activated, allowing entry into mitosis (21). The activity of cdks is tightly regulated throughout the cell cycle. Four major types of regulation are common to the cdk family (8 –10, 12). First, the activation of cdks is strictly de-

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

pendent on the binding of its partner cyclin. Second, cdk activity is regulated by phosphorylation, both positively as with activation loop phosphorylation and negatively as with tyrosine phosphorylation. Third, some cdks are regulated by the binding of cyclin-dependent kinase inhibitors (CKIs), which associate with the cdk or cyclin/cdk complex, preventing activation. The two classes of CKIs are the p21/p27 family, whose members associate with both cdk2 and cdk4, and the p15/p16 family, whose members associate only with cdk4/cdk6. Fourth, many of the cyclin/cdk complexes are regulated by their subcellular localization with nuclear localization, allowing them access to their targets. Although all cdks are regulated by these general mechanisms, we will specifically discuss the regulatory mechanisms of cyclin D/cdk4 complexes after growth factor stimulation (Fig. 2) (12, 14, 22). Cyclin D1 expression is strictly dependent on the presence of growth factors and its accu-

FIG. 2. Schematic diagram of cdk4 activation. The activity of cdk4 complexes is regulated in four distinct manners: 1) cyclin D binding, 2) CKI binding, 3) nuclear localization, and 4) phosphorylation. Initially, cdk4 is present in one of two complexes, a chaperone complex containing hsp90 and cdc37 or a dimeric, inactive complex with p15/ p16. Upon mitogenic stimulation, cyclin D mRNA and protein levels increase dramatically, which enables the assembly of cyclin D and cdk4 complexes. Although the p21/p27 family of CKIs inhibit cdk2 activity, they are essential for the proper assembly of cyclin D/cdk4 complexes. Because neither cyclin D nor cdk4 have a canonical NLS, the nuclear import of these complexes is also dependent on p21/p27 proteins present in the complex. After nuclear accumulation of cyclin D/cdk4, CAK (cdk activating kinase) phosphorylates cdk4 on Thr172, resulting in full activation of cdk4 complexes.

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

mulation after mitogenic stimulation is regulated at the levels of transcription, translation, and protein stability. However, before cyclin D accumulation, cdk4 exists in two major complexes. Because cdk4 is unstable in its monomeric form, cdk4 associates with heat shock protein 90 (hsp90) and cdc37 in a large chaperone complex. cdk4 Also exists in an inactive, dimeric complex with p15/p16 proteins. Cyclin D1 accumulation ultimately leads to assembly with cdk4. Although the family of p21/p27 proteins bind cdk2 complexes inhibiting kinase activity, low concentrations of p21/p27 bound to cyclin D/cdk4 do not significantly inhibit kinase activity. Indeed, p21/p27 proteins actually appear to promote the activation of cyclin D/cdk4 by three mechanisms: complex assembly, nuclear import, and cyclin D stabilization. In mouse embryonic fibroblasts (MEFs) null for both p21 and p27, the amount of assembled cyclin D/cdk4 complexes is at least 10-fold lower than their wild-type counterparts. Neither cyclin D nor cdk4 possess a canonical nuclear localization signal (NLS), and a second role for p21/p27 proteins is to provide an NLS to mediate nuclear entry of the complex. When cyclin D is overexpressed in p21/p27 double-null MEFs, it remains cytoplasmic. Finally, cyclin D is stabilized via its association into a complex with cdk4 and p21/p27. Importantly, ectopic expression of either p21 or p27 into the double-null p21/p27 MEFs restores these defects in cyclin D/cdk4 assembly and nuclear import. After nuclear accumulation of cyclin D/cdk4 complexes, cdk4 is phosphorylated on Thr172 by the nuclear enzyme cdk-activating kinase to promote full kinase activation. This multileveled regulation of cdk4 allows exquisite control over the activation of cyclin D/cdk4 in G1. Therefore, in addition to phosphorylating pRb during G1, cyclin D/cdk4 complexes also act to sequester p21/p27 proteins in late G1, promoting the activation of cdk2 complexes. Importantly, disruptions in the regulation of cyclin/cdk complexes are found in a wide variety of endocrine tumors. For example, cyclin D1 overexpression occurs in some breast cancers, and the cyclin D1 gene, previously referred to as the PRAD1 oncogene, was initially identified for its role in some parathyroid adenomas (23). In those tumors, a chromosomal rearrangement placed the cyclin D1 locus adjacent to the PTH locus. This rearrangement leads to dramatic increases in cyclin D1 expression. Regulation of cell proliferation is common to a wide variety of hormones, growth factors, and cytokines. For a comprehensive description of the hormonal regulation of cell cycle-regulatory proteins, we refer readers to a recent review by Pestell et al. (24). Both steroid hormones and peptide hormones alter the expression and/or activity of proteins that are components of the cyclin/cdk complexes. Common to all the regulatory transitions of the cell cycle is the ubiquitous second messenger, Ca2⫹, and the universally important Ca2⫹ intracellular receptor, calmodulin (CaM). In the past several years, progress has been made in understanding how Ca2⫹/CaM regulates cell cycle transitions and affects the activation state of cdk complexes. In this review, we will discuss the requirements for Ca2⫹ and CaM during cell proliferation. Then, we will focus on two classes of Ca2⫹/CaMdependent enzymes that have been implicated in cell cycle regulation in eukaryotes.

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Endocrine Reviews, December 2003, 24(6):719 –736

721

II. Role of Calcium (Ca2ⴙ) in Cell Proliferation A. Requirement of Ca2⫹ for cell proliferation

In all eukaryotic cells, Ca2⫹ is required in both the extracellular environment and intracellular stores for cell growth and division. In mammalian cells, lowering of extracellular Ca2⫹ from 1.0 mm to 0.1 mm led to a gradual decrease in the rate of proliferation (25). Extracellular Ca2⫹ is required at multiple distinct points in the cell cycle in mammalian cells. When proliferating mouse or human fibroblasts were placed into media containing low Ca2⫹, they ceased cellular division and accumulated in G1 (26 –28). In BALBc/3T3 fibroblasts, this G1 arrest was reversible, and returning the extracellular Ca2⫹ content to normal levels enabled cells to undergo DNA synthesis within hours (28). Cells were most sensitive to the depletion of extracellular Ca2⫹ at two points during the cell cycle, in early G1 and near the G1/S boundary (29). When human fibroblasts were stimulated by growth factors, depletion of extracellular Ca2⫹ anytime during the first 8 h after stimulation resulted in an inhibition of DNA synthesis (30). At later times, depletion of extracellular Ca2⫹ had no effect on the ability of the cells to enter S phase. Again, these arrests due to low Ca2⫹ were fully reversible as cells continue to proliferate after the addition of normal Ca2⫹ levels to the media. This requirement for extracellular Ca2⫹ in growth and proliferation is modulated by the degree of cellular transformation. Indeed, neoplastic or transformed cells continued to proliferate in Ca2⫹-deficient media (31, 32). In contrast to their normal counterparts, human fibroblasts transformed with the simian virus 40 proliferated normally in very low extracellular Ca2⫹ concentrations (29, 33). Examination of primary cells, preneoplastic cells, and neoplastic cells revealed a gradient for extracellular Ca2⫹ levels required for proliferation (34). Primary C3H mouse skin cells have reduced rates of DNA synthesis when extracellular Ca2⫹ is lowered to 0.05– 0.1 mm, whereas preneoplastic C3H/ 10T1/2 and MCA-C3H/10T1/2 type I mouse fibroblasts required a reduction to 0.01 mm extracellular Ca2⫹ to inhibit DNA synthesis. Finally, the neoplastic MCA-C3H/1-T1/2 type III fibroblasts continued to proliferate with very low extracellular Ca2⫹ levels. Additionally, the proliferative responses of liver tumor cell lines in low Ca2⫹ reflected their tumorigenic potential (35). Therefore, the strict requirement for extracellular Ca2⫹ is lost during neoplastic transformation, but how this change in extracellular Ca2⫹ dependence affects intracellular Ca2⫹-dependent pathways is unknown. In addition to the requirement for extracellular Ca2⫹, intracellular Ca2⫹ stores are also required for cellular proliferation in mammalian cells. Depletion of intracellular inositol 1,4,5-triphosphate-sensitive Ca2⫹ stores with pharmacological agents, such as thapsigargin or 2,5-di-tert-butylhydroquinone, resulted in a cessation of cell division (36). These agents block the Ca2⫹ pumping ATPase present in the endoplasmic reticulum and result in a depletion of Ca2⫹ stores in the endoplasmic reticulum. The consequences of intracellular Ca2⫹ pool depletion included inhibition of DNA synthesis, protein synthesis, and nuclear transport (36 –38). Depletion of intracellular Ca2⫹ stores resulted in the accu-

722

Endocrine Reviews, December 2003, 24(6):719 –736

mulation of cells in a quiescent state, and upon removal of thapsigargin or 2,5-di-tert-butyl-hydroquinone, cells reentered S phase with the same kinetics as cells released from quiescence (36). Furthermore, depletion of intracellular Ca2⫹ stores at any point during G1 to S resulted in an accumulation of cells in a G0-like state even when cells have partially replicated DNA (F. Riberio-Neto and A. R. Means, unpublished data). Therefore, normal cells require both extracellular and intracellular Ca2⫹ for proliferation, with cells being the most sensitive to Ca2⫹ depletion during G1. B. Ca2⫹ signals and the cell cycle

In cells, cytoplasmic Ca2⫹ transients are generated by release of Ca2⫹ from intracellular pools or by entry of Ca2⫹ from the extracellular environment via Ca2⫹ channels in the plasma membrane. Ca2⫹ transients come in a variety of categories including elemental “blips/quarks,” slightly larger “puffs/sparks,” which are restricted to small areas, or “waves,” which involve the whole cell (1– 4, 39). In addition to spatial difference in Ca2⫹ transients, both the amplitude and frequency of Ca2⫹ transients can be modulated. Even though Ca2⫹ is a ubiquitous second messenger, the temporal and spatial complexity of Ca2⫹ transients enables a cell to use Ca2⫹ signaling for a wide variety of physiological responses. Although many hormones and growth factors cause intracellular Ca2⫹ transients, the nature of the Ca2⫹ signal allows a cell to decode stimuli from a wide variety of hormones. Although these Ca2⫹ signals act to regulate innumerable cellular pathways, we will focus the discussion on the role of Ca2⫹ transients during the cell cycle. During the cell cycle, Ca2⫹ transients have been characterized during G1 and mitosis (40, 41). In rat liver epithelial cells (T51B), epidermal growth factor stimulation resulted in a rise in intracellular Ca2⫹, and this rise required Ca2⫹ in the extracellular environment (42). When mouse C127 cells, which are a nontransformed cell line derived from a mammary tumor, were synchronized in mitosis, multiple Ca2⫹ transients were observed as they entered early G1. Whereas in mid-G1 there were no detectable transients, Ca2⫹ transients resumed near the G1/S boundary (Christenson, M., M. Poenie, and A. R. Means, unpublished observations). Therefore, Ca2⫹ transients within a cell correspond to the same cell cycle points in which extracellular Ca2⫹ is required, namely early G1 and at the G1/S boundary. Ca2⫹ transients are also evident during several stages of mitotic progression, particularly at the metaphase/anaphase transition and during cytokinesis (43, 44). In sea urchin eggs, Ca2⫹ transients occurred at pronuclear migration, nuclear envelope breakdown, the metaphase to anaphase transition, and during cleavage (45). These transients at the metaphase to anaphase transition have also been demonstrated in mammalian Ptk1 cells (46). Regardless of the size and duration of a Ca2⫹ transient, the Ca2⫹ signal is transduced into cellular consequences by direct binding of Ca2⫹ to targets or by Ca2⫹ binding to intracellular receptors, such as CaM, which relay the signal to Ca2⫹/CaM-dependent targets.

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

C. Calmodulin (CaM), an intracellular Ca2⫹ receptor

In mammalian cells, CaM is a 148-amino acid, highly conserved Ca2⫹ binding protein that contains four EF-hand Ca2⫹ binding motifs (47). Based on nuclear magnetic resonance and crystal structures of CaM in the apo and Ca2⫹-bound state, we know that Ca2⫹-bound CaM has a dumbbell shape with two EF-hand motifs on either end connected by a central helix (47, 48). Ca2⫹ binding exposes hydrophobic patches, promoting interaction with target enzymes. A number of crystal structures of Ca2⫹/CaM bound to target peptides demonstrate that CaM wraps around the target peptide, engulfing it (48 –52). For targets of Ca2⫹/CaM, this binding has enormous consequences. As in the cases of calcineurin and Ca2⫹/CaM-dependent protein kinase II (CaMKII), one general mechanism by which Ca2⫹/CaM-binding activates its target enzymes is through the relief of autoinhibition. CaM regulates numerous intracellular enzymes that include phosphodiesterases, adenylyl cyclases, ion channels, protein kinases, and protein phosphatases (47). Ca2⫹/CaMdependent pathways are involved in the regulation of a wide variety of cellular processes including secretion, cell motility, ion homeostasis, gene transcription, neurotransmission, and metabolism. Hormone and neurotransmitter stimulation of cells leads to a variety of Ca2⫹/CaM-mediated responses (53). Initially, hormone receptors are activated, leading to an intracellular Ca2⫹ rise. Ca2⫹ regulates some targets directly and other targets indirectly through Ca2⫹ binding proteins, such as CaM. Persistent stimulation can cause changes in the subcellular distribution of CaM, which leads to changes in Ca2⫹/CaM responsiveness in a given area of the cell. Longterm stimulation can also lead to changes in the total amount of CaM making a cell more or less sensitive to Ca2⫹ signals (53). III. Regulation of Cell Proliferation by CaM A. CaM expression and the cell cycle

Intracellular CaM levels are regulated as cells progress through the cell cycle. In Chinese hamster ovary-K1 cells, CaM levels fell within the first hour after release from plateau phase and then doubled at the G1/S boundary due to increased protein synthesis (54, 55). Similarly, in normal human fibroblasts, CaM levels decreased in the first few hours after mitogenic stimulation and then increased 2- to 4-fold at the G1/S boundary (56). However, as these cells approached senescence, these changes in CaM levels during G1 were lost and CaM levels remained relatively constant. In addition to these cell culture experiments, changes in CaM levels were also found in in vivo models of proliferative responses. During liver regeneration, there was a wave of increased CaM in the early prereplicative period, which was also due to new protein synthesis (57). However, another group found an increase in CaM mRNA as well as protein levels after partial hepatectomy (58). Similar to findings in mammalian cells, in Aspergillus nidulans, CaM levels also increased 2-fold just before DNA synthesis (59). Therefore, the rise in CaM levels just before S phase may be universal, but why CaM must increase at this point in the cell cycle or what the target of CaM is at the G1/S boundary remains unknown.

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

Indeed, manipulation of CaM expression affects the proliferative capacity of cells. In mammalian cells, overexpression of CaM by 2- to 4-fold using a bovine-papilloma-virusbased expression vector caused an acceleration of cell proliferation (60). In A. nidulans, overexpression of CaM accelerated cell cycle reentry from sporulation and shortened cell cycle length (61). In mammalian cells, the acceleration of cell proliferation was due to a shortening of G1. Examination of six independent cell lines that overexpressed CaM demonstrated a linear relationship between CaM concentration and rate of G1 progression (60, 62). Because a relationship between the amount of CaM and cell cycle timing exists in both A. nidulans and murine C127 cells, it suggests that this correlation was not merely secondary to a transformed phenotype. In contrast, reduction of CaM levels using antisense CaM resulted in a transient inhibition of cell proliferation (62). Deletion of one of two functional CaM genes, CaMII, from chicken DT40 lymphoma B cells reduced CaM expression by 60%, and these cells exhibited slower growth (63). Therefore, the amount of CaM in a cell regulates proliferative rates, primarily due to effects on G1. The association between CaM levels and proliferation is reflected in the relationships between CaM levels and cellular transformation. Transformation of Swiss 3T3 cells with simian virus 40 or normal rat kidney (NRK) cells with Rous sarcoma virus resulted in increased CaM levels (64). Additionally, in normal chicken embryo fibroblasts, transformation with Rous sarcoma virus resulted in increased CaM levels due to increased protein synthesis (65). NRK cells infected with a temperature-sensitive, transformation defective mutant avian sarcoma virus (tsLA23) possessed more CaM than uninfected controls (66). However, upon shift to 40 C, the temperature at which they behaved more like nontransformed cells, CaM levels dropped to levels similar to nontransformed cells. Rat fibroblasts transformed with a variety of oncogenes in combination with protein kinase C have increased CaM levels even in the presence of an overall reduction in CaM mRNA transcripts (67). These studies raise the question: do increases in CaM contribute to the transformed phenotype or do increases in CaM merely reflect the growth advantages of transformed cells? Furthermore, how relevant are studies of Ca2⫹/CaM-dependent signaling cascades in transformed cells to their function in normal cells? Studies involving transformed cells and in A. nidulans indicate a reciprocal regulation between CaM levels and Ca2⫹. In transformed rat fibroblasts, a reduction in extracellular Ca2⫹ resulted in an increase in CaM expression, whereas an increase in extracellular Ca2⫹ resulted in a decrease in CaM expression (68). In chicken DT40 lymphoma cells, deletion of CaMII caused an increase in resting Ca2⫹ levels (63). In A. nidulans, induction of CaM expression using the alcA (alcohol dehydrogenase A) gene promoter led to a 10-fold reduction in the level of extracellular Ca2⫹ required for growth (61). Although CaM and Ca2⫹ appear to be reciprocally regulated, how these changes regulate downstream Ca2⫹/CaM pathways remains unknown.

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Endocrine Reviews, December 2003, 24(6):719 –736

723

B. Genetic analysis of CaM function

Organisms that facilitate genetic analysis, such as S. cerevisiae, Schizosaccharomyces pombe, A. nidulans, and Drosophila melanogaster, all contain a unique CaM gene, which is essential (59, 69 –71). The requirement for CaM has been characterized in both budding and fission yeast. In yeast, CaM was localized to sites of cell growth and the SPB (spindle pole body) (72, 73). Based on this localization of CaM, it is not surprising that studies involving the disruption of CaM function have revealed a role for CaM in nuclear division and the maintenance of cell polarity in yeast (74). In S. cerevisiae, CaM is required for mitotic progression. When the expression of the CaM gene, CMD1, was regulated using the GAL1 promoter, repression of CaM resulted in nuclear division defects with cells having short mitotic spindles and increased chromosomal loss (75). Additionally, some cells also demonstrated bud growth inhibition. Using a temperature-sensitive allele of CaM, Davis (76) synchronized cells in G1 and followed their progression through the cell cycle. These yeast cells were viable until mitosis and demonstrated defects in both chromosomal segregation and cytokinesis. In studying multiple temperature-sensitive CaM mutants, Ohya and Botstein (77) found that they sorted into four complementation groups, each of which exhibited a defect in actin organization, CaM localization, nuclear division, or bud formation. Similar to budding yeast, CaM is required for mitotic progression in fission yeast. When cells with a temperature-sensitive allele of CaM were synchronized in S phase and released, they progressed through DNA synthesis and then lost viability in mitosis, with defects in chromosomal segregation (72). In the filamentous fungi A. nidulans, CaM is also critical for the progression through the G2/M transition. Repression of CaM expression using the alcA promoter arrested the majority of cells in G2 (61). When cells were released from a temperature-sensitive block in late G2, cells with low levels of CaM failed to activate NIMXcdc2 and progress through mitosis (78). The results from yeast and A. nidulans demonstrate a critical role for CaM for entry into and progression through mitosis. However, in mammalian cells, the requirement for Ca2⫹ is clearly linked to G1 progression as well as mitosis. Therefore, why do unicellular eukaryotes, such as yeast and fungi, not demonstrate G1 defects when CaM function is disrupted? One possibility is that mammalian cells have evolved complex mechanisms to regulate G1 progression that are dependent on Ca2⫹, and those pathways may not exist in yeast and fungi. Another possibility is that it may be more difficult to study defects in Ca2⫹/CaM-dependent pathways during G1 in unicellular eukaryotes. C. Requirement of CaM for cell growth in culture

The importance of CaM for mammalian cell survival is reflected by both the number of CaM genes in higher eukaryotes and its degree of evolutionary conservation (79). Rodents and humans have three CaM genes on three separate chromosomes (80 – 82). Strikingly, the encoded amino acid sequences are not merely conserved among these sep-

724

Endocrine Reviews, December 2003, 24(6):719 –736

arate genes and species, they are identical. Because mammals have three CaM genes that encode identical proteins and CaM is essential in genetic model systems, deletion of CaM in mice would be not only labor intensive but also could be uninformative as it is predicted to result in very early embryonic lethality. Therefore, studies to examine the role of CaM in the cell cycle in mammalian cells have focused on the use of antagonists of CaM function. Microinjection of monoclonal antibodies against CaM inhibited DNA replication in a dose-dependent manner (83). A series of pharmacological inhibitors of CaM have been developed to probe its functions in cells. W-7 and its derivatives (W-13) bind CaM and subsequently, prevent activation of Ca2⫹/CaM-dependent target enzymes. These compounds are cell permeable and distribute through the cell (84). Treatment of a wide variety of cells with W-7 or its derivatives inhibited proliferation (30, 55, 83– 86). Both W-7 and W-13 also prevented proliferation and colony formation of breast cancer cell lines (87). When Chinese hamster ovary-K1 cells were released from plateau phase, W-13 prevented cell cycle reentry. CaM appeared to be required at two points, early after mitogenic stimulation and late in G1 near the G1/S boundary (55). In human fibroblasts, addition of W-7 up to 8 h after mitogenic stimulation completely inhibited DNA synthesis, but if W-7 was added later in G1 after pRb hyperphosphorylation, there was no inhibition of DNA synthesis (30, 33). It is intriguing that both Ca2⫹ and CaM appear to be required at two points during reentry, early after mitogenic stimulation and in late G1. Based on the work of Takuwa et al. (33), the point in late G1 when Ca2⫹/CaM is required is before the restriction point and pRb phosphorylation. Recently, the effects of W-13 on proteins that control pRb phosphorylation in G1, such as cyclin D/cdk4, have been investigated in NRK cells. Although W-13 did not affect the accumulation of cyclin D after mitogenic stimulation, it prevented the entry of cyclin D/cdk4 complexes into the nucleus (85). Taules et al. isolated cyclin D/cdk4 complexes from NRK cells using CaM Sepharose and suggested that the interaction between CaM and cyclin D/cdk4 was mediated by hsp90 and/or p21 (85, 88). However, how and if the association between CaM and cyclin D-cdk4 regulates the nuclear import of cyclin D/cdk4 remains unknown. In any case, it is clear that Ca2⫹/CaM regulates pRb hyperphosphorylation in late G1, most likely via effects on cyclin D/cdk4 (Fig. 1). Interestingly, there are some striking similarities between the studies of CaM function and cyclin D1 function in mammalian cells. Overexpression of both CaM and cyclin D1 specifically accelerated the G1 phase of the cell cycle (60, 62, 89, 90). Inhibition of CaM or cyclin D1 function by antibody microinjection arrested cells in G1 (83, 91). One logical explanation is that cyclin D1 and its cdk complexes are the ultimate target of Ca2⫹/CaM-dependent cascades during G1 progression. If the downstream target of Ca2⫹/CaM during G1 is the cyclin D/cdk4/pRb pathway, it may explain some of the changes in the Ca2⫹ requirement during transformation. One hypothesis is that a human cancer must subvert the cyclin D/cdk4/pRb pathway by one of several methods: overexpression of cyclin D1 or cdk4, loss or mutation of cdk4 inhibitors (p15/p16 family), or loss of pRb function (13).

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

Indeed, more than 80% of human cancers have a defect in the cyclin D/cdk4/pRb/E2F pathway (22). Importantly, a cancer that has an alteration in one component of this pathway does not have defects in the other components. Therefore, in one scenario, a cancer cell, containing a disruption in the cyclin D/cdk4/pRb regulatory pathway, may no longer require Ca2⫹/CaM to regulate the activation of this pathway because it is already activated or unnecessary for G1 progression in the tumor cell. D. In vivo studies of CaM function during cell growth and proliferation

Because most of the studies examining CaM in the cell cycle have relied on manipulation of cultured cells, one would like to know whether some of the same findings are true in a whole animal. One model system in which requirement of Ca2⫹/CaM in vivo has been tested is cardiomyocytes. Cardiomyocytes possess several distinct stages of cell proliferation. Normal cell division is limited to embryogenesis in myocytes (92). In the perinatal period, myocytes continue to undergo DNA synthesis in the absence of cytokinesis, resulting in polynucleate cells. After this process of polynucleation, myocytes no longer synthesize DNA but often undergo hypertrophy in response to certain signals as those that occur in heart failure. As with most cells, the proliferation of primary or secondary rat ventricular myocytes required extracellular Ca2⫹, and chelation of extracellular Ca2⫹ resulted in a G1 arrest (93). To test the effects of CaM on myocyte proliferation, our laboratory generated transgenic mice that overexpress CaM in the heart using the atrial naturetic factor promoter. The ventricles of these mice were both hypertrophic and hyperplasic (94). At birth, there was a 40% increase in the number of ventricular myocytes over control animals (Colomer, J., and A. R. Means, unpublished data). After birth, these mice demonstrated increased DNA synthesis without cell division or polynucleation, resulting in increased polyploidy of the nuclei. Finally, the myocytes overexpressing CaM became hypertrophic, and this hypertrophy receded as the CaM levels fell. Therefore, CaM overexpression affected all stages of myocyte proliferation and growth. However, the effect of CaM overexpression on myocyte proliferation is limited to, and does not extend beyond, the developmental period during which myocyte numbers are expanded. E. Targets of CaM and cell proliferation

Ca2⫹/CaM bind and regulate numerous intracellular proteins involved in a myriad of pathways. Therefore, identification of conserved Ca2⫹/CaM binding proteins that regulate cell cycle progression remains difficult. Genetic systems, such as yeast and fungi, have proved essential in the isolation of key cell cycle regulators, in particular cyclins and cdks. Therefore, one could hypothesize that the Ca2⫹/CaMdependent target proteins involved in cell cycle regulation should be essential or, at the very least, loss of function should result in growth defects in genetic model systems. However, whereas CaM is essential in yeast, fungi, and flies,

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

only a handful of essential CaM binding proteins have been identified. Although CaM function has been extensively characterized in S. cerevisiae, this organism is unique relative to its regulation of Ca2⫹ and function of CaM (95). Whereas CaM from vertebrate systems binds four Ca2⫹ ions with each EF-hand motif, CaM from S. cerevisiae binds only three Ca2⫹ ions, with the fourth EF-hand motif being nonfunctional for Ca2⫹ binding (96). Although CaM is essential, high-affinity Ca2⫹ binding to CaM is not essential because expression of a mutant CaM, which does not bind Ca2⫹ with high affinity, supported growth in budding yeast (97). Based on these findings, it is not surprising that no essential Ca2⫹/CaMdependent target enzymes have been identified in S. cerevisiae (Table 1). Budding yeast does possess essential Ca2⫹independent CaM targets, such as the spindle pole bodyassociated protein Nuf1p/Spc110p and the unconventional (class II) myosin Myo2p (95). Previously, Nuf1p/Spc110p has been classified as a Ca2⫹-independent CaM target because it was shown to interact with the mutant CaM, which does not bind Ca2⫹ with high affinity in a yeast two-hybrid assay (98). However, the CaM binding region of Nuf1p/ Spc110p possesses a Ca2⫹-dependent, not Ca2⫹-independent, consensus sequence and using an in vitro CaM overlay assay, CaM binding to Nuf1p/Spc110p was Ca2⫹-dependent (99, 100). For a comprehensive discussion of CaM targets in S. cerevisiae, we refer readers to a recent review by Cyert (95). Therefore, studies in S. cerevisiae may be more useful in understanding the Ca2⫹-independent functions rather than the Ca2⫹-dependent functions of CaM. In contrast to S. cerevisiae, S. pombe and A. nidulans required high-affinity Ca2⫹ binding to CaM to support growth (101, 102). Although several Ca2⫹/CaM-dependent targets have been isolated from S. pombe, these targets are either not essential or have yet to be characterized (Table 1). On the other hand, both Ca2⫹-dependent and Ca2⫹-independent targets have been identified and shown to be essential in A. nidulans. In terms of Ca2⫹-independent targets,

Endocrine Reviews, December 2003, 24(6):719 –736

725

both an unconventional myosin, myosin A, and a Nuf1/ Spc110 homolog, An110, have been isolated from A. nidulans, but only the myosin A gene has been silenced and demonstrated to be essential (102, 103). In addition, A. nidulans contains at least three genes that encode essential Ca2⫹/ CaM-dependent enzymes with homologs in mammalian cells. These genes produce two Ca2⫹/CaM-dependent protein kinases (CMKA and CMKB) and the Ca2⫹/CaM-dependent protein phosphatase 2B, calcineurin (104 –106). Although yeast contains homologs of both calcineurin and CaMK, neither calcineurin nor any CaMK is essential for growth under normal conditions. However, null strains for these genes either demonstrated mild growth defects or impaired growth under certain environmental conditions. Additionally, cultured mammalian cells show proliferative defects when calcineurin or CaMK function is pharmacologically inhibited. Therefore, we will focus on the possible roles of calcineurin and CaMKs as downstream Ca2⫹/CaM target enzymes that regulate proliferation in a variety of circumstances. IV. Role of Calcineurin in Cell Proliferation A. Calcineurin structure and biochemistry

Calcineurin is a heterodimer composed of a catalytic subunit, calcineurin A, and a Ca2⫹-binding regulatory subunit, calcineurin B (107–110). The two subunits are tightly bound, only being dissociated by denaturation, and both subunits are essential for calcineurin function. Calcineurin A contains an amino-terminal catalytic domain followed by the calcineurin B binding domain and a regulatory domain (Fig. 3). The regulatory domain contains a CaM binding region and autoinhibitory domain. Binding of Ca2⫹/CaM to the regulatory domain leads to dramatic enzyme activation through the relief of autoinhibition. The calcineurin B subunit consists of four EF-hand Ca2⫹ binding motifs, similar to CaM, and a conserved amino-terminal myristylation site.

TABLE 1. CaM targets in yeast and A. nidulans Target

Mammalian homologs

Organism

Essential

CaM binding 2⫹

a

Nuf1p/Spc110p Pcp1 An110 Myo2p MYOA

Kendrin Kendrin Kendrin Class V myosins Myosin I

S. cerevisiae S. pombe A. nidulans S. cerevisiae A. nidulans

Yes Yes Unknown Yes Yes

Ca -independent Ca2⫹-independenta Ca2⫹-independenta Ca2⫹-independent Ca2⫹-independent

Cna1p, Cna2p (Cnb1p)

Calcineurin A (calcineurin B)

S. cerevisiae

No

Ca2⫹-dependent

Ppb1

Calcineurin A

S. pombe

No

Ca2⫹-dependent

CNA Cmk1p, Cmk2p Cmk1 Cmk2 CMKA CMKB CMKC

Calcineurin A CaMKII CaMKI CaMK CaMKII CaMKI CaMKK

A. nidulans S. cerevisiae S. pombe S. pombe A. nidulans A. nidulans A. nidulans

Yes No No No Yes Yes No

Ca2⫹-dependent Ca2⫹-dependent Ca2⫹-dependent Ca2⫹-dependent Ca2⫹-dependent Ca2⫹-dependent Ca2⫹-dependent

Function

Ref.

SPB regulation SPB regulation Unknown Polarized growth Polarized growth Secretion Stress response Ca2⫹ homeostasis G2/M transition Mating Cell shape and polarity G1 progression Stress response Cell cycle Oxidative stress response G2/M transition Reentry from sporulation Reentry from sporulation

Reviewed in 95 102 102 Reviewed in 95 103 Reviewed in 95 119 106, 125 163–165 167 168, 183 105, 169 104 104

SPB, Spindle pole body. Although Nuf1p/Spc110p has been previously characterized as a Ca2⫹-independent target of CaM based on two-hybrid studies, it has been shown to bind CaM in a Ca2⫹-dependent manner in vitro, and Ca2⫹ has been implicated in its in vivo functions in S. cerevisiae (98 –100). a

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

726

Endocrine Reviews, December 2003, 24(6):719 –736

FIG. 3. Schematic diagrams of calcineurin and CaMK. A, The catalytic subunit of the serine/threonine protein phosphatase calcineurin, calcineurin A, contains an amino-terminal catalytic domain followed by a calcineurin B-binding domain (CnB), a CaM-binding domain (CaM), and an autoinhibitory domain (AI). Calcineurin B contains four EF-hand Ca2⫹ binding motifs and an amino-terminal myristylation site. B, The multifunctional CaMKs contain an amino-terminal catalytic domain followed by a regulatory domain containing the overlapping CaM binding and autoinhibitory domains. CaMKII also contains a carboxy-terminal association domain, and some isoforms have an alternatively spliced NLS. CaMKI and CaMKIV do not contain association domains but have homologous Thr phosphorylation sites in their activation loops.

The Ca2⫹-dependent phosphatase activity is controlled by both calcineurin B and CaM (107–110). Although calcineurin remains inactive when the high-affinity Ca2⫹-binding site in calcineurin B is occupied, Ca2⫹ binding to the low-affinity sites enables some activation of the enzyme. However, addition of Ca2⫹/CaM results in a 10- to 100-fold activation due to changes in Vmax. Importantly, calcineurin is activated after small changes in intracellular Ca2⫹ concentration following cell stimulation due to the highly cooperative nature of Ca2⫹ binding to CaM and the very high affinity of calcineurin for Ca2⫹/CaM. In mammalian systems, calcineurin is well known for its role in T cell activation, and two clinically useful immunosuppressants, cyclosporin A and tacrolimus (FK506), prevent calcineurin function. Calcineurin dephosphorylates the transcription factor nuclear factor of activated T cells (NFAT), allowing it to enter the nucleus and promote gene transcription (111–113). However, calcineurin is not limited to T cells but is found in all cell types. More recently, the function of calcineurin in a wide variety of cells has been investigated. B. Genetic analysis of calcineurin function

Although none of the three genes encoding calcineurin subunits (CNA1, CNA2/CMP2, and CNB1) in S. cerevisiae

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

are essential, yeast calcineurin functions in the cellular response to stress (95). When yeast cells were grown in stressful environmental conditions, such as high ion concentrations or high temperature, the expression of calcineurin-activated genes was induced. Similar to the regulation of NFAT, calcineurin dephosphorylated the transcription factor Crz1p/Tcn1p, promoting its nuclear accumulation and activity (114, 115). DNA microarray analysis revealed that the induced genes are involved in signaling pathways, ion and small molecule transport, cell wall maintenance, and vesicular transport (116). Therefore, calcineurin-deleted strains were sensitive to both high pH and high osmolarity (95). Exposure to ␣-factor also activated calcineurin-dependent gene expression, and prolonged exposure to ␣-factor in the absence of calcineurin function led to cell death (117). Additional functions of calcineurin in S. cerevisiae include the regulation of Ca2⫹ homeostasis and Swe1p activity at the G2/M transition (95). In S. pombe, the catalytic subunit of calcineurin, ppb1, is not essential, but null mutants displayed several defects (118, 119). First, the ppb1 null mutants showed impaired cytokinesis at low temperature as well as defects in maintaining cell shape and polarity. They also exhibited a mating defect and were sterile. Interestingly, calcineurin mRNA expression peaked during S phase and was induced under nitrogenstarvation conditions (120). Although calcineurin is not essential in yeast, the evidence suggests that it plays critical roles in the response to stressful environmental conditions and during mating. The requirement for calcineurin for cell growth under stressful environmental conditions has been recently strengthened by studies in pathogenic fungi. Cryptococcus neoformans causes meningoencephalitis in humans, particularly in immunocompromised hosts such as those with AIDS. Although not essential for growth under normal conditions, both the calcineurin A and B genes were required for growth at 37 C and for virulence (121–123). Similar to yeast, calcineurin A was also required for mating in this fungi (124). In contrast to yeast, in A. nidulans, the calcineurin A gene is essential (106). To study the function of calcineurin in this fungi, the endogenous calcineurin A gene was placed under control of the conditional alcA promoter (125). In repressing media, which reduced calcineurin expression, germlings underwent only one round of DNA replication and then arrested primarily in G1, with some cells arrested at G2 and mitosis. These results suggested that calcineurin was critical for G1 progression, but not for initial cell cycle reentry from sporulation. However, there may have been enough calcineurin present in the spores to allow passage through the first G1 but then, as expression fell due to protein turnover, cells were unable to transit through a second G1. Interestingly, endogenous calcineurin mRNA was maximally expressed at the G1/S boundary, suggesting that it may be regulated in a cell cycle-dependent manner. Therefore, calcineurin is crucial for G1 transition in this filamentous fungi.

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

Endocrine Reviews, December 2003, 24(6):719 –736

C. Calcineurin function in mammalian systems

The importance of calcineurin in mammalian cells was illuminated by the investigations into the immunosuppressive actions of cyclosporin A. Cyclosporin A blocks the activation and proliferation of quiescent T cells after T cell receptor engagement (111–113). Calcineurin was identified as the target of cyclosporin A. Its enzymatic activity was not inhibited directly by cyclosporin A but rather by a complex of cyclosporin A and cyclophilin, an intracellular prolyl isomerase (126 –129). In T cells, Ca2⫹ transients lead to the activation of calcineurin, which dephosphorylates NFAT and leads to its nuclear import and subsequent transcriptional induction of IL-2 (111). Therefore, calcineurin activity is essential to T cell activation and reentry into the cell cycle after T cell receptor engagement. Although the T cell represents a very specialized system of reentry from quiescence, cyclosporin A has antiproliferative effects in a wide variety of cells, including adenocarcinoma cell lines, lymphoma and leukemia cell lines, keratinocytes, fibroblasts, and smooth muscle cells (130 –134). Where investigated, the cell cycle arrest induced by cyclosporin A has been in G1, although distinct mechanisms have been proposed (Table 2). For example, studies in both lymphoid and nonlymphoid cells have demonstrated that cyclosporin A induced TGF␤ expression that, in turn, led to increased levels of p21. This increase in p21 levels induced a G1 arrest, and the effects of cyclosporin A were blocked with neutralizing antibodies to TGF␤ (135, 136). Interestingly, Tomono et al. (137) demonstrated that growth factor stimulated Swiss 3T3 cells arrested in G1 with cyclosporin A treatment and showed a reduction in cyclins A and E, but not

727

cyclin D1. Both of these studies suggest that cyclosporin A inhibits G1 progression by preventing cdk2 activation. In contrast, recent studies have implicated calcineurin function earlier in G1 through the regulation of cyclin D/cdk4 activation. These studies have implicated calcineurin in the regulation of the transcription, and therefore expression, of both cyclin D and cdk4. Cyclosporin A treatment induced a G1 arrest in pancreatic acinar cells (AR42J) with low levels of cyclin D protein (138). This reduction in protein was associated with low levels of cyclin D1 mRNA, and this transcriptional effect was mapped to the cAMP response element present in the 5⬘-regulatory region of cyclin D1. Interestingly, the steady-state levels of cAMP response element binding protein were reduced in cyclosporin Atreated cells, but how calcineurin regulated cAMP response element binding protein levels was unclear. Next, calcineurin and NFATc2 have been implicated in the regulation of the cdk4 promoter (139). Evidence suggested that NFATc2 inhibits the basal activity of the cdk4 promoter; therefore, cells lacking calcineurin A␣ or NFATc2 had higher levels of cdk4 protein. Taken together, these results suggest that calcineurin function promotes cyclin D1 expression and inhibits cdk4 expression. Because both cyclin D and cdk4 are transcriptionally induced during G1, these results seem at odds. However, one possibility is that the regulation of cyclin D/cdk4 by calcineurin is cell type specific or is dependent on the proliferative state of the cell. In T lymphocyctes, Baksh et al. (140) demonstrated a more direct relationship between calcineurin and cdk4. They found that calcineurin could dephosphorylate and inactivate cdk4 directly, suggesting that it may play a role in the in-

TABLE 2. Cell cycle effects of CaM antagonists, KN-93, cyclosporin A, and FK506 in mammalian cells Inhibitor

Target

Cell line

W-7 W-13

CaM CaM

CHO-K1 CHO-K1

W-7

CaM

WI-38 IMR-90

W-13

CaM

NRK

W-7, W-13, TFP KN-93

CaM CaMK

MDA-MB-231 HeLa

KN-93

CaMK

NIH 3T3

KN-93

CaMK

HeLa

CsA/FK506 CsA/FK506

CNA CNA

A541 Swiss 3T3

CsA

CNA

CsA CsA

CNA CNA

CsA/FK506 CsA CsA/FK506

CNA CNA CNA

T cells A-549 Keratinocytes Smooth muscle cells Dermal fibroblasts Jurkat cells AR42J Lymphocytes

Results

Inhibits proliferation Arrests at two points upon release from plateau phase: Early in G0/G1 and near G1/S transition Arrests in G1 after mitogenic stimulation: Low histone H1 activity Hypophosphorylated Rb Arrests in G1 after mitogenic stimulation: Low cyclin D/cdk4 activity Cyclin D/cdk4 remains cytoplasmic Inhibits colony formation Arrests in G1: Elevated histone H1 activity Arrests in G1 after mitogenic stimulation: Decreased cdk4 activity and decreased cyclin D1 Decreased cdk2 activity and elevated p27 Arrests at G2/M: Prevents cdc25c phosphorylation and activation Inhibits DNA synthesis after mitogenic stimulation Inhibits DNA synthesis after mitogenic stimulation: Decreased cyclins E and A Inhibition of cell cycle progression: Induction of p21 via TGF␤ Inhibits proliferation and DNA synthesis Inhibits DNA synthesis after mitogenic stimulation Increased cdk4 activity Decreased cyclin D1 mRNA and protein Increased cdk4 expression

CsA, Cyclosporin A; CNA, calcineurin A; CHO, Chinese hamster ovary.

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Ref.

84 55 30, 33 85 87 170 171, 172 175 133 134, 137 136 130 132 140 138 139

728

Endocrine Reviews, December 2003, 24(6):719 –736

activation of cdk4 in mitosis (140). In our laboratory, we have examined the requirement for calcineurin in normal human fibroblasts (WI-38). We chose diploid fibroblasts, which are strictly dependent on Ca2⫹/CaM for proliferation, because they are unlikely to possess mutations or alterations in key cell cycle-regulatory pathways. Similar to Schneider et al. (138), we found that cyclosporin A induces a G1 arrest that is characterized by low levels of cyclin D1 protein (our unpublished data). In contrast to their results in pancreatic acinar cells, we found normal levels of cyclin D1 mRNA). In WI-38 cells, cyclosporin A dramatically reduced the amount of newly synthesized cyclin D1 protein, and expression of constitutively active calcineurin promoted cyclin D1 synthesis during mid-G1. Clearly, the regulation of cyclin D/cdk4 by calcineurin becomes a complicated issue with calcineurin potentially regulating the transcription and translation of cyclin D1 as well as the transcription and phosphorylation status of cdk4. Although cyclosporin A prevents G1 progression in several cell types, the mechanisms by which it induces a cell cycle arrest are diverse; therefore, one wonders whether these effects are related or merely distinct pathways in each cell type. Although it is clear that cyclosporin A possesses antiproliferative effects in numerous cell types, what is not clear is whether these effects are specifically due to calcineurin inhibition. Both cyclosporin A and FK506 bind cyclophilins and FK506 binding proteins (FKBPs) respectively (collectively known as immunophilins), and the resulting drugimmunophilin complexes inhibit both the Ca2⫹ and Ca2⫹/ CaM-stimulated activity of calcineurin (128). The antiproliferative effects of cyclosporin A require higher drug concentrations than does inhibition of T cell activation, and FK506 is less potent than cyclosporin A in its antiproliferative effects, although FK506 is a more potent immunosuppressive agent (141, 142). Therefore, the question arises whether calcineurin, immunophilins, or both are the targets of cyclosporin A and FK506 in cells other than T cells. Although both immunosuppressive compounds inhibit the prolyl isomerase activity of immunophilins, in T cells, the concentration of immunophilins far exceeds that of calcineurin and, as a result, the low drug concentrations used for immunosuppression inhibit the majority of calcineurin while binding a relatively small fraction of the total pool of immunophilins. Other cells, such as neurons and cardiomyocytes, have approximately 40 times more calcineurin than T cells and, therefore, higher drug concentrations should be required to inhibit the majority of calcineurin and a greater fraction of the immunophilin pool is bound to drug and therefore inhibited (141, 142). Additionally, only some forms of cyclophilins and FKBPs are able to form active drugimmunophilin complexes capable of inhibiting calcineurin (143). In most cases, the cellular complement of cyclophilins and FKBPs is unknown and the active pool may be limited. Evidence suggests that immunophilins may limit the degree of calcineurin inhibition. At high concentrations, these drugs are unable to inhibit all calcineurin activity, and cyclosporin A inhibits a greater percentage of calcineurin than FK506. Addition of exogenous immunophilins increases the degree of calcineurin inhibition, suggesting that immunophilins

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

may be limiting for calcineurin inhibition (144). This idea is supported by the fact that transfection of T cells with cyclophilins A or B as well as FKBP12 renders the cells more sensitive to cyclosporin A and FK506, respectively (143). In T cells, 100% calcineurin inhibition is not required to fully inhibit NFAT dephosphorylation, but 100% calcineurin inhibition may be necessary in other cellular contexts (144). Importantly, examples exist for both cyclosporin A and FK506 having cellular effects that are independent of calcineurin inhibition (110, 142). One must realize that inhibition or disruption of immunophilins, or other unknown proteins, may contribute to effects seen with cyclosporin A and FK506. Although cyclosporin A and FK506 represent the best pharmacological inhibitors for probing calcineurin function in cells and animals, using these agents does not guarantee that any results are due solely to disruption of calcineurin function. V. Role of Ca2ⴙ/CaM-Dependent Kinases (CaMKs) in Cell Proliferation A. Structure and biochemistry of CaMKs

The multifunctional CaMKs are a family of serine/threonine protein kinases that include CaMKI, CaMKII, and CaMKIV (145–147). These kinases have an amino-terminal catalytic domain followed by a carboxy-terminal regulatory domain (Fig. 3). The regulatory domain consists of overlapping autoinhibitory and Ca2⫹/CaM binding domains. Similar to calcineurin, the autoinhibition is relieved upon Ca2⫹/ CaM binding. CaMKII, unlike CaMKI and CaMKIV, has an additional association domain carboxy terminal from the regulatory domain (148 –150). This domain enables CaMKII to form multimeric structures, whereas CaMKI and CaMKIV are monomeric enzymes (145, 147). Although all these kinases are regulated by phosphorylation, the mechanism and enzymatic consequences differ between the kinases (Table 3). After Ca2⫹/CaM binding, CaMKII autophosphorylates in an intraholoenzyme, intersubunit reaction (148 –150). Phosphorylation of Thr286 has two important consequences. First, the enzyme becomes autonomous, or Ca2⫹/CaM independent, after the dissociation of Ca2⫹/CaM. Second, the enzyme acquires a property called “CaM-trapping,” in which the affinity of the enzyme for CaM is increased more than 1000-fold. Recently, these changes in CaMKII after autophosphorylation have been shown to be mimicked by CaMKII association with the N-methyl-d-aspartate receptor NR2B subunit in the absence of phosphorylation (151). In contrast to CaMKII, both CaMKI and CaMKIV are phosphorylated on an activation-loop threonine by an upstream kinase, Ca2⫹/CaM-dependent protein kinase kinase (CaMKK) (147, 152). This phosphorylation results in maximal enzyme activation. For CaMKIV, phosphorylation allows a considerable degree of autonomous activity. However, recent work on CaMKI suggests that this phosphorylation may not strictly regulate enzyme activity. Rather, the peptide substrate specificity changes between the dephosphorylated and phosphorylated enzyme, resulting in activation-independent and -dependent peptide substrates (153). Although this idea is provocative, the identification of

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

Endocrine Reviews, December 2003, 24(6):719 –736

729

TABLE 3. Properties of the mammalian, multifunctional CaMKs CaMKI

Tissue distribution

Ubiquitous

CaMKIV

CaMKII

Limited (testis, thymus, and brain)

Subcellular localization Subunit composition Phosphorylation

Cytoplasmic

Predominantly nuclear

Monomeric Thr177 phosphorylation by CaMKK

Autonomous activity

None

Monomeric Thr196 phosphorylation by CaMKK and autophosphorylation Partial

Inhibition by KN-62 (KI) Substrate consensus sequence

0.8 ␮M

3 ␮M

Hyd-X-R-X-X-S/T-X-X-X-Hyd

Hyd-X-R-X-X-S/T

endogenous protein substrates that are activation independent or -dependent remains an important challenge. Another distinction between the multifunctional CaMKs is tissue expression and subcellular distribution. There are four genes encoding multiple isoforms of CaMKII. Two genes, CaMKII␣ and CaMKII␤, are expressed predominantly in neurological tissues, and two genes, CaMKII␦ and CaMKII␥, are expressed predominantly in somatic tissues (145, 150). Additionally, these CaMKII gene products are processed into numerous splice variants that are differentially expressed in tissues. Although most splice variants of CaMKII are cytoplasmic enzymes, an alternatively spliced NLS enables at least one isoform encoded by each of the four genes to be a predominantly nuclear enzyme (145). Interestingly, tumor cells expressed different variants of CaMKII, although the functional relevance of changes in CaMKII variants during tumorigenesis is unknown (154, 155). In terms of protein structure and enzymatic activation, CaMKI and CaMKIV are fairly similar. However, these enzymes distinguish themselves by tissue distribution and subcellular localization as well as by substrate preference (147). CaMKI is ubiquitously expressed and localized to the cytoplasm. In contrast, CaMKIV expression is more limited, with the highest levels of protein found in brain, thymus, and testis. This enzyme is largely nuclear. Due to its limited distribution, CaMKIV is unlikely to be universally required for proliferation. However, CaMKIV expression is up-regulated in several types of tumors, including lung, endometrial, and ovarian cancers (156 –158). Whereas CaMKI and CaMKIV have similar substrate preferences based on peptide studies with the consensus sequence Hyd-X-R-X-X-S/T, our laboratory has recently demonstrated that CaMKI and CaMKIV phosphorylate different sites within an aminoterminal fragment of p300 (159). CaMKI phosphorylated Ser89 [84LLRSGSSPNL (93)], which corresponds to the expected consensus sequence, whereas CaMKIV phosphorylated Ser24 [19SSPALSASAS (28)], which represents a novel site with little similarity to the proposed CaMKIV consensus site based on peptide phosphorylation studies. In mammalian cells, the CaMKK enzymes, which phosphorylate CaMKI and CaMKIV, consist of two enzymes, CaMKK␣ and CaMKK␤ (147, 152). These enzymes share a similar structure to other CaMKs with an amino-terminal

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Ubiquitous CaMKII␣/␤–neuronal CaMKII␦/␥–somatic Cytoplasmic and nuclear (if NLS present) Homo- or heteromultimeric Intrasubunit autophosphorylation on Thr286 Yes via autophosphorylation or NR2 binding 0.8 ␮M Hyd-X-R-NB-X-S/T

Ref.

Reviewed in 145, 147

Reviewed in 145, 147 Reviewed in 145, 147 Reviewed in 145, 147 Reviewed in 145, 147 184 185, 186

catalytic domain and a carboxy-terminal regulatory domain. Both kinases phosphorylate and activate CaMKI and CaMKIV equally well. At the current time, the major difference shown to exist between the two forms is the degree of Ca2⫹/CaM dependence. Although CaMKK␣ was dependent on Ca2⫹/CaM binding to relieve autoinhibition, CaMKK␤ possessed significant activity in the absence of Ca2⫹/CaM (160 –162). CaMKK␣ and CaMKK␤ are highly expressed in neurological tissues with variable expression in other tissues. Interestingly, all cells that expressed CaMKIV also expressed CaMKK␤, leading to speculation that CaMKI and CaMKIV might be regulated by different CaMKKs in the cell (160). However, both kinases were localized to the cytoplasm. Therefore, a major dilemma in the field arises: how does a cytoplasmic CaMKK phosphorylate the nuclear enzyme, CaMKIV, or could there be other nuclear kinases capable of phosphorylating CaMKIV? B. Genetic analysis of CaMK function

In S. cerevisiae, two genes homologous to CaMKII, cmk1 and cmk2, have been identified (163–165). Neither gene is essential when deleted alone or in combination. However, deletion of cmk1 and cmk2 lowered the LD50 of pheromone, suggesting that both calcineurin and CaMK have a role in the response of yeast cells to pheromone (166). In S. pombe, two CaMK genes have also been identified. Unfortunately, the effects of deleting cmk1, a CaMKI homolog, has yet to be characterized in fission yeast. However, its mRNA expression was regulated in a cell cycle-dependent manner, peaking at G1/S (167). A second CaMK homologous gene, cmk2, was not essential, but its mRNA also peaked at G1/S (168). Therefore, examination of the effects of cmk1 deletion, either alone or in combination with cmk2, on S. pombe growth will be informative. In A. nidulans, three CaMK homologs have been identified: CMKA, a CaMKII homolog; CMKB, a CaMKI or CaMKIV homolog; and, CMKC, a CaMKK homolog (104, 105, 169). In contrast to yeast, two of these genes are essential in A. nidulans. CmkA was essential and required for the G2/M transition (105). When constitutively active CMKA was overexpressed, spores failed to enter the first S phase and prematurely activated NIMXcdc2. CmkB was also essential but

730

Endocrine Reviews, December 2003, 24(6):719 –736

appeared to be required sometime between sporulation and initial S phase entry. Strains with delayed and reduced CMKB expression demonstrated a delay in reentry from sporulation as well as in NIMXcdc2 activation as demonstrated by histone H1 phosphorylation assays (104). Although not essential, cmkC null strains also showed a delay in NIMXcdc2 activation after sporulation. These results suggested that the CaMKI homolog, CMKB (and perhaps its upstream activating kinase, CMKC), regulated NIMXcdc2 activation in late G1 before DNA replication, and that the CaMKII homolog, CMKA, regulated the G2/M transition. C. CaMK function in mammalian systems

Similar to the findings in A. nidulans, studies in mammalian cells suggest that one or more CaMK functions in G1 as well as at the G2/M transition. One drawback to these studies is that the majority of them relied on the use of the selective CaMK inhibitors, KN-62 or KN-93. Although often suggested to be CaMKII specific, KN-62 actually inhibits all three multifunctional CaMKs with similar efficacy (Table 3). KN-62 or KN-93 demonstrated antiproliferative effects in a variety of cells. In HeLa cells, KN-93 caused a G1 arrest (170). At the arrest point, p13-precipitable histone H1 kinase activity was elevated 4-fold, suggesting the arrest was downstream of cdk2 activation. However, the activities of neither cyclin E/cdk2 nor cyclin A/cdk2 were evaluated individually. In NIH 3T3 cells, KN-93 arrested both asynchronous cells and mitogen-stimulated cells in G1 (171). With asynchronous cells, the arrest was reversible after 2 d of drug treatment, but by 3 d of treatment, the cells began to undergo apoptosis. In subsequent experiments, KN-93 treatment was found to prevent cdk4 and cdk2 activation as well as pRb phosphorylation (172). The authors concluded that cdk4 was inactive due to reduced levels of cyclin D, although cyclins E and A were expressed normally. The reduction of cdk2 activity was associated with elevated levels of p27, but the amount of p27 associated with cdk complexes was not evaluated. These results in NIH 3T3 cells differ dramatically from the results in HeLa cells in which cdk activity was elevated. One way to rationalize these results is to speculate that KN-93 may arrest these cells at different points in G1 with NIH 3T3 cells arresting at the first CaM-dependent step in G1 early after mitogenic stimulation and HeLa cells arresting later in G1 at the second CaM-dependent step near the G1/S boundary. Another possibility is that HeLa, a transformed cell line, has lost a CaM-dependent step in G1 which is still present in NIH 3T3 cells, which are immortalized but not transformed. To learn more about the function of CaMKs in G1 and to identify which CaMK may be required for G1 progression, we examined the requirements for CaMK function in G1 progression in normal fibroblasts (WI-38). In these cells, KN-93 treatment arrested cells in G1 with low levels of cdk4 activity and hypophosphorylated pRb (our unpublished results). Unlike results of Morris et al. (172) in which cyclin D levels were reduced by KN-93 treatment, cyclin D was expressed at normal levels in WI-38 cells treated with KN-93. Indeed, cyclin D/cdk4 kinase complexes were assembled, phosphorylated, and localized to the nucleus in KN-93-

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

treated cells (Fig. 2). Therefore, CaMK inhibition appeared to block a very late, uncharacterized step in cdk4 activation, suggesting that a CaMK may be the late G1 target of Ca2⫹/ CaM. To evaluate the relevant CaMK, we expressed mutant forms of the two multifunctional CaMKs expressed in WI-38 cells (CaMKI and CaMKII). These kinase-deficient enzymes have the lysine in the ATP-binding loop mutated to prevent ATP binding. We asked whether overexpression of either protein could act as a “dominant negative” by mimicking the KN-93 arrest. Expression of kinase-deficient CaMKI, but not CaMKII, during G1 prevented cdk4 activation similar to KN93. As with the studies in A. nidulans, these results suggest that CaMKI, rather than CaMKII, regulates G1 progression. One potential role for CaMKII during G1/S is centrosome duplication. In 1990, CaMKII was localized to the centrosome in mammalian cells (173). Recently, Matsumoto and Maller (174) implicated CaMKII function in centrosome duplication in Xenopus egg extracts. Both Ca2⫹ chelation and CaMKII inhibition blocked centrosome duplication in egg extracts. Importantly, readdition of CaMKII and CaM to the extracts restored centrosome duplication in this system. To date, the function of CaMKII in regulating centrosome duplication in mammalian cells is unknown, but it will be critical to further investigate this pathway because many tumors show excess numbers of centrosomes due to aberrant duplication. Whether or not CaMKII is essential for G1 progression, this enzyme probably acts to regulate the G2/M transition and mitotic progression in mammalian systems. When HeLa cells were synchronized in S phase and released into KN-93, they arrested at G2/M without detectable cdc25C hyperphosphorylation (175). At the G2/M transition, cdc25C, a dual-specificity phosphatase, is activated by phosphorylation. Once activated, it removes the inhibitory tyrosine phosphorylation on cdc2, leading to its activation (176, 177). In vitro, CaMKII phosphorylated inactive cdc25C and marginally increased its activity, suggesting that CaMKII may be one relevant cdc25C kinase in cells (175). Again, these results differ dramatically from earlier results in which HeLa cells arrested in G1, not in G2, upon KN-93 treatment. Another group found that KN-93 treatment delayed mitotic progression in HeLa cells, but those cells did progress through mitosis (178). Although all these groups used HeLa cells, the major difference between the studies was the proliferative state of cells when KN-93 was added, one being asynchronously growing cells and the other being cells synchronized in S phase. One explanation could be that cells in mid-G1 contain about 50% less CaM than in S phase and, therefore, the cells are more sensitive to KN-93 in G1 (55, 56). By using an S phase synchronization, the G1 arrest point was avoided, which enabled evaluation of the G2/M transition. However, a role for CaMKII in G2/M progression is supported by the work in A. nidulans, in which the CaMKII homolog was required for this transition, and expression of constitutively active CaMKII prematurely activated NIMXcdc2 (105, 179). CaMKII is clearly the target of the Ca2⫹ signal required for the metaphase to anaphase transition. Unfertilized marine eggs are arrested at metaphase of meiosis II by cytostatic factor, the product of the c-mos protooncogene. Upon fertilization, intracellular Ca2⫹ increases result in the inactivation of cytostatic factor and M phase-promoting factor, the

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

cyclin/cdc2 complex (180 –182). First, it was demonstrated that CaM was required for cyclin degradation, followed by the demonstration that CaMKII was the target of CaM at this point. Although inhibitors of CaMKII blocked cyclin degradation and cdc2 inactivation after a Ca2⫹ signal, constitutively active CaMKII promoted cyclin degradation and cdc2 inactivation in the absence of a Ca2⫹ signal. Therefore, CaMKII is a critical regulator of the metaphase to anaphase transition in egg extracts, but this pathway still needs to be investigated during a mitotic, rather than meiotic, cell cycle. VI. Conclusions and Perspectives

In summary, cells require Ca2⫹/CaM-dependent pathways to grow and divide. Although Ca2⫹/CaM-dependent pathways probably act at innumerable points during cell cycle progression, the best-characterized pathways to date are during G1 and G2/M progression. Unfortunately, conserved Ca2⫹/CaM-dependent pathways common to both unicellular and multicellular eukaryotes that regulate progression through these cell cycle transitions remain ambiguous. Although not absolutely required for all types of cell proliferation, calcineurin and the family of CaMKs represent two potential Ca2⫹/CaM-dependent enzymes that regulate

Endocrine Reviews, December 2003, 24(6):719 –736

731

cell cycle transitions. Importantly, experimental evidence demonstrates that these homologous enzymes regulate similar cell cycle transitions in A. nidulans and mammalian cells. Both Ca2⫹ and CaM are required at least two points in G1, and both points are prior to the activation of cyclin D/cdk4 and pRb hyperphosphorylation (Fig. 4). Available evidence strongly suggests that these Ca2⫹/CaM-dependent pathways directly or indirectly regulate cyclin D1/cdk4 activity. Cyclosporin A, an inhibitor of calcineurin function, regulates the expression of both cyclin D1 and cdk4. Intriguingly, cyclosporin A appears to inhibit cyclin D1 accumulation, whereas it appears to promote cdk4 expression. At first, these results seem incompatible, but the studies were carried out by different groups in different cell types. Additional experiments will be necessary to determine whether calcineurin plays a more general role in regulating cyclin D1/cdk4 expression in multiple tissues and cell types. At this point, we favor a function of calcineurin in promoting cyclin D1 accumulation in early G1, which would be consistent with the early G1 requirement for Ca2⫹/CaM. One or more of the CaMKs must also be required for G1 progression because KN-93, an inhibitor of the multifunctional CaMKs, prevents G1 progression in both normal and transformed cells (Fig. 4). Whereas the nature of this arrest

FIG. 4. Schematic model of how calcineurin and CaMKs regulate mammalian cell cycle transitions. In early/mid G1, inhibitors of both calcineurin and CaMK arrest cells before the activation of cyclin D1/cdk4. Cyclosporin A, an inhibitor of calcineurin function, arrests cells with low levels of cyclin D1 protein, which may be due to transcriptional or translation effects or both. Calcineurin also appears to regulate cdk4 transcription as well as the phosphorylation status of cdk4 in certain cell types. KN-93, an inhibitor of the multifunctional CaMKs, also arrests cells before cyclin D1/cdk4 activation, but the nature of this G1 arrest varies between cell types. Some studies suggest an early G1 arrest with low levels of cyclin D1 protein, whereas others suggest a much later arrest after cyclin D1/cdk4 complex assembly. In late G1 or S phase, inhibition of calcineurin or CaMK leads to p21 and p27 accumulation, respectively. Increased levels of p21 or p27 are sufficient to lead to cdk2 inactivation and a cell cycle block. Finally, CaMKII is the most likely target of Ca2⫹/CaM-dependent pathways at the G2/M and anaphase to metaphase transitions. Inhibition or loss of CaMKII function leads to a G2 arrest with inactive cdc2 in both mammalian and fungal systems.

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

732

Endocrine Reviews, December 2003, 24(6):719 –736

varies between cell types, KN-93 arrests nontransformed cells before cyclin D1/cdk4 activation. Unfortunately, the mechanism by which CaMK inhibition prevents cdk4 activation differs among cell types, and no previous studies have addressed which CaMK is required at this point in G1. We found that kinase-deficient CaMKI, but not CaMKII, acts like a “dominant negative,” and its expression arrested cells with low cdk4 activity and hypophosphorylated pRb. Could CaMKI, not CaMKII, be the target of Ca2⫹/CaM-dependent pathways in G1? Studies in A. nidulans support a role for CaMKI function during G1. CMKA, the CaMKI homolog, is essential and reduction of its expression delayed the activation of NIMXcdc2 after sporulation and before DNA synthesis. Taken together, CaMKI probably represents one target of Ca2⫹/CaM-dependent signaling during G1 progression and acts to regulate the activation of the G1 cdks. Some reports implicate both calcineurin and CaMK in the progression through late G1 and S phase. Cyclosporin A results in p21 accumulation secondary to TGF␤ induction, and KN-93 causes a rise in p27 levels. An increase in either p21 or p27 is sufficient to inhibit cdk2 complexes in late G1 or S phase. One problem that arises from these studies is the fact that it becomes unclear whether the accumulation of p21/p27 proteins represents a “direct” target or is merely the cellular response to a prolonged arrest earlier in G1. Currently, all the experimental evidence in mammalian cells, A. nidulans, and Xenopus extracts points to CaMKII and its homologs as the target of Ca2⫹/CaM-dependent pathways at the G2/M and metaphase to anaphase transitions (Fig. 4). In HeLa cells, KN-93 blocked G2 progression with low cdc2 activity. In vitro, CaMKII directly phosphorylated and increased the activity of cdc25C, the phosphatase responsible for cdc2 activation at the G2/M transition. In A. nidulans, CMKA, the CaMKII homolog, is essential and required for G2/M progression. Overexpression of a constitutively active CMKA prematurely activated NIMXcdc2, suggesting that CMKA may also phosphorylate and activate NIMTcdc25 in this organism. Therefore, at the G2/M transition, Ca2⫹/CaM regulates CaMKII, which may promote cdc2 activation. Finally, CaMKII is the target of Ca2⫹/CaM at the metaphase to anaphase transition during a meiotic cell cycle. In Xenopus egg extracts, CaMKII clearly regulates the metaphase to anaphase transition, and its activity led to the degradation of cyclin and inactivation of cdc2. Unfortunately, this pathway has yet to be characterized in cultured mammalian cells. The fact that KN-93 delayed the metaphase to anaphase transition in HeLa cells suggests that CaMKII may play a role in the regulation of this transition in a mitotic cell cycle, but is not absolutely required. Hormones, growth factors, and cytokines act as critical regulators of cell cycle progression. Both steroid and peptide hormones are known to regulate the activity of cyclin/cdk complexes. Hormones function at multiple levels of cdk regulation including the expression of cyclins and CKIs, the subcellular localization of cyclin/cdk complexes, and the subunit composition of cdk complexes. Hormones and growth factors elicit cellular responses by inducing intracellular Ca2⫹ transients. Ca2⫹ and its intracellular receptor,

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

CaM, are universally required for cell cycle progression. Based on our current understanding, Ca2⫹/CaM-dependent pathways are also involved at multiple levels of cdk regulation, similar to hormone action. Future experimental studies designed for understanding how hormones affect the Ca2⫹/CaM-dependent pathways involved in cell cycle progression are greatly needed as well as an understanding of how these pathways differ in various tissues and cell types within those tissues. Additionally, both hormonal regulation and Ca2⫹/CaM-dependent regulation of cell proliferation are often disrupted in human tumors. By investigating how hormones and Ca2⫹/CaM regulate normal cell proliferation in different tissues, we ultimately may gain insight into how these pathways are disrupted in human cancer. Acknowledgments We thank James Joseph for valuable discussions and critical reading of the manuscript. We also thank Josep Colomer for discussions regarding his unpublished data and Libby MacDougall for technical assistance. Address all correspondence and requests for reprints to: Dr. Anthony R. Means, Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, North Carolina 27710. Email: [email protected] This work was supported by NIH Grants HD-07503 and GM-33976 (to A.R.M.) and by an NIH Medical Scientist Training Program award (to C.R.K.).

References 1. Berridge MJ, Lipp P, Bootman MD 2000 The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1:11–21 2. Carafoli E 2002 Calcium signaling: a tale for all seasons. Proc Natl Acad Sci USA 99:1115–1122 3. Petersen OH, Petersen CC, Kasai H 1994 Calcium and hormone action. Annu Rev Physiol 56:297–319 4. Taylor CW 1995 Why do hormones stimulate Ca2⫹ mobilization? Biochem Soc Trans 23:637– 642 5. Hanahan D, Weinberg RA 2000 The hallmarks of cancer. Cell 100:57–70 6. Elledge SJ 1996 Cell cycle checkpoints: preventing an identity crisis. Science 274:1664 –1672 7. Nigg EA 2001 Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2:21–32 8. Morgan DO 1995 Principles of CDK regulation. Nature 374: 131–134 9. Morgan DO 1997 Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 13:261–291 10. Morgan DO, Fisher RP, Espinoza FH, Farrell A, Nourse J, Chamberlin H, Jin P 1998 Control of eukaryotic cell cycle progression by phosphorylation of cyclin-dependent kinases. Cancer J Sci Am 4(Suppl 1):S77–S83 11. Lew DJ, Weinert T, Pringle JR 1997 Cell cycle control in Saccharomyces cerevisiae. In: Pringle JR, Broach JR, Jones EW, eds. The yeast Saccharomyces: cell cycle and cell biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 607– 695 12. Sherr CJ, Roberts JM 1999 CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512 13. Sherr CJ 1996 Cancer cell cycles. Science 274:1672–1677 14. Ekholm SV, Reed SI 2000 Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle. Curr Opin Cell Biol 12:676 – 684 15. Lundberg AS, Weinberg RA 1998 Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol Cell Biol 18:753–761 16. Weinberg RA 1995 The retinoblastoma protein and cell cycle control. Cell 81:323–330

Kahl and Means • Calcium/Calmodulin and the Cell Cycle 17. Nevins JR, DeGregori J, Jakoi L, Leone G 1997 Functional analysis of E2F transcription factor. Methods Enzymol 283:205–219 18. Nevins JR, Leone G, DeGregori J, Jakoi L 1997 Role of the Rb/E2F pathway in cell growth control. J Cell Physiol 173:233–236 19. Yam CH, Fung TK, Poon RY 2002 Cyclin A in cell cycle control and cancer. Cell Mol Life Sci 59:1317–1326 20. Stillman B 1996 Cell cycle control of DNA replication. Science 274:1659 –1664 21. Kishimoto T, Okumura E 1997 In vivo regulation of the entry into M-phase: initial activation and nuclear translocation of cyclin B/Cdc2. Prog Cell Cycle Res 3:241–249 22. Ortega S, Malumbres M, Barbacid M 2002 Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 1602: 73– 87 23. Hall M, Peters G 1996 Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv Cancer Res 68:67–108 24. Pestell RG, Albanese C, Reutens AT, Segall JE, Lee RJ, Arnold A 1999 The cyclins and cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and differentiation. Endocr Rev 20:501–534 25. Hickie RA, Wei JW, Blyth LM, Wong DY, Klaassen DJ 1983 Cations and calmodulin in normal and neoplastic cell growth regulation. Can J Biochem Cell Biol 61:934 –941 26. Hazelton B, Mitchell B, Tupper J 1979 Calcium, magnesium, and growth control in the WI-38 human fibroblast cell. J Cell Biol 83: 487– 498 27. Whitfield JF, Boynton AL, MacManus JP, Sikorska M, Tsang BK 1979 The regulation of cell proliferation by calcium and cyclic AMP. Mol Cell Biochem 27:155–179 28. Boynton AL, Whitfield JF, Isaacs RJ 1976 The different roles of serum and calcium in the control of proliferation of BALB/c 3T3 mouse cells. In Vitro 12:120 –123 29. Boynton AL, Whitfield JF, Isaacs RJ, Tremblay R 1977 The control of human WI-38 cell proliferation by extracellular calcium and its elimination by SV-40 virus-induced proliferative transformation. J Cell Physiol 92:241–247 30. Takuwa N, Zhou W, Kumada M, Takuwa Y 1992 Ca2⫹/calmodulin is involved in growth factor-induced retinoblastoma gene product phosphorylation in human vascular endothelial cells. FEBS Lett 306:173–175 31. Boynton AL 1988 Calcium and epithelial cell proliferation. Miner Electrolyte Metab 14:86 –94 32. Whitfield JF 1992 Calcium signals and cancer. Crit Rev Oncog 3:55–90 33. Takuwa N, Zhou W, Kumada M, Takuwa Y 1993 Ca2⫹-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. J Biol Chem 268:138 –145 34. Boynton AL, Whitfield JF, Isaacs RJ, Tremblay RG 1977 Different extracellular calcium requirements for proliferation of nonneoplastic, preneoplastic, and neoplastic mouse cells. Cancer Res 37:2657– 2661 35. Swierenga SH, Whitfield JF, Karasaki S 1978 Loss of proliferative calcium dependence: simple in vitro indicator of tumorigenicity. Proc Natl Acad Sci USA 75:6069 – 6072 36. Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL, Gill DL 1993 Intracellular Ca2⫹ pool content is linked to control of cell growth. Proc Natl Acad Sci USA 90:4986 – 4990 37. Greber UF, Gerace L 1995 Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus. J Cell Biol 128:5–14 38. Hussain A, Garnett C, Klein MG, Tsai-Wu JJ, Schneider MF, Inesi G 1995 Direct involvement of intracellular Ca2⫹ transport ATPase in the development of thapsigargin resistance by Chinese hamster lung fibroblasts. J Biol Chem 270:12140 –12146 39. Bootman MD, Collins TJ, Peppiatt CM, Prothero LS, Mackenzie L, De Smet P, Travers M, Tovey SC, Seo JT, Berridge MJ, Ciccolini F, Lipp P 2001 Calcium signalling–an overview. Semin Cell Dev Biol 12:3–10 40. Santella L 1998 The role of calcium in the cell cycle: facts and hypotheses. Biochem Biophys Res Commun 244:317–324

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Endocrine Reviews, December 2003, 24(6):719 –736

733

41. Berridge MJ 1995 Calcium signalling and cell proliferation. Bioessays 17:491–500 42. Hill TD, Kindmark H, Boynton AL 1988 Epidermal growth factorstimulated DNA synthesis requires an influx of extracellular calcium. J Cell Biochem 38:137–144 43. Whitfield JF, Bird RP, Chakravarthy BR, Isaacs RJ, Morley P 1995 Calcium-cell cycle regulator, differentiator, killer, chemopreventor, and maybe, tumor promoter. J Cell Biochem Suppl 22:74 –91 44. Whitaker M, Larman MG 2001 Calcium and mitosis. Semin Cell Dev Biol 12:53–58 45. Poenie M, Alderton J, Tsien RY, Steinhardt RA 1985 Changes of free calcium levels with stages of the cell division cycle. Nature 315:147–149 46. Poenie M, Alderton J, Steinhardt R, Tsien R 1986 Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science 233:886 – 889 47. Chin D, Means AR 2000 Calmodulin: a prototypical calcium sensor. Trends Cell Biol 10:322–328 48. Tjandra N, Bax A, Crivici A, Ikura M 1999 Calmodulin structure and target interaction. In: Carafoli E, Klee CB, eds. Calcium as a cellular regulator. New York: Oxford University Press; 152–169 49. Meador WE, Means AR, Quiocho FA 1992 Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science 257:1251–1255 50. Meador WE, Means AR, Quiocho FA 1993 Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. Science 262:1718 –1721 51. Roth SM, Schneider DM, Strobel LA, VanBerkum MF, Means AR, Wand AJ 1991 Structure of the smooth muscle myosin lightchain kinase calmodulin-binding domain peptide bound to calmodulin. Biochemistry 30:10078 –10084 52. Roth SM, Schneider DM, Strobel LA, Van Berkum MF, Means AR, Wand AJ 1992 Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide. Biochemistry 31:1443–1451 53. Gnegy ME 1993 Calmodulin in neurotransmitter and hormone action. Annu Rev Pharmacol Toxicol 33:45–70 54. Chafouleas JG, Bolton WE, Hidaka H, Boyd III AE, Means AR 1982 Calmodulin and the cell cycle: involvement in regulation of cell-cycle progression. Cell 28:41–50 55. Chafouleas JG, Lagace L, Bolton WE, Boyd III AE, Means AR 1984 Changes in calmodulin and its mRNA accompany reentry of quiescent (G0) cells into the cell cycle. Cell 36:73– 81 56. Brooks-Frederich KM, Cianciarulo FL, Rittling SR, Cristofalo VJ 1993 Cell cycle-dependent regulation of Ca2⫹ in young and senescent WI-38 cells. Exp Cell Res 205:412– 415 57. Piol MR, Berchtold MW, Bachs O, Heizmann CW 1988 Increased calmodulin synthesis in the pre-replicative phase of rat liver regeneration. FEBS Lett 231:445– 450 58. Agell N, Pujol MJ, Lopez-Girona A, Bosch M, Rosa JL, Bachs O 1994 Calmodulin expression during rat liver regeneration. Hepatology 20:1002–1008 59. Rasmussen CD, Means RL, Lu KP, May GS, Means AR 1990 Characterization and expression of the unique calmodulin gene of Aspergillus nidulans. J Biol Chem 265:13767–13775 60. Rasmussen CD, Means AR 1987 Calmodulin is involved in regulation of cell proliferation. EMBO J 6:3961–3968 61. Lu KP, Rasmussen CD, May GS, Means AR 1992 Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans. Mol Endocrinol 6:365–374 62. Rasmussen CD, Means AR 1989 Calmodulin is required for cellcycle progression during G1 and mitosis. EMBO J 8:73– 82 63. Schmalzigaug R, Ye Q, Berchtold MW 2001 Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B-cell receptor stimulation. Immunology 103:332–342 64. Chafouleas JG, Pardue RL, Brinkley BR, Dedman JR, Means AR 1981 Regulation of intracellular levels of calmodulin and tubulin in normal and transformed cells. Proc Natl Acad Sci USA 78:996 –1000 65. Zendegui JG, Zielinski RE, Watterson DM, Van Eldik LJ 1984 Biosynthesis of calmodulin in normal and virus-transformed chicken embryo fibroblasts. Mol Cell Biol 4:883– 889 66. Durkin JP, Whitfield JF, MacManus JP 1983 The role of calmod-

734

67.

68. 69.

70. 71. 72.

73. 74. 75. 76. 77. 78. 79.

80. 81. 82.

83. 84.

85.

86.

87. 88.

89.

Endocrine Reviews, December 2003, 24(6):719 –736 ulin in the proliferation of transformed and phenotypically normal tsASV-infected rat cells. J Cell Physiol 115:313–319 Ye Q, Wei Y, Fischer R, Borner C, Berchtold MW 1997 Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes. Biochim Biophys Acta 1359:89 –96 Klug M, Blum JK, Ye Q, Berchtold MW 1994 Intracellular Ca2⫹ and Ca2⫹-binding proteins in chemically transformed rat fibroblasts. Exp Cell Res 213:313–318 Heiman RG, Atkinson RC, Andruss BF, Bolduc C, Kovalick GE, Beckingham K 1996 Spontaneous avoidance behavior in Drosophila null for calmodulin expression. Proc Natl Acad Sci USA 93:2420 – 2425 Davis TN, Urdea MS, Masiarz FR, Thorner J 1986 Isolation of the yeast calmodulin gene: calmodulin is an essential protein. Cell 47:423– 431 Takeda T, Yamamoto M 1987 Analysis and in vivo disruption of the gene coding for calmodulin in Schizosaccharomyces pombe. Proc Natl Acad Sci USA 84:3580 –3584 Moser MJ, Flory MR, Davis TN 1997 Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation. J Cell Sci 110:1805– 1812 Brockerhoff SE, Davis TN 1992 Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae. J Cell Biol 118: 619 – 629 Ohya Y, Anraku Y 1992 Yeast calmodulin: structural and functional elements essential for the cell cycle. Cell Calcium 13:445– 455 Ohya Y, Anraku Y 1989 A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division. Curr Genet 15:113–120 Davis TN 1992 A temperature-sensitive calmodulin mutant loses viability during mitosis. J Cell Biol 118:607– 617 Ohya Y, Botstein D 1994 Diverse essential functions revealed by complementing yeast calmodulin mutants. Science 263:963–966 Lu KP, Osmani SA, Osmani AH, Means AR 1993 Essential roles for calcium and calmodulin in G2/M progression in Aspergillus nidulans. J Cell Biol 121:621– 630 Toutenhoofd SL, Strehler EE 2000 The calmodulin multigene family as a unique case of genetic redundancy: multiple levels of regulation to provide spatial and temporal control of calmodulin pools? Cell Calcium 28:83–96 Nojima H 1989 Structural organization of multiple rat calmodulin genes. J Mol Biol 208:269 –282 Nojima H, Sokabe H 1989 Structural organization of calmodulin genes in the rat genome. Adv Exp Med Biol 255:223–232 Berchtold MW, Egli R, Rhyner JA, Hameister H, Strehler EE 1993 Localization of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to chromosomes 14q24 – q31,2p21.1-p21.3, and 19q13.2-q13.3. Genomics 16:461– 465 Reddy GP, Reed WC, Sheehan E, Sacks DB 1992 Calmodulinspecific monoclonal antibodies inhibit DNA replication in mammalian cells. Biochemistry 31:10426 –10430 Hidaka H, Sasaki Y, Tanaka T, Endo T, Ohno S, Fujii Y, Nagata T 1981 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci USA 78:4354 – 4357 Taules M, Rius E, Talaya D, Lopez-Girona A, Bachs O, Agell N 1998 Calmodulin is essential for cyclin-dependent kinase 4 (Cdk4) activity and nuclear accumulation of cyclin D1-Cdk4 during G1. J Biol Chem 273:33279 –33286 Lopez-Girona A, Colomer J, Pujol MJ, Bachs O, Agell N 1992 Calmodulin regulates DNA polymerase ␣ activity during proliferative activation of NRK cells. Biochem Biophys Res Commun 184:1517–1523 Wei JW, Hickie RA, Klaassen DJ 1983 Inhibition of human breast cancer colony formation by anticalmodulin agents: trifluoperazine, W-7, and W-13. Cancer Chemother Pharmacol 11:86 –90 Taules M, Rodriguez-Vilarrupla A, Rius E, Estanyol JM, Casanovas O, Sacks DB, Perez-Paya E, Bachs O, Agell N 1999 Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization. J Biol Chem 274:24445–24458 Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Rous-

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

90.

91. 92. 93. 94.

95. 96. 97. 98.

99.

100. 101. 102.

103. 104.

105. 106.

107. 108. 109. 110. 111. 112.

sel MF, Sherr CJ 1993 Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev 7:1559 –1571 Musgrove EA, Lee CS, Buckley MF, Sutherland RL 1994 Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle. Proc Natl Acad Sci USA 91:8022– 8026 Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G 1993 Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev 7:812– 821 Li F, Wang X, Capasso JM, Gerdes AM 1996 Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 28:1737–1746 Swierenga SH, MacManus JP, Whitfield JF 1976 Regulation by calcium of the proliferation of heart cells from young adult rats. In Vitro 12:31–36 Gruver CL, DeMayo F, Goldstein MA, Means AR 1993 Targeted developmental overexpression of calmodulin induces proliferative and hypertrophic growth of cardiomyocytes in transgenic mice. Endocrinology 133:376 –388 Cyert MS 2001 Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae. Annu Rev Genet 35:647– 672 Luan Y, Matsuura I, Yazawa M, Nakamura T, Yagi K 1987 Yeast calmodulin: structural and functional differences compared with vertebrate calmodulin. J Biochem (Tokyo) 102:1531–1537 Geiser JR, van Tuinen D, Brockerhoff SE, Neff MM, Davis TN 1991 Can calmodulin function without binding calcium? Cell 65: 949 –959 Geiser JR, Sundberg HA, Chang BH, Muller EG, Davis TN 1993 The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae. Mol Cell Biol 13:7913–7924 Stirling DA, Stark MJ 2000 Mutations in SPC110, encoding the yeast spindle pole body calmodulin-binding protein, cause defects in cell integrity as well as spindle formation. Biochim Biophys Acta 1499:85–100 Stirling DA, Welch KA, Stark MJ 1994 Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body. EMBO J 13:4329 – 4342 Moser MJ, Lee SY, Klevit RE, Davis TN 1995 Ca2⫹ binding to calmodulin and its role in Schizosaccharomyces pombe as revealed by mutagenesis and NMR spectroscopy. J Biol Chem 270:20643–20652 Flory MR, Morphew M, Joseph JD, Means AR, Davis TN 2002 Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe. Cell Growth Differ 13: 47–58 McGoldrick CA, Gruver C, May GS 1995 myoA Of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth. J Cell Biol 128:577–587 Joseph JD, Means AR 2000 Identification and characterization of two Ca2⫹/CaM-dependent protein kinases required for normal nuclear division in Aspergillus nidulans. J Biol Chem 275:38230 – 38238 Dayton JS, Means AR 1996 Ca2⫹/calmodulin-dependent kinase is essential for both growth and nuclear division in Aspergillus nidulans. Mol Biol Cell 7:1511–1519 Rasmussen C, Garen C, Brining S, Kincaid RL, Means RL, Means AR 1994 The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans. EMBO J 13:2545–2552 Guerini D 1997 Calcineurin: not just a simple protein phosphatase. Biochem Biophys Res Commun 235:271–275 Klee CB, Wang X, Ren H 1999 Calcium-regulated protein dephosphorylation. In: Carafoli E, Klee CB, eds. Calcium as a cellular regulator. New York: Oxford University Press; 344 –363 Klee CB, Ren H, Wang X 1998 Regulation of the calmodulinstimulated protein phosphatase, calcineurin. J Biol Chem 273:13367–13370 Aramburu J, Rao A, Klee CB 2000 Calcineurin: from structure to function. Curr Top Cell Regul 36:237–295 Cardenas ME, Heitman J 1995 Role of calcium in T-lymphocyte activation. Adv Second Messenger Phosphoprotein Res 30:281–298 Ho S, Clipstone N, Timmermann L, Northrop J, Graef I, Fiorentino D, Nourse J, Crabtree GR 1996 The mechanism of

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

113. 114. 115. 116.

117. 118. 119. 120.

121.

122. 123. 124. 125. 126. 127. 128. 129.

130. 131. 132.

133. 134. 135.

action of cyclosporin A and FK506. Clin Immunol Immunopathol 80:S40 –S45 Schreiber SL, Crabtree GR 1992 The mechanism of action of cyclosporin A and FK506. Immunol Today 13:136 –142 Boustany LM, Cyert MS 2002 Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site. Genes Dev 16:608 – 619 Stathopoulos-Gerontides A, Guo JJ, Cyert MS 1999 Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. Genes Dev 13:798 – 803 Yoshimoto H, Saltsman K, Gasch AP, Li HX, Ogawa N, Botstein D, Brown PO, Cyert MS 2002 Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae. J Biol Chem 277:31079 –31088 Withee JL, Mulholland J, Jeng R, Cyert MS 1997 An essential role of the yeast pheromone-induced Ca2⫹ signal is to activate calcineurin. Mol Biol Cell 8:263–277 Sugiura R, Sio SO, Shuntoh H, Kuno T 2002 Calcineurin phosphatase in signal transduction: lessons from fission yeast. Genes Cells 7:619 – 627 Yoshida T, Toda T, Yanagida M 1994 A calcineurin-like gene ppb1⫹ in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning. J Cell Sci 107:1725–1735 Plochocka-Zulinska D, Rasmussen G, Rasmussen C 1995 Regulation of calcineurin gene expression in Schizosaccharomyces pombe. Dependence on the ste11 transcription factor. J Biol Chem 270: 24794 –24799 Fox DS, Cruz MC, Sia RA, Ke H, Cox GM, Cardenas ME, Heitman J 2001 Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans. Mol Microbiol 39:835– 849 Odom A, Muir S, Lim E, Toffaletti DL, Perfect J, Heitman J 1997 Calcineurin is required for virulence of Cryptococcus neoformans. EMBO J 16:2576 –2589 Fox DS, Heitman J 2002 Good fungi gone bad: the corruption of calcineurin. Bioessays 24:894 –903 Cruz MC, Fox DS, Heitman J 2001 Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans. EMBO J 20:1020 –1032 Nanthakumar NN, Dayton JS, Means AR 1996 Role of Ca⫹⫹/ calmodulin binding proteins in Aspergillus nidulans cell cycle regulation. Prog Cell Cycle Res 2:217–228 Clipstone NA, Crabtree GR 1992 Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357: 695– 697 Fruman DA, Klee CB, Bierer BE, Burakoff SJ 1992 Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proc Natl Acad Sci USA 89:3686 –3690 Liu J, Farmer Jr JD, Lane WS, Friedman J, Weissman I, Schreiber SL 1991 Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66:807– 815 Liu J, Albers MW, Wandless TJ, Luan S, Alberg DG, Belshaw PJ, Cohen P, Mackintosh C, Klee CB, Schreiber SL 1992 Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry 31:3896 – 3901 Furue M, Gaspari AA, Katz SI 1988 The effect of cyclosporin A on epidermal cells. II. Cyclosporin A inhibits proliferation of normal and transformed keratinocytes. J Invest Dermatol 90:796 – 800 Sharpe GR, Fisher C 1990 Time-dependent inhibition of growth of human keratinocytes and fibroblasts by cyclosporin A: effect on keratinocytes at therapeutic blood levels. Br J Dermatol 123:207–213 Thyberg J, Hansson GK 1991 Cyclosporine A inhibits induction of DNA synthesis by PDGF and other peptide mitogens in cultured rat aortic smooth muscle cells and dermal fibroblasts. Growth Factors 4:209 –219 Richter A, Davies DE, Alexander P 1995 Growth inhibitory effects of FK506 and cyclosporin A independent of inhibition of calcineurin. Biochem Pharmacol 49:367–373 Tomono M, Toyoshima K, Ito M, Amano H 1996 Calcineurin is essential for DNA synthesis in Swiss 3T3 fibroblasts. Biochem J 317:675– 680 Hojo M, Morimoto T, Maluccio M, Asano T, Morimoto K, Lag-

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Endocrine Reviews, December 2003, 24(6):719 –736

136. 137.

138.

139.

140. 141. 142. 143.

144. 145. 146. 147. 148. 149. 150. 151. 152. 153.

154. 155. 156.

157. 158.

735

man M, Shimbo T, Suthanthiran M 1999 Cyclosporine induces cancer progression by a cell-autonomous mechanism. Nature 397: 530 –534 Khanna AK, Hosenpud JD 1999 Cyclosporine induces the expression of the cyclin inhibitor p21. Transplantation 67:1262–1268 Tomono M, Toyoshima K, Ito M, Amano H, Kiss Z 1998 Inhibitors of calcineurin block expression of cyclins A and E induced by fibroblast growth factor in Swiss 3T3 fibroblasts. Arch Biochem Biophys 353:374 –378 Schneider G, Oswald F, Wahl C, Greten FR, Adler G, Schmid RM 2002 Cyclosporine inhibits growth through the activating transcription factor/cAMP-responsive element-binding protein binding site in the cyclin D1 promoter. J Biol Chem 277:43599 – 43607 Baksh S, Widlund HR, Frazer-Abel AA, Du J, Fosmire S, Fisher DE, DeCaprio JA, Modiano JF, Burakoff SJ 2002 NFATc2-mediated repression of cyclin-dependent kinase 4 expression. Mol Cell 10:1071–1081 Baksh S, DeCaprio JA, Burakoff SJ 2000 Calcineurin regulation of the mammalian G0/G1 checkpoint element, cyclin dependent kinase 4. Oncogene 19:2820 –2827 Crabtree GR 1999 Generic signals and specific outcomes: signaling through Ca2⫹, calcineurin, and NF-AT. Cell 96:611– 614 Vogel KW, Briesewitz R, Wandless TJ, Crabtree GR 2001 Calcineurin inhibitors and the generalization of the presenting protein strategy. Adv Protein Chem 56:253–291 Bram RJ, Hung DT, Martin PK, Schreiber SL, Crabtree GR 1993 Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location. Mol Cell Biol 13:4760 – 4769 Kung L, Halloran PF 2000 Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations. Transplantation 70:327–335 Schulman H, Braun A 1999 Calcium/calmodulin-dependent protein kinases. In: Carafoli E, Klee CB, eds. Calcium as a cellular regulator. New York: Oxford University Press; 311–335 Means AR 2000 Regulatory cascades involving calmodulin-dependent protein kinases. Mol Endocrinol 14:4 –13 Hook SS, Means AR 2001 Ca2⫹/CaM-dependent kinases: from activation to function. Annu Rev Pharmacol Toxicol 41:471–505 Soderling TR, Stull JT 2001 Structure and regulation of calcium/ calmodulin-dependent protein kinases. Chem Rev 101:2341–2352 Soderling TR, Chang B, Brickey D 2001 Cellular signaling through multifunctional Ca2⫹/calmodulin-dependent protein kinase II. J Biol Chem 276:3719 –3722 Hudmon A, Schulman H 2002 Structure-function of the multifunctional Ca2⫹/calmodulin-dependent protein kinase II. Biochem J 364:593– 611 Bayer KU, De Koninck P, Leonard AS, Hell JW, Schulman H 2001 Interaction with the NMDA receptor locks CaMKII in an active conformation. Nature 411:801– 805 Soderling TR 1999 The Ca-calmodulin-dependent protein kinase cascade. Trends Biochem Sci 24:232–236 Hook SS, Kemp BE, Means AR 1999 Peptide specificity determinants at P-7 and P-6 enhance the catalytic efficiency of Ca2⫹/ calmodulin-dependent protein kinase I in the absence of activation loop phosphorylation. J Biol Chem 274:20215–20222 Tombes RM, Mikkelsen RB, Jarvis WD, Grant S 1999 Downregulation of ␦ CaM kinase II in human tumor cells. Biochim Biophys Acta 1452:1–11 Tombes RM, Krystal GW 1997 Identification of novel human tumor cell-specific CaMK-II variants. Biochim Biophys Acta 1355: 281–292 Shang S, Takai N, Nishida M, Miyazaki T, Nasu K, Miyakawa I 2003 CaMKIV expression is associated with clinical stage and PCNA-labeling index in endometrial carcinoma. Int J Mol Med 11:181–186 Takai N, Miyazaki T, Nishida M, Nasu K, Miyakawa I 2002 Ca2⫹/calmodulin-dependent protein kinase IV expression in epithelial ovarian cancer. Cancer Lett 183:185–193 Williams CL, Phelps SH, Porter RA 1996 Expression of Ca2⫹/ calmodulin-dependent protein kinase types II and IV, and reduced DNA synthesis due to the Ca2⫹/calmodulin-dependent protein

736

159.

160.

161. 162. 163. 164.

165. 166.

167. 168. 169. 170. 171.

172.

173.

Endocrine Reviews, December 2003, 24(6):719 –736 kinase inhibitor KN-62 (1-[N, O-bis(5-isoquinolinesulfonyl)-Nmethyl-l-tyrosyl]-4-phenyl piperazine) in small cell lung carcinoma. Biochem Pharmacol 51:707–715 Corcoran EE, Joseph JD, MacDonald JA, Kane CD, Haystead TA, Means AR 2003 Proteomic analysis of calcium/calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic preferences. J Biol Chem 278:10516 –10522 Anderson KA, Means RL, Huang QH, Kemp BE, Goldstein EG, Selbert MA, Edelman AM, Fremeau RT, Means AR 1998 Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2⫹/calmodulin-dependent protein kinase kinase ␤. J Biol Chem 273:31880 –31889 Tokumitsu H, Iwabu M, Ishikawa Y, Kobayashi R 2001 Differential regulatory mechanism of Ca2⫹/calmodulin-dependent protein kinase kinase isoforms. Biochemistry 40:13925–13932 Tokumitsu H, Muramatsu M, Ikura M, Kobayashi R 2000 Regulatory mechanism of Ca2⫹/calmodulin-dependent protein kinase kinase. J Biol Chem 275:20090 –20095 Pausch MH, Kaim D, Kunisawa R, Admon A, Thorner J 1991 Multiple Ca2⫹/calmodulin-dependent protein kinase genes in a unicellular eukaryote. EMBO J 10:1511–1522 Ohya Y, Kawasaki H, Suzuki K, Londesborough J, Anraku Y 1991 Two yeast genes encoding calmodulin-dependent protein kinases. Isolation, sequencing and bacterial expressions of CMK1 and CMK2. J Biol Chem 266:12784 –12794 Miyakawa T, Oka Y, Tsuchiya E, Fukui S 1989 Saccharomyces cerevisiae protein kinase dependent on Ca2⫹ and calmodulin. J Bacteriol 171:1417–1422 Moser MJ, Geiser JR, Davis TN 1996 Ca2⫹-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2⫹-calmodulin-dependent protein kinase. Mol Cell Biol 16:4824 – 4831 Rasmussen CD 2000 Cloning of a calmodulin kinase I homologue from Schizosaccharomyces pombe. J Biol Chem 275:685– 690 Alemany V, Sanchez-Piris M, Bachs O, Aligue R 2002 Cmk2, a novel serine/threonine kinase in fission yeast. FEBS Lett 524:79 – 86 Bartelt DC, Fidel S, Farber LH, Wolff DJ, Hammell RL 1988 Calmodulin-dependent multifunctional protein kinase in Aspergillus nidulans. Proc Natl Acad Sci USA 85:3279 –3283 Rasmussen G, Rasmussen C 1995 Calmodulin-dependent protein kinase II is required for G1/S progression in HeLa cells. Biochem Cell Biol 73:201–207 Tombes RM, Grant S, Westin EH, Krystal G 1995 G1 cell cycle arrest and apoptosis are induced in NIH 3T3 cells by KN-93, an inhibitor of CaMK-II (the multifunctional Ca2⫹/CaM kinase). Cell Growth Differ 6:1063–1070 Morris TA, DeLorenzo RJ, Tombes RM 1998 CaMK-II inhibition reduces cyclin D1 levels and enhances the association of p27 kip1 with Cdk2 to cause G1 arrest in NIH 3T3 cells. Exp Cell Res 240: 218 –227 Ohta Y, Ohba T, Miyamoto E 1990 Ca2⫹/calmodulin-dependent

Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212 by guest on 28 April 2018

Kahl and Means • Calcium/Calmodulin and the Cell Cycle

174. 175.

176. 177. 178.

179.

180.

181.

182.

183.

184.

185.

186.

protein kinase II: localization in the interphase nucleus and the mitotic apparatus of mammalian cells. Proc Natl Acad Sci USA 87:5341–5345 Matsumoto Y, Maller JL 2002 Calcium, calmodulin, and CaMKII requirement for initiation of centrosome duplication in Xenopus egg extracts. Science 295:499 –502 Patel R, Holt M, Philipova R, Moss S, Schulman H, Hidaka H, Whitaker M 1999 Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells. J Biol Chem 274:7958 –7968 Jackman MR, Pines JN 1997 Cyclins and the G2/M transition. Cancer Surv 29:47–73 Nilsson I, Hoffmann I 2000 Cell cycle regulation by the Cdc25 phosphatase family. Prog Cell Cycle Res 4:107–114 Petzelt CP, Kodirov S, Taschenberger G, Kox WJ 2001 Participation of the Ca2⫹-calmodulin-activated kinase II in the control of metaphase-anaphase transition in human cells. Cell Biol Int 25: 403– 409 Dayton JS, Sumi M, Nanthakumar NN, Means AR 1997 Expression of a constitutively active Ca2⫹/calmodulin-dependent kinase in Aspergillus nidulans spores prevents germination and entry into the cell cycle. J Biol Chem 272:3223–3230 Lorca T, Galas S, Fesquet D, Devault A, Cavadore JC, Doree M 1991 Degradation of the proto-oncogene product p39 mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca2⫹-calmodulin dependent event. EMBO J 10: 2087–2093 Lorca T, Abrieu A, Means A, Doree M 1994 Ca2⫹ is involved through type II calmodulin-dependent protein kinase in cyclin degradation and exit from metaphase. Biochim Biophys Acta 1223: 325–332 Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, Mery J, Means A, Doree M 1993 Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs. Nature 366:270 –273 Sanchez-Piris M, Posas F, Alemany V, Winge I, Hidalgo E, Bachs O, Aligue R 2002 The serine/threonine kinase Cmk2 is required for oxidative stress response in fission yeast. J Biol Chem 277:17722– 17727 Hidaka H, Yokokura H 1996 Molecular and cellular pharmacology of a calcium/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, and proposal of CaM kinase phosphorylation cascades. Adv Pharmacol 36:193–219 White RR, Kwon YG, Taing M, Lawrence DS, Edelman AM 1998 Definition of optimal substrate recognition motifs of Ca2⫹-calmodulin-dependent protein kinases IV and II reveals shared and distinctive features. J Biol Chem 273:3166 –3172 Dale S, Wilson WA, Edelman AM, Hardie DG 1995 Similar substrate recognition motifs for mammalian AMP-activated protein kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1, and mammalian calmodulin-dependent protein kinase I. FEBS Lett 361:191–195