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