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Regulation of Cell Cycle Progression by Calcium/Calmodulin-Dependent Pathways

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Correspondence: 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. E-mail: means001{at}mc.duke.edu

	Abstract

Top Abstract I. Introduction II. Role of Calcium... III. Regulation of Cell... IV. Role of Calcineurin... V. Role of Ca2+/CaM-Dependent... VI. Conclusions and Perspectives References

 
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 (Ca²⁺). The major intracellular receptor for Ca²⁺ is calmodulin (CaM). CaM is a small protein that contains four EF-hand Ca²⁺ binding sites and is highly conserved among eukaryotes. In all organisms in which the CaM gene has been deleted, it is essential. Although Ca²⁺/CaM is required for proliferation in both unicellular and multicellular eukaryotes, the essential targets of Ca²⁺/CaM-dependent pathways required for cell proliferation remain elusive. Potential Ca²⁺/CaM-dependent targets include the serine/threonine phosphatase calcineurin and the family of multifunctional Ca²⁺/CaM-dependent protein 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 Ca²⁺/CaM and its downstream targets, particularly calcineurin and the Ca²⁺/CaM-dependent protein kinases, regulate key cell cycle-regulatory proteins, remains enigmatic. By understanding how Ca²⁺/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 Ca²⁺ and its downstream targets to proliferate.

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

	I. Introduction

Top Abstract I. Introduction II. Role of Calcium... III. Regulation of Cell... IV. Role of Calcineurin... V. Role of Ca2+/CaM-Dependent... VI. Conclusions and Perspectives References

 
CALCIUM (Ca²⁺) IS a universal second messenger that regulates a number of diverse cellular processes including cell proliferation, development, motility, secretion, 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 unicellular eukaryotes, such as yeast. Importantly, homologous pathways have been identified in multicellular eukaryotes that also serve to control cell cycle progression.

View larger version (23K): [in this window] [in a new window]   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.

Mammalian cells contain multiple cdks and cyclins, which act at different phases of the cell cycle (Fig. 1) (8, 9, 10). As cells progress through G₁, cyclin D/cdk4 complexes are first activated and phosphorylate the tumor suppressor protein, retinoblastoma (pRb) (12, 13, 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, 17, 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, 9, 10, 12). First, the activation of cdks is strictly dependent 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 accumulation 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.

View larger version (29K): [in this window] [in a new window]   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.

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⁺/CaM-dependent enzymes that have been implicated in cell cycle regulation in eukaryotes.

	II. Role of Calcium (Ca2+) in Cell Proliferation

Top Abstract I. Introduction II. Role of Calcium... III. Regulation of Cell... IV. Role of Calcineurin... V. Role of Ca2+/CaM-Dependent... VI. Conclusions and Perspectives References

 
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, 27, 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-butyl-hydroquinone, 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, 37, 38). Depletion of intracellular Ca²⁺ stores resulted in the accumulation 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, 2, 3, 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.

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, 49, 50, 51, 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⁺/CaM-dependent 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. Long-term 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

Top Abstract I. Introduction II. Role of Calcium... III. Regulation of Cell... IV. Role of Calcineurin... V. Role of Ca2+/CaM-Dependent... VI. Conclusions and Perspectives References

 
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.

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-virus-based 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.

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, 70, 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 NIMX^cdc2 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, 81, 82). Strikingly, the encoded amino acid sequences are not merely conserved among these separate 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, 84, 85, 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). 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⁺/CaM-dependent 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, 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⁺/CaM-dependent target enzymes have been identified in S. cerevisiae (Table 1). Budding yeast does possess essential Ca²⁺-independent CaM targets, such as the spindle pole body-associated 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.

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, 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, 105, 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

Top Abstract I. Introduction II. Role of Calcium... III. Regulation of Cell... IV. Role of Calcineurin... V. Role of Ca2+/CaM-Dependent... VI. Conclusions and Perspectives References

 
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, 108, 109, 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.

View larger version (23K): [in this window] [in a new window]   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.

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, 112, 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 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 nitrogen-starvation 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, 122, 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.

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, 112, 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, 127, 128, 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, 131, 132, 133, 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 TGFß 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 TGFß (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 cyclin D1. Both of these studies suggest that cyclosporin A inhibits G₁ progression by preventing cdk2 activation.

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 inactivation 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 drug-immunophilin 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 drug-immunophilin 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 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 Ca2+/CaM-Dependent Kinases (CaMKs) in Cell Proliferation

Top Abstract I. Introduction II. Role of Calcium... III. Regulation of Cell... IV. Role of Calcineurin... V. Role of Ca2+/CaM-Dependent... VI. Conclusions and Perspectives References

 
A. Structure and biochemistry of CaMKs
The multifunctional CaMKs are a family of serine/threonine protein kinases that include CaMKI, CaMKII, and CaMKIV (145, 146, 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, 149, 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, 149, 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 endogenous protein substrates that are activation independent or -dependent remains an important challenge.

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, 157, 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 amino-terminal 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 CaMKKß (147, 152). These enzymes share a similar structure to other CaMKs with an amino-terminal 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, CaMKKß possessed significant activity in the absence of Ca²⁺/CaM (160, 161, 162). CaMKK and CaMKKß are highly expressed in neurological tissues with variable expression in other tissues. Interestingly, all cells that expressed CaMKIV also expressed CaMKKß, 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, 164, 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 NIMX^cdc2. CmkBwas also essential but 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 NIMX^cdc2 activation as demonstrated by histone H1 phosphorylation assays (104). Although not essential, cmkC null strains also showed a delay in NIMX^cdc2 activation after sporulation. These results suggested that the CaMKI homolog, CMKB (and perhaps its upstream activating kinase, CMKC), regulated NIMX^cdc2 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-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 KN-93. 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 NIMX^cdc2 (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 cyclin/cdc2 complex (180, 181, 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

Top Abstract I. Introduction II. Role of Calcium... III. Regulation of Cell... IV. Role of Calcineurin... V. Role of Ca2+/CaM-Dependent... VI. Conclusions and Perspectives References

 
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 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.

View larger version (27K): [in this window] [in a new window]   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.

Some reports implicate both calcineurin and CaMK in the progression through late G₁ and S phase. Cyclosporin A results in p21 accumulation secondary to TGFß 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 NIMX^cdc2, 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, 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.

	Footnotes

 
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.).

Abbreviations: Ca²⁺, Calcium; CaM, calmodulin; CaMK, Ca²⁺/CaM-dependent kinase; CAMKK, Ca²⁺/CaM-dependent protein kinase kinase; cdk, cyclin-dependent kinase; CKI, cdk inhibitor; FKBP, FK506-binding protein; hsp, heat shock protein; MEF, mouse embryonic fibroblast; NFAT, nuclear factor of activated T cells; NLS, nuclear localization sequence; NRK, normal rat kidney.

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Top Abstract I. Introduction II. Role of Calcium... III. Regulation of Cell... IV. Role of Calcineurin... V. Role of Ca2+/CaM-Dependent... VI. Conclusions and Perspectives References

 

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