Regulatory Cascades Involving Calmodulin-Dependent Protein Kinases

Anthony R. Means, With Illustrations by Katharine E. Winkler

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. 1Go)(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. 1Go, 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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Figure 1. Calcium Signaling.

IP3-mediated release of Ca2+ from intracellular stores is triggered by liganding of a Gq-coupled receptor. Membrane depolarization opens voltage-gated Ca2+ channels. Intracellular Ca2+ spikes may have a variety of downstream effects. G, Gq protein; PLC, phospholipase C; PKC, protein kinase C.

 
THE INTRACELLULAR CALCIUM RECEPTOR, CALMODULIN (CaM)

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. 2Go). 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 {alpha}-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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Figure 2. Ca2+/Calmodulin-Dependent Kinase Activation

Clockwise from top: Calmodulin undergoes a conformational change upon Ca2+ binding. Ca2+/CaM docks at a hydrophobic site adjacent to the autoinhibitory domain of a CaM kinase. Kinase activation is achieved by Ca2+/CaM binding and steric displacement of the autoinhibitory domain. Stepwise activation, potentiation, and inactivation of CaMKII are represented schematically in blue.

 
Much of what is known concerning the molecular mechanisms by which CaM binds to and activates target enzymes comes from the study of four protein kinases (1 ). One of these kinases, myosin light chain kinase (MLCK, including the nonmuscle, smooth muscle, and skeletal muscle forms) is considered to be the simplest model enzyme in that it appears to be exclusively associated with actomyosin and phosphorylates a single substrate, the regulatory light chain of myosin. The other three enzymes, CaM kinases I, II, and IV, can be found in the cytoplasm associated with a variety of membrane or cytoskeletal elements or in the nucleus. These enzymes phosphorylate a number of different substrates and also are regulated in a more complicated manner.

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. 2Go. 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. 3Go. First, the Rho-associated protein kinase (RAK in Fig. 3Go) 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. 3Go) (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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Figure 3. Regulation of Myosin Light Chain Phosphorylation

MLCK-mediated and Rho-mediated signals (rho) are shown. PAK, the p21-activated kinase, PAK1(8 ); RAK, the Rho-activated protein kinase (7 ); ERK, the MAP kinases ERK1/2 (9 10 ).

 
A second signaling pathway has also been reported to regulate the activity of MLCK in a manner that does not require the binding of Ca2+/CaM to MLCK. Klemke et al. (10 ) demonstrated that the mitogen-activated protein (MAP) kinase ERK1 can phosphorylate and activate MLCK in vitro (ERK in Fig. 3Go). This reaction occurs in the absence of Ca2+/CaM and seems to increase the activity of the enzyme when activated by Ca2+/CaM. Nguyen et al. (11 ) have reported that this reaction occurs in cells as a component of the Ras-mediated pathway by which urokinase-type plasminogen activator promotes migration. These authors propose that the sequence of reactions after the binding of uPA to its receptor is Ras – Raf – MEK – ERK – MLCK – MLC – cellular migration. These observations, although not thoroughly understood physiologically, demonstrate that the prevailing theory of Ca2+ and CaM being sufficient components for regulation of MLC phosphorylation in nonmuscle cells may be an oversimplification and must be modified accordingly (Fig. 3Go). They also point out how cross-talk between intracellular signaling pathways can fine tune physiological responses to agents that act at the surface of cells.

CaMKII REGULATORY PATHWAYS

Unlike the MLCKs, CaMKII is a multimeric enzyme composed of 10–12 catalytic subunits (12 ). The interaction domains of the 10–12 subunits present in the holoenzyme complex form the spokes of a wheel, and the catalytic domains form the outside of the wheel (Fig. 2Go). 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{alpha}) 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 {gamma} and {delta} 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{alpha}, 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{alpha} have been confirmed by knocking out the gene in mice. The molecular mechanisms involved in the effects of CaMKII{alpha} on LTP have been elucidated using gene-targeting strategies that reconstitute various mutants of CaMKII{alpha} 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, {alpha} 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. 4Go) (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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Figure 4. Nuclear Calcium Signaling

The CaM kinase cascade induces transcription by phosphorylation of transcription factors including CREB. Both CaMKK and CaMKIV require Ca2+/CaM binding for activity. CaMKK activates CaMKIV by phosphorylation; CaMKIV is dephosphorylated by the associated protein phosphatase, PP2A, to extinguish the activity. CBP, The CREB binding protein

 
Functions of CaMKIV in Transcription
CaMKIV is much more tissue restricted than CaMKI. It is most abundant in brain and T lymphocytes but also exists in testis, ovary, adrenal, skin, and bone marrow (39 ). To date, the only convincing homologs based on amino acid sequence alignment are in mammals. Unlike the other CaM kinases, CaMKIV seems to reside in the nucleus of all cells in which it is expressed. Even when the protein is overexpressed in cells (whether or not they normally express it), the bulk of CaMKIV is found in the nucleus. This intracellular localization of the full-length protein implies the presence of a nuclear localization sequence although one has not been demonstrated experimentally. CaMKIV has been implicated in regulation of transcription of a number of genes including those encoding interleukin 2 (24 39 ), members of the immediate early gene family such as c-fos (16 ), TNF family members such as CD40L, FasL, and TNF{alpha} (40 ), the neurotrophin, BDNF (41 ), an Epstein-Barr virus gene involved in the switch to the lytic cycle called BZLF1 (42 ), and orphan members of the steroid receptor superfamily such as ROR{alpha} and COUP-TF (43 ). However, the only direct substrates for CaMKIV involved in transcription that have been defined are CREB and CREM{tau} (12 39 ) although CBP/p300 has been indirectly implicated as a possible substrate (Fig. 4Go) (44 ). CaMKIV seems to regulate both CREB and CBP activity in the transcriptional responses required to generate the late phase of LTP in hippocampal neurons (45 ) and long-term depression in cerebellar Purkinje cells (46 ).

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. 4Go). 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 q21–23 (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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