Department of Pharmacology and Cancer Biology Duke University Medical Center Durham, North Carolina 27710
INTRODUCTION
Elucidation of the components that comprise
signaling cascades in cells is a primary theme in molecular
endocrinology. These signal transduction pathways are frequently
initiated at the plasma membrane by the interaction of a ligand with a
cell surface receptor. This interaction can result in activation of a
number of pathways that are initiated by stimulation of signaling
proteins such as JAK/STAT, small G proteins (such as Ras), or adenylyl
cyclase (to generate cAMP). However, when the receptor is coupled to
heterotrimeric G proteins such as Gq, phospholipase C is stimulated
leading to activation of protein kinase C (PKC) and the generation of
inositol tris-phosphate (IP3) which, in turn,
releases Ca2+ from intracellular stores (Fig. 1)(1 ). Alternatively, excitable cells can
respond to membrane depolarizing stimuli by altering the gating of
voltage-dependent Ca2+ channels. In either case,
as depicted in Fig. 1
, the increase in intracellular
Ca2+ initiates signaling cascades that lead to
essential biological processes such as secretion, cell proliferation,
differentiation, and movement. Calcium concentrations are only
transiently increased, and various signals can result in changes in
either the amplitude and/or frequency of such fluxes. The diffusion of
calcium in the cell is severely restricted due to the large number of
proteins and organelles that can bind to or otherwise sequester
calcium. Thus, global intracellular Ca2+ signals
result from the coordination and summation of elementary release events
that have been called "Ca2+ puffs" and
influence the concentration of free Ca2+ in both
cytoplasm and nucleus (2 ).
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Among the plethora of Ca2+ binding
proteins in cells, the most ubiquitous and abundant one is CaM (1 ).
This is an essential protein that serves as a receptor to sense changes
in calcium concentrations and, in this fashion, mediates the second
messenger role of this ion. Calcium binds to CaM by means of a
structural motif called an EF-hand, and a pair of these structures is
located in both globular ends of the protein (Fig. 2). The binding affinity to each site is
approximately 1 uM, although the pair of EF-hands in the N
terminus is of slightly lower affinity than the pair in the C terminus.
When the four binding sites are filled, CaM undergoes a conformational
change exposing a flexible eight-turn
-helix, which separates the
hydrophobic pockets that form in each of the globular ends of the
protein. Calmodulin thus becomes "loaded" to interact with one of
its many target proteins in the cell. This target protein interaction,
while usually of high affinity [dissociation constant
(Kd) = low
nM], is rapidly reversible upon a decline in the
Ca2+ concentration. Among the several CaM binding
targets relevant to hormone-mediated signaling cascades are protein
kinases and phosphatases, adenylyl cyclases and cyclic nucleotide
phosphodiesterases, calcium pumping ATPases, ion channel and receptor
proteins, and NO synthases.
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MECHANISMS OF CaM ACTION
Binding of Ca2+/CaM to the interacting
region of any of the protein kinases causes a remarkable conformational
change whereby the central helix acts as a flexible tether as
schematically depicted in Fig. 2. The central helix bends and twists so
that the globular domains of CaM completely engulf the target peptide
(3 ). This interaction removes an inhibitory region of the kinase, which
relieves an intrasteric autoinhibition, exposes the active site, and
allows substrate binding to occur (4 ). The current conventional wisdom
suggests that, in the native enzyme, activation is accomplished by a
two-step mechanism (5 ). The first step is a hydrophobic interaction
between the C-terminal hydrophobic pocket of CaM and the N-terminal
half of the CaM-binding domain of the protein kinase to form an
inactive complex. Next, a transient increase in
Ca2+ promotes the formation of additional
contacts between both domains of CaM and the CaM-binding domain of the
enzymes. Since the CaM-binding domain and the autoinhibitory domain of
the kinases partially overlap, it is predicted that the intermolecular
contacts between CaM and the kinase CaM-binding domain result in a
simultaneous disruption of the intramolecular contacts between the
overlapping autoinhibitory domain and active site of the enzyme. Such
multiple interactions result in the removal of the entire
autoinhibitory domain from the active site, which generates an active
enzyme. It is proposed that this activating mechanism is common to all
of the Ca2+/CaM-dependent protein kinases (5 ).
However, the subsequent events that govern maintenance and inactivation
of protein kinase activity vary in a dramatic way between the four
CaM-dependent protein kinases as will be detailed below.
REGULATORY CASCADES INVOLVING MLCK
The MLCKs are derived from two genes, and an isoform is present in
all vertebrate (and many invertebrate) cells (6 ). In vitro,
these enzymes are entirely dependent on the binding of
Ca2+/CaM for activity, and a decrease in the
Ca2+ concentration results in enzyme inactivation
(7 ). Smooth muscle MLCK is rate limiting for contraction of this muscle
type, whereas it plays a much less prominent role in the contraction of
striated muscle. In nonmuscle cells, MLCK is involved in the regulation
of motility, mitosis, actin-based cytoskeleton dynamics, and secretion
(7 ). All of the biological roles of MLCK seem to involve the actomyosin
system and require phosphorylation of the regulatory myosin light chain
(MLC) on Ser 19. However, it has recently been shown that nonmuscle
MLCK can be regulated by mechanisms that appear to be independent of
changes in the intracellular Ca2+
concentration. These surprising observations raise the
possibility that cross-talk between different signal transduction
pathways may play a role in controlling the phosphorylation of the
regulatory MLCs as illustrated in Fig. 3.
First, the Rho-associated protein kinase (RAK in Fig. 3
) has been shown
to directly phosphorylate MLC on Ser 19, and this seems to be the
mechanism by which thrombin (acting through a Gq-coupled receptor)
stimulates rounding of astrocytoma cells in culture (8 ). This effect of
thrombin does not require Ca2+ mobilization or
activation of PKC, phosphatidyl inositol 3 kinase, or tyrosine
kinases, but is prevented by the ADP-ribosylation of Rho, a
modification that inhibits its function. Interestingly, inhibition of
MLCK has also been reported to occur in response to activation of the
Rho/Rac/Cdc42 pathway due to phosphorylation of MLCK by another
Rho-activated protein kinase called PAK1 (p21-activated kinase; PAK in
Fig. 3
) (9 ). This phosphorylation event is independent of
Ca2+/CaM binding to MLCK but, in cells, is
sufficient to decrease phosphorylation of MLC on Ser 19 by almost 90%.
Thus, two protein kinases that are activated by Rho have opposing
effects on MLC phosphorylation in a manner that is independent of
changes in intracellular Ca2+. On the one hand,
Rho-kinase activates the actomyosin system by phosphorylating MLC,
whereas PAK1 inhibits MLCK due to a direct phosphorylation of the
enzyme. Perhaps the overall cellular consequences of activating Rho
might depend on the timing of GTPase activation as well as on the
extent of MLC phosphorylation, which might be influenced by the
intracellular localization of actomyosin.
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CaMKII REGULATORY PATHWAYS
Unlike the MLCKs, CaMKII is a multimeric enzyme composed of 1012
catalytic subunits (12 ). The interaction domains of the 1012 subunits
present in the holoenzyme complex form the spokes of a wheel, and the
catalytic domains form the outside of the wheel (Fig. 2). Activation of
a single catalytic subunit requires that Ca2+/CaM
bind to it and to the adjacent subunit. This dual interaction allows
the catalytic domain of one subunit to phosphorylate a single Thr
residue (Thr-286 in CaMKII
) of the other. Once a subunit becomes
phosphorylated, its kinase activity becomes independent of
Ca2+/CaM binding. In addition, the phosphorylated
subunit shows a 1000-fold increase in the affinity for CaM due to a
marked change in the off rate (12 ). This unique mechanism allows CaMKII
to molecularly potentiate transient increases in
Ca2+ and apparently enables detection of the
frequency of such transients. It follows that dephosphorylation of
Thr-286 must accompany the release of Ca2+/CaM to
return the enzyme to its inactive state. Both protein phosphatases 1
and 2A (PP1 and PP2A) can effectively dephosphorylate Thr-286 in
vitro, and both enzymes appear to play physiologically relevant
roles in a manner that depends on the subcellular localization of
CaMKII (13 ).
Interestingly, CaMKII isoforms also exist in all cells of vertebrate
and invertebrate species. The isoform present in a given cell depends
on which of the four genes encoding the protein is transcribed and
which of a considerable number of alternatively spliced forms of each
primary transcript is translated (12 ). It is also possible to have
multiple isoforms in a single cell, and the ratio of the isoforms
regulates heteromultimer formation. Some of the isoforms produced from
the and
genes contain a nuclear localization sequence (NLS)
that interacts with importin and directs the enzyme to the nucleus
where it has been implicated in the regulation of transcription.
Interestingly, phosphorylation of the nuclear localization sequence
(NLS) in these forms of CaMKII by either CaMKI or IV when the CaMKII is
in the cytoplasm prevents association with importin and nuclear entry
(14 ).
Most (if not all) of CaMKII in a cell is associated with organelles and
not free in the cytosol (12 ). The first of the CaMKII isoforms to be
identified, CaMKII, is expressed exclusively in the brain and is a
major component of the postsynaptic membrane (PSD) in pyramidal
neurons. In the PSD, CaMKII is thought to increase synaptic strength by
phosphorylating ion channels and signaling proteins such as glutamate
receptors and N-methyl D-aspartate (NMDA)
receptors (12 ). In hippocampal neurons, it has been suggested
that stimulation of the NMDA receptor regulates the association of
CaMKII between F-actin and the PSD in a dynamic manner. Thus, CaMKII is
involved both in the maintenance of dendritic architecture and synaptic
plasticity. In addition, CaMKII is required for long lasting changes in
synaptic strength such as long-term potentiation (LTP), a process that
is thought to be involved in learning and memory. These functions of
CaMKII
have been confirmed by knocking out the gene in mice. The
molecular mechanisms involved in the effects of CaMKII
on LTP have
been elucidated using gene-targeting strategies that reconstitute
various mutants of CaMKII
into nullizygous animals (15 ).
Whereas the membrane and cytoskeletal roles of CaMKII are beginning to be clarified, the nuclear functions of CaMKII remain obscure (12 ). Most of the studies that have addressed nuclear function of CaMKII have relied on the ability of C-terminally truncated, Ca2+/CaM-independent forms of the enzyme to alter transcription when overexpressed in cells along with reporter gene constructs. One of the first such paradigms to be examined was the Ca2+ stimulation of immediate early genes that are regulated by cAMP response elements (CREs) such as c-fos (16 ). Phosphorylation of CREB (CRE binding protein) on Ser-133 is essential for transactivation because it is required for binding of the ubiquitously expressed CREB binding proteins CBP and p300, which function as transcriptional cointegrators (17 ). Ser-133 was originally identified as the target of protein kinase A (PKA), thus explaining the role of cAMP in transcriptional activation. However, CaMKII can also phosphorylate this residue leading to the speculation that CaMKII mediates the Ca2+ requirement for expression of the immediate early genes (16 ). Thus CREB was proposed as a target at which cAMP- and Ca2+-mediated signal transduction cascades converged to regulate transcription. However, while the truncated form of CaMKII can stimulate CREB-mediated transcription in some cells, it is inhibitory in others. Sun et al. (18 ) discovered that one mode of inhibition was that, in addition to Ser-133, CaMKII also readily phosphorylated a second residue on CREB, Ser-142. Indeed, phosphorylation of Ser-142 was not only inhibitory, but this modification was also dominant and could reverse the activation of CREB resulting from its phosphorylation on Ser-133 by PKA. The mechanism by which phosphorylation of Ser-142 inhibits CREB-mediated transcription seems to be by destabilizing the association between CREB and CBP (19 ). Interestingly, the nature of the effect of CaMKII on transcription seems to be both cell and promoter dependent. Whereas CaMKII inhibits transcription of the interleukin 2 gene in a human T cell line (20 ), it was found to stimulate atrial natriuretic factor (ANF) gene expression in ventricular myocytes (21 ). Regulatory elements sensitive to CaMKII include C/EBPß, CRE, SRE, and AP1 (12 ).
SPECIFICITY OF CaMKII INHIBITORS
The main problem with assigning specific roles in gene regulation to CaMKII is that the diagnosis is frequently based on the effects of inhibitors. The most popular CaMKII inhibitors are KN-62 and its more water- soluble cousin KN-93 (22 ). Both of these drugs are isoquinolonesulfonamides and would be expected to function as competitive ATP antagonists. The original paper describing the properties of both drugs touted them as CaMKII specific. However, the subsequent studies demonstrating that these drugs also inhibit CaMKI and CaMKIV have been largely ignored, even though the inhibition constant (Ki) for all three CaM kinases varies only by a factor of 2 (23 ). Typically, once KN-93 is shown to inhibit a cellular response and the cell is shown to contain one or more isoforms of CaMKII, it is presumed that CaMKII is the enzyme responsible for the cellular response. The next step in the analysis is to demonstrate that the Ca2+/CaM-independent form of the specific CaMKII isoform can alter transcription of a reporter gene when transfected into the cells. This, too, is dangerous as CaMKII, CaMKI, and CaMKIV have broadly overlapping substrate specificities so, at the very least, all three CaM kinases should be evaluated. Thus, with exception of the ANF gene in ventricular myocytes (21 ) it is difficult to point to an unequivocal demonstration that the Ca2+/CaM-dependent protein kinase responsible for a specific transcriptional event is CaMKII.
CASCADES INVOLVING BROAD SUBSTRATE CaM-DEPENDENT PROTEIN KINASES
As mentioned above, the other two broad-substrate, CaM-dependent
protein kinases that have been the subjects of considerable
experimental attention are CaMKI and CaMKIV (24 ). These enzymes share a
common requirement for phosphorylation by an upstream protein kinase
(CaMKK), but the details of the activating mechanisms differ
considerably. Two CaMKKs, and ß, have been identified in mammals
and are the products of individual genes (25 26 ). These upstream
kinases are also Ca2+/CaM binding proteins, which
has led to the recognition that a CaM kinase cascade roughly analogous
to a mitogen-activated protein (MAP) kinase cascade exists in cells.
Indeed, the CaMKKs phosphorylate the CaM kinase on a single Thr residue
that is positioned in the activation loop (27 28 ) (between protein
kinase subdomains VII and VIII) of the CaM kinase. This is a reaction
that is similar to that by which a MAP kinase kinase phosphorylates and
activates a MAP kinase.
CaMKI Activation and Substrates
CaMKI is present in all mammalian cells (12 ), and homologs exist
in a variety of organisms. It is largely cytoplasmic in mammalian
cells, but, at least in Caenorhabditis elegans, it appears
to be nuclear and contains a nuclear localization sequence at its N
terminus (29 ). Whether the vertebrate enzyme can be stimulated to
translocate into the nucleus alone or in combination with another
protein remains to be established. However, overexpressed full-length
CaMKI does not appear in the nucleus when cells are cultured either
under normal conditions or when stimulated to increase the
intracellular Ca2+ concentration. Therefore, if
nuclear translocation does occur, it is likely mediated by another
signaling cascade. The absence of a canonical NLS makes this
possibility a remote one.
Binding of Ca2+/CaM is required for activity of CaMKI but, unlike CaMKII and CaMKIV, it does not develop autonomous activity (27 ). In the absence of activation loop phosphorylation, CaMKI exhibits a low level of Ca2+/CaM-dependent activity when using the optimized peptide substrates such as those derived from site 1 of synapsin. The Thr-177 in the activation loop of CaMKI cannot be phosphorylated by the CaMKK until Ca2+/CaM is bound. Phosphorylation of Thr-177 decreases the Km of the enzyme for synapsin 40-fold (30 ). Removal of Ca2+/CaM inactivates CaMKI even when Thr-177 is phosphorylated but, in this state, the KCaM is decreased 4-fold suggesting the possibility that activation loop phosphorylation might sensitize the enzyme to subsequent transient increases in the Ca2+ concentration. This also suggests that dephosphorylation of Thr-177 might serve as an inactivating mechanism under certain cellular conditions. However, whereas both PP1 and PP2A will dephosphorylate Thr-177 in vitro, it is not known which phosphatase might accomplish this in vivo.
Interestingly, it has been possible to generate peptide substrates that are equally well phosphorylated by the Thr-177-phosphorylated or nonphosphorylated forms of CaMKI (and also for CaMKIV +/- phosphorylation of Thr-196) raising the intriguing possibility that activation loop-independent protein substrates for CaMKI/IV might exist in cells (30 ). If this is the case, although CaMKI and IV absolutely require Ca2+/CaM for activation, they might not require the action of a CaMKK. This was one observation that raised the possibility that the CaMKKs might have cellular substrates other than CaMKI and CaMKIV and, thus, might constitute the sole target of Ca2+/CaM in some instances rather than functioning as an upstream component in a CaM kinase cascade. A specific example of this postulate was provided by Yano et al. (31 ) who showed that a CaMKK can phosphorylate and activate protein kinase B (PKB or AKT) in vitro and when overexpressed in a neuronal cell line. Similarly, CaMKK can phosphorylate and activate the AMP-dependent protein kinase, although not nearly as well as does the AMPKK (32 ). Analysis of the amino acid sequences of the various protein kinases phosphorylated on an activation loop Thr by a CaMKK suggests that a loose consensus sequence may exist. When this sequence is used to search protein sequence databases, several more protein kinases surface as potential substrates (E. Corcoran and A. R. Means, unpublished observations). This should be a fruitful avenue of research and, if additional substrates are identified, determination of the biological significance of each CaMKK/kinase interaction should present a considerable challenge.
Even though CaMKI is present in virtually all mammalian cells and tissues, nothing is known about its physiologically relevant substrates. Certainly in vitro it will phosphorylate synapsin on site 1 and CREB on Ser-133 (12 ). Expression vectors encoding a C-terminally truncated, Ca2+/CaM-independent fragment of CaMKI will phosphorylate and activate CREB in the cell, whereas those encoding the full-length protein will not, because full-length CaMKI does not readily enter the nucleus (33 ). However, increased levels of intracellular Ca2+ in PC12 cells activates CaMKK and CaMKI, suggesting that a CaMKI cascade may exist in this cell type (34 ). In C. elegans, homologs to both CaMKK and CaMKI have been cloned. In this organism CaMKI has a NLS at its N terminus, and the full-length protein will phosphorylate and activate mammalian CREB when expressed in mammalian cells (29 ). This nematode also has a CREB gene that seems to be involved in pheromone response, a pathway that also is known to have a requirement for Ca2+ (information derived from the on-line C. elegans newsletter). Perhaps in C. elegans Ca2+, CaM, CaMKK, CaMKI, and CREB indeed constitute a functional signal transduction cascade.
CaMK Cascades In Fungi
Fungal species also contain homologs of CaMKK and CaMKI, an
observation supporting the possibility that CaM kinase cascades
may be evolutionarily conserved. The SspI gene of
Saccharomyces pombe is involved in the regulation of actin
depolymerization and, although not appreciated in the original
publications, sequence similarity identifies SspI as a CaMKK
homolog (35 ). It can be activated independently of and partially
compensate for another gene called Spc1, which encodes a
stress-activated MAPK. These observations suggest the potential of
cross-talk between two well known signaling cascades, the MAPK and
Ca2+ kinase pathways. The data with
SspI point to a cytoplasmic role for this CaMKK and imply
that its target should be cytoplasmic and involved in cytoskeletal
dynamics. A CaMKI homolog has also been cloned from S. pombe
(C. Rasmussen, University of Saskatchewan, personal communication).
However, neither the role of the CaMKI in S. pombe nor its
relationship to the SspI gene is currently known. The
filamentous fungus Aspergillus nidulans also contains
homologs to CaMKK and CaMKI (J. Joseph and A. R. Means,
unpublished observations). In this species the CaMKI gene is essential
and required for progression through the nuclear division cycle.
Both enzymes seem to be involved in the regulation of entry into DNA
synthesis. The fact that the temporal requirement for these enzymes
precedes the activation of cdc2/cyclin B in G1
(single cyclin and cyclin-dependent kinase genes are required for both
G1/S and G2/M in
Aspergillus) suggests that they may play a nuclear role.
Thus, although much more work needs to be done, fungi seem to contain
CaM kinase cascades that function in both cytoplasm and nucleus. If
this proves to be true, then it follows that simpler organisms might
contain a single CaMKI/IV-like protein that can act in either the
cytoplasm or the nucleus. The evolutionary appearance of two unique
genes encoding CaMKI and CaMKIV might herald more complex regulation
requiring cytoplasmic and nuclear CaM kinases with similar substrate
specificity to coexist in the same cell.
Regulation of CaMKIV
In contrast to the relatively simple mechanism by which the
activity of CaMKI is regulated, at least three steps are required to
activate CaMKIV, and inactivation requires dephosphorylation (Fig. 4) (36 ). First,
Ca2+/CaM must bind to CaMKIV, which exposes
Thr-196 in its activation loop and allows a low level of protein kinase
activity. Second, the low level of protein kinase activity is
sufficient to intramolecularly autophosphorylate a number of Ser
residues in the extreme N terminus of the protein which is required to
relieve a novel form of autoinhibition. Third, Thr-196 is
phosphorylated by a CaMKK that increases protein kinase activity by
decreasing the Km for peptide substrate about
12-fold. When activated in this fashion, CaMKIV exhibits an activity
that is independent of Ca2+/CaM. Precisely what
is responsible for generation of this autonomous activity has not been
elucidated although it can be abrogated in vitro by
dephosphorylation of the protein by PP2A (37 ). Again the specific
residues dephosphorylated by PP2A have yet to be defined. However,
dephosphorylation seems to be a mechanism by which CaMKIV is
inactivated in the cell. At least in brain extracts and T lymphocytes,
CaMKIV and PP2A can be isolated as a complex by the use of a series of
chromatographic steps or by immunoprecipitation (38 ). The ratio of the
heterotrimeric PP2A and the monomeric CaMKIV in the complex is 1:1,
and, although it is not known which regions of the proteins are
required for complex formation, association does not require the
activity of either enzyme. On the other hand, in purified complexes
that contain both proteins in an enzymatically active state, the
phosphatase will dephosphorylate and inactivate CaMKIV. This mechanism
for inactivating CaMKIV also seems to occur in cells. When T cells are
stimulated through the T cell receptor, CaMKIV is rapidly
activated (it reaches a maximum by 2 min) but the inactivation is just
as rapid (it is complete by about 5 min). In addition, inactivation
occurs in the face of a high nuclear concentration of
Ca2+ and, as mentioned above, activated CaMKIV
does not require the continued presence of
Ca2+/CaM anyway. However, inactivation of CaMKIV
can be prevented by the expression of the SV40 virus small t antigen,
which is known to bind to and inactivate PP2A (38 ). Thus, both
activation of CaMKIV by a rise in intracellular
Ca2+ and inactivation by PP2A-mediated
dephosphorylation have been demonstrated to occur in a cellular
context.
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What might be the rate-limiting step in a signal transduction cascade
responsible for activating CaMKIV in the nucleus? In analyzing the
possibilities, we must remember that a considerable proportion of the
total amount of CaMKIV in a cell apparently exists in a nuclear complex
with PP2A even at resting Ca2+ concentrations
(Fig. 4). What else might be in this complex? Does it contain CaM,
CaMKK, and components of the transcription apparatus that might be
phosphorylated by CaMKIV and/or dephosphorylated by PP2A such as CREB
or CBP? In this case, a change in the nuclear
Ca2+ concentration might be sufficient to trigger
a transcriptional response. A second possibility suggested from
experiments in hippocampal neurons is that CaM might be translocated
into the nucleus (45 ). An alternate explanation of these results is
that since CaM can enter and exit the nucleus and nuclear targets for
CaM might be activated by the increase in Ca2+,
the increase in nuclear CaM might be due more to nuclear retention than
translocation (47 ). A third possibility, but one that has yet to be
tested experimentally, is that the CaMKK might be subject to nuclear
translocation [it has been reported to exist in both cytoplasm and
nucleus (12 )]. No canonical NLS exists in the mammalian CaMKKs
characterized to date. However, the arrangement of the basic residues
in the putative CaM binding region is reminiscent of a bipartite NLS.
Maybe this motif serves a dual purpose: to allow the kinase to
translocate into the nucleus and then to bind CaM in response to a rise
in the nuclear concentration of Ca2+. Another
provocative possibility is that PP2A might be the target of some
phantom signaling pathway and regulation of the phosphatase could, in
turn, regulate the kinase.
PERSPECTIVES
The field of CaM kinase cascades is just beginning to be mined. The CaMKK/CaMKIV interaction should be of considerable interest to molecular endocrinologists. On the one hand, this cascade is implicated in the control of transcription, and, on the other hand, it is present in a number of cells and tissues that are regulated by hormones, growth factors, and cytokines. Additional substrates for the CaMKK must exist, and the bona fide cellular substrates of CaMKIV remain to be identified. Evidence suggests that a CaM kinase cascade might impact MAP kinase pathways and be modified by the cAMP pathway. Overexpression of CaMKIV in mammalian cells has been reported to increase the activity of several members of the MAP kinase family, implying positive cooperativity between the CaMKIV and MAP kinase signaling cascades (48 ). Conversely, PKA has been shown to phosphorylate and decrease the activity of CaMKIV and CaMKI, suggesting negative cross-talk between the Ca2+ and cAMP pathways (49 50 ). Another provocative observation is that the unique human gene encoding CaMKIV is localized on the long arm of chromosome 5, in the region between q2123 (51 ). The adjacent regions of 5q also contain genes encoding a number of cytokines, growth factors, and receptors involved in hematopoiesis that include M-CSF, GM-CSF, IL-3, IL-4, IL-9, IL-13, and the platelet-derived growth factor receptor. In an attempt to elucidate the physiological roles of CaMKIV in mammals, the unique mouse gene encoding CaMKIV has been silenced by homologous recombination in our laboratory. Indeed, phenotypic consequences include defects in hematopoiesis as well as in T cell function, motor control, behavior, learning and memory, and spermatogenesis. It will be enlightening to determine the mechanism by which CaMKIV is involved in each of these processes and also to examine the consequences of silencing the two genes known to encode CaMKKs.
ACKNOWLEDGMENTS
I am indebted to several members of my laboratory for their research efforts and for allowing me to mention unpublished data and critique of the manuscript. Without the efforts of Shannon Lemrow, Jim Joseph, Ethan Corcoran, Chris Kane, David Chin, Kristin Anderson, Beth Harvat, and Joy Wu, this minireview would not have been possible. I am also particularly grateful to Kate Winkler, who is responsible for the artwork and, in her other life, is an excellent graduate student. Finally, I appreciate Mary Greenway, who ensured that this manuscript was appropriately referenced and formatted.
FOOTNOTES
Address requests for reprints to: Dr. Anthony R. Means, Department of Pharmacology, Duke University Medical Center, C238 Levine Science Research Center, Durham, North Carolina 27710.
Our research efforts are supported by NIH Grants HD-07503 and GM-33976.
Received for publication September 10, 1999. Revision received October 19, 1999. Accepted for publication October 21, 1999.
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