Regulatory Cascades Involving Calmodulin-Dependent Protein Kinases

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). 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. 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 α-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. 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. 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. 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. 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. 3). 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. 3). 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. 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. 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 CaMKIIα) 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 γ and δ genes a localization sequence that with and the enzyme to the nucleus it has been in the regulation of Interestingly, phosphorylation of the localization sequence in these forms of CaMKII by either or when the CaMKII is in the cytoplasm with and not of CaMKII in a cell is associated with organelles and not free in the (12). The first of the CaMKII isoforms to be is exclusively in the and is a component of the membrane in In the CaMKII is to increase by phosphorylating ion and signaling proteins such as and (12). In it has been that stimulation of the receptor regulates the of CaMKII between and the in a manner. Thus, CaMKII is involved both in the maintenance of and In addition, CaMKII is for changes in such as a that is to be involved in and These of have been by out the in The molecular mechanisms involved in the effects of on have been that various of the membrane and cytoskeletal roles of CaMKII are to be the of CaMKII (12). of the that have of CaMKII have on the of forms of the enzyme to when in cells with One of the first such to be the Ca2+ stimulation of genes that are regulated by response elements such as Phosphorylation of binding on is essential for it is for binding of the binding proteins and which as as the target of protein kinase A thus the role of in However, CaMKII can also phosphorylate this residue leading to the that CaMKII mediates the Ca2+ for of the genes proposed as a target at which and signal transduction cascades to regulate However, while the form of CaMKII can in cells, it is inhibitory in et al. that one of inhibition that, in to CaMKII also phosphorylated a second residue on phosphorylation of not only but this modification also and the activation of from its phosphorylation on by The mechanism by which phosphorylation of inhibits seems to be by the between and Interestingly, the of the effect of CaMKII on seems to be both cell and CaMKII inhibits of the in a cell it found to in elements to CaMKII and (12). The with roles in regulation to CaMKII is that the is frequently on the effects of The most CaMKII are and its more Both of these are and be to as The the of both as CaMKII However, the subsequent that these also and have been the inhibition constant for all three CaM kinases only by a of is shown to a cellular response and the cell is shown to one or more isoforms of it is that CaMKII is the enzyme for the cellular The step in the is to demonstrate that the form of the CaMKII isoform can of a when the cells. is as and have overlapping substrate at the all three CaM kinases be Thus, with of the in it is to point to an that the Ca2+/CaM-dependent protein kinase for a event is the other two CaM-dependent protein kinases that have been the of considerable are and These enzymes a common for phosphorylation by an protein kinase but the of the activating mechanisms and have been in and are the of genes These kinases are also Ca2+/CaM binding proteins, which has to the that a CaM kinase to a mitogen-activated protein (MAP) kinase in cells. the phosphorylate the CaM kinase on a single Thr residue that is in the activation protein kinase and of the CaM kinase. This is a reaction that is to that by which a MAP kinase kinase phosphorylates and activates a MAP kinase. is present in all cells and exist in a variety of It is in cells, but, at in it appears to be and a localization sequence at its N terminus the vertebrate enzyme can be stimulated to the nucleus or in with another protein to be However, does not appear in the nucleus when cells are either or when stimulated to increase the intracellular Ca2+ concentration. does it is by another signaling The absence of a this possibility a Binding of Ca2+/CaM is for activity of but, CaMKII and it does not activity In the absence of activation phosphorylation, a low of Ca2+/CaM-dependent activity when the peptide substrates such as derived from site 1 of The in the activation of be phosphorylated by the Ca2+/CaM is Phosphorylation of the of the enzyme for of Ca2+/CaM when is phosphorylated but, in this the is the possibility that activation phosphorylation might the enzyme to subsequent transient increases in the Ca2+ concentration. This also suggests that dephosphorylation of might as an mechanism cellular However, whereas both and will dephosphorylate in vitro, it is not known which might this in Interestingly, it has been possible to generate peptide substrates that are well phosphorylated by the or forms of (and also for phosphorylation of the possibility that activation protein substrates for might exist in cells this is the case, although and require Ca2+/CaM for activation, might not require the of a This one that the possibility that the might have cellular substrates other than and and, might the target of Ca2+/CaM in than as an component in a CaM kinase A of this by et al. that a can phosphorylate and activate protein kinase or in vitro and when in a cell can phosphorylate and activate the protein kinase, although not as well as does the of the of the various protein kinases phosphorylated on an activation Thr by a suggests that a sequence may When this sequence is to protein sequence several more protein kinases surface as substrates and This be a of and, additional substrates are of the biological of each interaction present a considerable is present in all cells and is known its physiologically relevant in vitro it will phosphorylate on site 1 and on (12). encoding a of will phosphorylate and activate in the cell, whereas encoding the protein will does not the nucleus However, of intracellular Ca2+ in cells activates and that a may exist in this cell In C. to both and have been In this has a at its N and the protein will phosphorylate and activate when in cells This also has a that seems to be involved in a pathway that also is known to have a for Ca2+ derived from the C. Perhaps in C. and a signal transduction also of and an the possibility that CaM kinase cascades may be The of is involved in the regulation of and, although not in the sequence as a It can be activated of and partially for another called which a These observations the of cross-talk between two well known signaling the and Ca2+ kinase The with point to a role for this and that its target be and involved in cytoskeletal A has also been from of However, the role of the in its to the is The also to and and In this the is essential and for through the Both enzymes seem to be involved in the regulation of The that the for these enzymes the activation of in and kinase genes are for both and in suggests that may play a Thus, although much more to be seem to CaM kinase cascades that in both cytoplasm and nucleus. this to be it follows that might a single protein that can act in either the cytoplasm or the nucleus. The of two unique genes encoding and might more complex regulation and CaM kinases with substrate to in the cell. In to the mechanism by which the activity of is at three are to activate and inactivation requires dephosphorylation (Fig. First, Ca2+/CaM must bind to which exposes in its activation and allows a low of protein kinase the low of protein kinase activity is sufficient to a number of Ser in the N terminus of the protein which is to a form of is phosphorylated by a that increases protein kinase activity by the for peptide substrate When activated in this fashion, an activity that is independent of Ca2+/CaM. what is for generation of this activity has not been although it can be in vitro by dephosphorylation of the protein by the by have to be However, dephosphorylation seems to be a mechanism by which is in the cell. in and and can be as a complex by the of a of or by The ratio of the heterotrimeric and the in the complex is and, although it is not known which of the proteins are for complex does not require the activity of either enzyme. On the other hand, in that both proteins in an active the will dephosphorylate and This mechanism for also seems to occur in cells. When cells are stimulated through the cell is rapidly activated a by but the inactivation is as is by In addition, inactivation occurs in the of a high concentration of Ca2+ and, as activated does not require the of Ca2+/CaM However, inactivation of can be prevented by the of the small which is known to bind to and Thus, both activation of by a in intracellular Ca2+ and inactivation by dephosphorylation have been demonstrated to occur in a cellular Calcium The CaM kinase by phosphorylation of including Both and require Ca2+/CaM binding for activates by is by the associated protein to the The binding protein is much more restricted than It is most abundant in and but also in and the only on sequence are in Unlike the other CaM kinases, seems to in the nucleus of all cells in which it is when the protein is in cells or not the of is found in the nucleus. This intracellular localization of the protein the of a localization sequence although one has not been demonstrated has been in regulation of of a number of genes including encoding of the such as such as and the an involved in the to the called and of the receptor such as and However, the only direct substrates for involved in that have been are and although has been as a possible substrate (Fig. seems to regulate both and activity in the responses to generate the of in and in cells might be the step in a signal transduction for activating in the In the must that a considerable of the of in a cell apparently in a complex with at Ca2+ concentrations (Fig. might be in this it and components of the that might be phosphorylated by and/or by such as or In this case, a change in the Ca2+ concentration might be sufficient to a A second possibility from in is that CaM might be the nucleus of these results is that CaM can and the nucleus and targets for CaM might be activated by the increase in the increase in CaM might be due more to than A but one that has to be is that the might be to has been reported to exist in both cytoplasm and nucleus in the to However, the of the in the CaM binding region is of a this motif serves a dual to the kinase to the nucleus and to bind CaM in response to a in the concentration of Ca2+. possibility is that might be the target of signaling pathway and regulation of the in turn, regulate the kinase. The of CaM kinase cascades is to be The interaction be of considerable to molecular On the one hand, this is in the of and, on the other hand, it is present in a number of cells and that are regulated by and substrates for the must and the cellular substrates of to be suggests that a CaM kinase might MAP kinase pathways and be modified by the of in cells has been reported to increase the activity of several of the MAP kinase between the and MAP kinase signaling cascades has been shown to phosphorylate and decrease the activity of and cross-talk between the Ca2+ and pathways is that the unique encoding is on the of in the region between The adjacent of also genes encoding a number of and involved in that and the receptor. In an to the physiological roles of in the unique encoding has been by in consequences in as well as in cell and and It will be to the mechanism by which is involved in each of these processes and also to the consequences of the two genes known to to several of for and for to and of the the of and this not have been also to is for the and, in other is an that this and are by and