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calmodulin-dependent protein kinases
The multifunctional Ca 2 +/calmodulin-dependent protein kinases Howard Schulman Stanford University School Medicine, Stanford, USA
Ca2+ mediates the effect of many hormones, neurotransmitters and growth factors on contractility and motility, carbohydrate metabolism, cell cycle, gene expression and neuronal plasticity. Multifunctional Ca2+/calmodulin-dependent (CAM) kinase, CaM kinase la, CaM kinase Ib and CaM kinase IV are four of the kinases that mediate Ca2+-signaling pathways. Recent studies have clarified our understanding of their structure, regulation and function.
Current Opinion in Cell Biology 1993, 5:247-253 Introduction Physiologic elevation in intracellular free Ca 2 + initiates a host of cellular changes that include secretion, contraction, metabolism and gene expression. The broad scope of Ca 2 + activity necessitates a diversity in Ca 2 +-linked receptor and signal-transduction systems. Intracellular effects of Ca 2 + are transmitted by many effector systems that include ATPases, ion channels, proteases and phospholipases, in addition to protein kinases and phosphatases. Protein kinases that are dedicated to the regulation of a single important process as well as general or multifunctional kinases that transmit information from cell stimuli to multiple target substrates are found in the Ca2+-signaling system (Fig. 1). Multifunctional Ca2+/calmodulin-dependent (CAM) protein kinase, also referred to as calmodulin-dependent multiprotein kinase, CaM kinase II or type II CaM kinase, is a general protein kinase. This review focuses on multifunctional Ca 2 +/calmodulin-dependent protein kinase kinase II, which I will refer to as multifunctional CaM kinase or simply as CaM kinase, as well as on CaM kinases Ia, Ib and 1V [calmodulin kinase-Gr (granule cell)], which have been far less characterized, but may also be capable of phosphorylating multiple substrates. In attempting to synthesize recent findings in a short review I have had to select a limited set of topics and have therefore omitted a number of potentially important contributions to the field. However, these have been well covered in several recent reviews with extensive citations [1-4].
Multifunctional Ca 2 +/calmodulin-dependent kinase Several considerations justify a special interest in multifunctional CaM kinase. It is the only clearly es tablished multifunctional Ca2+-stimulated kinase. Like cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC), two other major second messenger regulated kinases, CaM kinase is widespread in nature, responds to many hormones and neurotransmitters, and orchestrates their effects on almost every major cellular activity. Its regulation by autophosphorylation is extremely interesting and may be designed to potentiate its response to brief elevations in Ca 2 + and to decode the frequency by which the cell is stimulated.
Ca2+/calmodulin-dependent kinase structure CaM kinase represents a growing isozyme family derived from four genes, consisting of homomultimers or heteromultimers of 6-12 kinase subunits each. Three isoforms are neuronal-specific: a (54kDa), [3 (60kDa), and 13' (59 kDa). The Y (59 kDa) and 8 (60 kDa) isoforms s h o w a broader tissue distribution that includes brain. Two new isoforms, YB and To have recently been cloned from human Jurkat T cells and are distributed in human T cells, but not B cells, as well as in epithelial, skeletal muscle and neuronal tissue [5"]. A distinct 0t-like isoform may be the predominant protein in the post-synaptic specialization, termed post-synaptic density (PSD) [6"]. The 54 kDa subunit purified from PSD shows some differences in isoelectric point and immunogenicity from 'authentic' soluble aCaM kinase and contains a sequence of eight amino acids
Abbreviations CAM kinase~Ca2+/calmodulin-dependent kinase; LTP--Iong-term potentiation; MLCK~myosin light chain kinase; PKA--protein kinase A; PKC--protein kinase C; PSD---post-synapticdensity. (~) Current Biology Ltd ISSN 0955-0674
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;;;; j Fig. 1. Both dedicated and multifunctional CaM kinases mediate the actions of diverse signal transduction systems [voltage-sensitive Ca2+ channels, ligand-gated receptor/channels and phospholipase C (PLC)-Iinkedreceptors] that elevate intracellular Ca2+. (a) Dedicated kinases,such as myosin light chain kinase (MLCK),phosphorylasekinase and CaM kinase III, selectively phosphorylatethe unique substrates myosin light chain, phosphorylase and eukaryotic elongation factor II, respectively. (b) Multifunctional CaM kinases la, Ib, II and IV are activated by these signals and respond by phosphorylation of multiple substrates localized in the nucleus, cytoskeleton, membrane and cytosol. not found in any known CaM kinase isoform. The at- and ]3-isoforms in brain may serve some special functions as they are highly abundant in brain (2 % of all protein in the hippocampus); this is probably 50-fold higher than the level of Y- and 8-isoforms in either non-neuronal or neuronal tissue. Electron microscopic analysis of CaM kinase suggests a 'flower-with-petals' structure where the association domains of all subunits in a holo-enzyme assemble into a single globfilar hub from which radiate 8-12 smaller particles containing the catalytic and regulatory domains of the individual subunits [7°°]. It is not known which isoforms co-assemble and in which ratios, although the enzyme from brain is reported to contain homomultimers of either a- or 13-subunits [7°°], suggesting that heteromultimers may not exist, The primary differences between the otherwise highly homologous isoforms are 'inserts' of 11-39 amino acids between the regulatory and association domains. The inserts in 13- and fl'-isoforms correspond to distinct exons [8°]. The finding of new y-isoforms suggest that additional variants of ~, ~, 7 and 8 may be generated by alternative splicing [5°,9]. Functional differences between isoforms with almost identical catalytic domains may arise from targeting of isoforms to distinct substrates. Chemical crosslinking was used to demonstrate
a complex between synapsin I, the major CaM kinase substrate on synaptic vesicles, and the regulatory domain of ¢z- but not ]3-CAMkinase [10o]. This arrangement may add both specificity and speed to the phosphorylation that modulates synaptic release.
Regulation by Caa+/calmodulin and autophosphorylation In this section, I will summarize the regulatory motif governing the activity states of CaM kinase before detailing the evidence supporting it. An auto-inhibitory segment is positioned in the active site, sterically blocking access to its substrates. Ca2+/calmodulin displaces this segment by wrapping around it and thereby activating the enzyme. The kinase can then lock itself into activated states by auto-phosphorylation on a conserved threonine in the auto-inhibitory segment of all isoforms (Thr286 in ~). This markedly reduces the dissociation rate of calmodulin at either high or low Ca2 +. Even after calmodulin dissociates, disruption of the auto-inhibitory domain by phospho-Thr286 produces a partially active or autonomous kinase. An extended segment encompassing approximately amino acids 281-309 may be needed to sterically restrict access of both ATP and protein substrate to the
The multifunctional Ca 2 +/calmodulin-dependent protein kinases Schulman active site of CaM kinase [11,12.,13.]. Important interactions at the amino-terminal end, particularly at His282 and Thr286, may be responsible for positioning a downstream segment to interfere with ATP binding while the carboxyl-terminal end of the segment resides in the protein substrate binding site [12.]. Thr306 may be closest to the phosphotransferase acceptor site in the basal state as it is selectively, although weakly, autophosphorylated when kinase is incubated with high ATP without Ca 2 +/calmodulin [ 14.]. Molecular modeling of CaM kinase using coordinates derived from the crystal structure of the catalytic subunit of PKA also suggests that Thr286 does not occupy the peptide-binding site like a substrate as previously assumed [13"]. In fact, its autophosphorylation is found to occur by an intermolecular reaction when monomers made by truncation of the association domain are examined [15"]. Thr286 may be critical for proper folding or alignment of the amino acids that block the active site in the basal state. The solution and crystal structures of calmodulin bound to a target peptide [16.%17"] provide insights into activation of CaM kinase. Calmodulin, whose structure is usually elongated with two lobes at each end, was found to bend and wrap around its ¢~-helical binding peptide. In order to wrap around the calmodulin-binding domain on CaM kinase it would have to 'peel' this segment away from the active cleft, thereby de-inhibiting the kinase. When calmodulin is bound it blocks autophosphorylation of Thr305 and Thr306, which are in the calmodulinbinding domain. They become rapidly phosphorylated in a Ca 2 +-independent manner, however, after calmodulin dissociates; phosphorylation of either threonine blocks rebinding of calmodulin (Fig. 2) [14.,18]. Once calmodulin has bound, CaM kinase autophosphorylates on Thr286 and is thereby converted from an enzyme with one of the weakest affinities for calmodulin (45 nM) to an enzyme with one of the highest att~nities (60pM) [15..]. This is largely due to a reduced rate of dissociation of calmodulin. At high Ca 2 + levels, the dissociation rate of fluorescently labeled calmodulin is reduced by 1000-fold, from 0.4s to several hundred seconds. Even when Ca 2+ concentration is reduced below the level needed for activation of CaM kinase (e.g. 100 nM), fluorescently labeled calmodulin remains trapped for a minimum of 10 s. The low affinity of the non-phosphorylated kinase for calmodulin may mean that one or both lobes of calmodulin cannot fully wrap around the autoinhibitory domain. Autophosphorylation may 'loosen' the auto-inhibitory domain, allowing tighter binding. When calmodulin dissociates, the kinase continues to phosphorylate substrates in an autonomous or Ca 2 + -independent fashion because the phosphorylated auto-inhibitory domain is not an effective inhibitor of the active site. One obvious consequence of trapping and autonomy would be to extend the activity of the kinase for several seconds after Ca 2 + declines to sub-threshold levels. An equally important effect may arise during repetitive stimuli or oscillations that do not saturate the kinase with Ca2+/calmodulin and produce submaximal stimulation. Trapping may then enable the kinase to retain
some calmodulin during brief inter-spike intervals and recruit more calmodulin with each successive spike. Its activity or read-out may therefore be affected by the fiequency and number of oscillations.
Cellular regulation Conversion of CaM kinase to a Ca 2+-independent enz y m e in situ can be used to assess which signal transduction systems are subserved by it. In adrenal-like PC12 cells, multiple signals converge on CaM kinase, including ligand-gated Ca 2 + influx via receptors for ATP and acetylcholine (nicotinic) and IPymediated intracellular release of Ca 2 + via receptors for acetylcholine (muscarinic) and bradykinin [19"]. PKC may crosstalk with CaM kinase and other calmodulin-dependent processes by increasing the availability of free calmodulin [20"]. Activation of CaM kinase is enhanced under conditions in which PKC stimulation increases the level of unbound calmodulin. PKC may reduce the ability of a cell to buffer calmodulin by phosphorylating the calmodulin-binding domains of proteins such as neuromodulin (GAP-43), MARCKS and neurogranin (RC-3) [21].
Functional studies Gene knockout and memory The most exciting findings on CaM kinase during the period covered by this review involve its role in a form of neuronal plasticity referred to as long-term potentiation (LTP) and in a spatial-based form of learning by use of gene-targeted knockout [22"',23"]. Brief repetitive stimulation of a neuronal circuit is followed by an enhanced synaptic transmission through the circuit or LTP which lasts for hours when performed on an isolated slice of hippocampal tissue, or days and weeks when induced in vivo. Micro-injection of an auto-inhibitory peptide of CaM kinase was previously shown to block the induction of LTP [24]. Induction of LTP also increases transcription of a CaM kinase mRNA, which is not essential for the first few hours of LTP but may, along with transcriptional changes of other genes, be importam for its long-term maintenance [25"].
Mutant mice lacking ¢~-CaM kinase were generated by gene targeting of embryonic stem cells. Mice developed normally and no gross morphological changes in the brain were detected. The other major isoform, [3-CaM kinase, was unaffected. In most experiments, hippocampal slices from mutant animals displayed no LTP, supporting a role for CaM kinase in LTP. Surprisingly, in two experiments an entire population of synapses fully overcame the cx-CaM kinase deficit and exhibited a normal level of LTP. At individual synapses one can imagine that [3CaM kinase may compensate for the ¢~-CaMkinase deficit. However, a simple mechanism cannot explain how successful LTP can be induced in an all-or-none fashion in a large population of proximate synapses. The mutant mice were impaired in learning a Morris hidden platform task in which they find a hidden platform submerged in a round pool of water that has been made
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Fig. 2. (a) The structure of multifunctional CaM kinase. The enzyme consists of 10 subunits; the catalytic and regulatory domains at the amino-terminal end of each subunit radiate from a central hub of association domains. The three phosphorylation sites in the aisoform are the autonomy site (Thr286) and two inhibitory sites (Thr305 and Thr306). The regulatory domain sequence is shown in single letter code. (b) Autophosphory[ation of CaM kinase. (i) Two kinase subunits in the inactive state. The auto-inhibitory segment blocks the active site of the enzyme. (ii) The Ca2+/calmodulin complex binds to the caimodulin-binding segment, including the inhibitory sites, which displaces the auto-inhibitory segment and activates the enzyme. Activation permits inter-subunit autophosphorylation of an autonomy site in one subunit (shaded arrow) which (iii) leaves that subunit in a highly active state by trapping bound calmodulin. (iv) After dissociation of calmodulin, the subunit is left in a partially active autonomous state. (v) Subsequent autophosphorylation of the inhibitory sites produces a calmoduiin-insensitive state in which activity is capped at the level of the autonomous activity until it is dephosphorylated. Phosphorylated sites are shaded. (Adapted from [4].)
opaque [23°°]. Normal mice are able to use an effective strategy involving multiple spatial relationships between the hidden platform and distal visual cues outside the pool. Mutant mice perform less well because they are unable to perform true spatial learning and use other, less
effective strategies, to eventually find the escape platform. The finding that mutant mice were defective in acquiring a form of 'real' memory strengthens the notion that LTP in the hippocampus is related to memory.
The multifunctional Ca2+/calmodulin-dependent protein kinases Schulman 251 Gene
expression
Multifunctional CaM kinase may be responsible for some Ca 2 +-induced gene expression that cannot be attributed to activation of PKC [26"*,27]. A DNA sequence corresponding to the binding site for C/EBP13, a m e m b e r of the bZip family of transcription factors, mediated the induction of a downstream reporter gene in response to elevated Ca2+ in a pituitary cell line. A reporter gene was induced 60-fold when co-transfected with DNA encoding a constitutive monomeric fragment of CaM kinase [a-CaM kinase(1-290)] and C/EBP13 [26--]. Ca 2+ influx stimulated C/EBPI3 phosphorylation, which was inhibited by KN-62, a cell-permeable inhibitor of CaM kinase. A serine residue in the leucine zipper dimerization domain was shown to be necessary for induction and to be phosphorylated by CaM kinase but not PKA. Thus, promoters with binding sites for C/EBP13, in addition to those with binding sites for CREB [28,29], may confer Ca2+-regu lated gene transcription in diverse cell systems via CaM kinase-mediated phosphorylation of the transcription factors.
Additional functions
New roles for CaM kinase in the cell cycle, smooth muscle contraction, secretion and growth factor desensitization have been demonstrated. Induction of a constitutive monomeric a-CaM kinase (1-291) stably transfected into a mammalian cell line led to a cessation of the cell cycle at the G2-M transition [30*]. The block occurred despite apparent activation of cdc2 kinase activity. Autoinhibitory peptides were utilized to demonstrate that CaM kinase mediates activation of a chloride current in neutrophils stimulated by tumor necrosis factor a [31"]. In smooth muscle, CaM kinase inhibitors blocked an important feedforward stimulation of Ca 2 + influx elicited by an initial rise in Ca 2+ [32"]. CaM kinase inhibits smooth muscle myosin light chain kinase (MLCK) by phosphorylation near its calmodulin-binding domain [33"]. Inhibitor KN-62 blocked phosphorylation of MLCK, thereby preventing the normal decrease in MLCK sensitivity to Ca 2+ . This suggests a role for CaM kinase in desensitizing MLCK following smooth muscle contraction. Secretagogue-stimulated acid secretion from parietal cells is blocked by KN-62, implicating a role for the enzyme in acid secretion [34"]. The kinase phosphorylates the epidermal growth factor receptor in vitro at a site necessary for desensitization of its tyrosine kinase activity and its down-regulation from the surface membrane [35"]. Phosphorylation is at the carboxyl-terminal end of the molecule and distinct from the site phosphorylated by PKC.
Other (possibly) multifunctional Ca2 +/calmodulin-dependent kinases Three other protein kinases, CaM kinases Ia, Ib and IV (CaM kinase-Gr) appear to have broad substrate speci-
ficity in vitro and may also serve to coordinate the phosphorylation of multiple substrates in response to elevated Ca 2 +. A final consensus about their physiological niche as dedicated or multifunctional kinases will have to await definitive demonstration of their action in situ.
Ca2 +/calmodulin-dependentkinases la and Ib CaM kinase I, a Ca2+/calmodulin-dependent kinase, is most abundant in brain but is also present in pancreas, heart and other rat tissues [36]. I n vitro substrates of the kinase are not numerous but include synapsin I and synapsin III at sites that are also phosphorylated by PICA. As a-CaM kinase is a large multimer of 20-30 nm that may have a difficult access to transcription factors in the nucleus, attention has begun to focus on CaM kinase I and IV because they are monomeric. Indeed, CaM kinase I is capable of phosphorylating CREB at a site shared by PKA and multifunctional CaM kinase [28]. An apparently similar activity has recently been resolved as two distinct monomeric protein kinases termed CaM kinases Ia (43kDa) and Ib (39kDa) [37",38"]. CaM kinase Ib is not simply a proteolytic fragment of CaM kinase Ia. Both are highly dependent on Ca2+/calmodulin for activity, although CaM kinase Ib is 20-fold more sensitive to calmodulin and autophosphorylates in its absence. The relationship between CaM kinase I used to phosphorylate CREB and CaM kinases Ia and Ib is uncertain. CaM kinase I most resembles CaM kinase Ib, but preparations of the enzyme may have included CaM kinase Ia as well. CaM kinase Ia exhibits two interesting regulatory features. First, Ca2+/calmodulin-stimulated autophosphorylation of the enzyme on threonine residues produces a timedependent increase in its maximal activity of up to 15fold, although the activity remains Ca2+/calmodulin-de pendent [38"]. Second, either the low or the high activity state of the enzyme requires a non-dialyzable 'activator', which is resolved from it on a calmodulin-atfinity column [37"]. Autophosphorylation of CaM kinase Ib does not affect its activity. However, dephosphorylation of the purified enzyme by protein phosphatase 2A reduces activity 10-fold [38"]. The dephosphorylated site(s) must be distinct from the autophosphorylation site as it cannot be reactivated by autophosphorylation. Thus, it appears to be subject to regulation by a distinct protein kinase.
Ca2+/calmodulin-dependent kinase IV (CaM kinase-Gr) CaM kinase 1V is a Ca2+/calmodulin-stimulated protein kinase derived from a gene that either produces the kinase in brain or is a calmodulin-binding protein termed calspermin in testes [39,40]. The catalytic domain of CaM kinase IV is 58 % homologous to multifunctional CaM kinase and lacking in calspermin. All of the carboxylterminal half of calspermin, a highly acidic domain, is present in CaM kinase IV. The acidic domain may regulate intracellular distribution of the kinase to nuclei and axons [41]. Its monomeric structure (53 kDa) and localization in nucleus as well as cytoplasm should enable it to participate in neuronal gene expression.
:252 Cell regulation Is CaM kinase IV a multifunctional kinase? In vitro, the kinase phosphorylates numerous substrates [42•]. However, it does so with a specific activity that is at best 5-10 % of that exhibited by multifunctional CaM kinase. Its localization is also more restricted. It is most enriched in the cerebellum, particularly in granule cells, but is also found in other brain regions [41]. If present, its level in non-neuronal tissues is only 1% of the level in cerebellum [42o]. Its phosphorylation of cerebellar protein in vitro suggests that it functions as a nmltifunctional kinase in restricted tissues.
Concluding remarks
The first indication that the major PSD protein may not be identical to wCaM kinase. 7. ••
KANASEKIT, IKEUCHI Y, SUGIURA H, YAMAUCHIT: Structural Features of Ca 2 +/Calmodulin-Dependent Protein Kinase II Revealed by Electron Microscopy. J Cell Biol 1991, 115:104~1060. Electron microscopy shows the multimeric structure of CaM kinase with 8-12 catalytic domain particles surrounding a core of association domains. 8.
KARTS U, MULLERU, GIIJ3ERT DJ, COPEIAND NG, JENVaNS NA, HARBERS K: Structure, Expression, and C h r o m o s o m e Location of the Gene for the [3 Subunit of Brain-Specific Ca2+/Calmodulin-Dependent Protein Kinase II Identified by Transgene Integration in an Embryonic Lethal Mouse Mutant. Mol Ceil Biol 1992, 12:3644-3652. Genomic cloning of [3-CaM kinase indicates that the variable domain of the [3- and [3'-isoforms are derived by alternative splicing. •
9.
We can now appreciate that multifunctional CaM kinase, CaM kinases Ia, Ib and 1V are integral components of Ca2+-based signal transduction systems that modulate many cell biological and physiological processes. Inhibitors must be refined to allow selective inhibition of these kinases. These, along with catalytic fragments and constitutive constructs of the enzymes will help to extend the list of functions mediated by them. In parallel, we must begin to examine how cells use the diversity of Ca2 + -dependent kinases to achieve specificity. These kinases and their multiple isoforms may be found to contain localization signals that target them to distinct intracellular sites. This would add specificity to the source of Ca 2 + that activates them and to the substrates that they effectively phosphorylate. It will be interesting to assess whether temporal aspects of Ca 2 + signals are also decoded by these kinases.
BULIEITRF, BENNETt MK, MOLLOY SS, HURLEYJB, KENNEDY MB: Conserved and Variable Regions in the Subunits of Brain Type II Ca 2 +/Calmodulin-Dependent Protein Kinase. Neuron 1988, 1:63-72.
10.
BENFENATI F, VALTORTA F, RUBENSTEIN JL, GORELICK FS, CZERNIK AJ: Synaptic Vesicle-Associated Ca2+/Calmodulin-Dependent Protein Kinase II is a Binding Protein for Synapsin I. Nature 1992, 359:417-420. First example of a CaM kinase that is physically associated with its sub strate. •
GREENGARD P,
11.
HAGrWARAT, OHSAKO S, YAMAUCHIT: Studies on the Regulatory Domain of Ca2+/Calmodulin-Dependent Protein Kinase II by Expression of Mutated cDNAs in Escherichia coll. J Biol Chem 1991, 266:16401-16408.
12. •
SMITH MK, COLBRAN RJ, BRICKEY DA, SODERLING TR: Functional Determinants in t h e Autoinhibitory Domain of Calcium/Calmodulin-Dependent Protein Kinase II. Role of His282 and Multiple Basic Residues. J Biol Chem 1992, 267:1761-1768. Detailed analysis of the auto-inhibitory domain reveals that it blocks the binding of both ATP and peptide. CRUZALEGUIFH, KAPILOFFMS, MOP,FIN J-P, KEMP BE, ROSENFELD MG, MEANS AR: Regulation of Intrasteric Inhibition of the Multifunctional Calcium/Calmodulin-Dependent Protein Kinase. Proc Natl Acad Sci USA 1993, 89:12127-12131. Describes site-directed mutagenesis and molecular modeling of the auto-inhibitory domain. 13.
•
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
DUNKLEYPR: Autophosphorylation of Neuronal Calcium/ Calmodulin-Stimulated Protein Kinase II. Mol Neurobiol 1991, 5:179-202.
2.
ROSTASJAP, DUNKLEYPR: Distribution of Multiple Forms of Calcium/Calmodulin Stimulated Protein Kinase II in Brain. J Neurochem 1992, 59:1191-1202.
3.
HANSONPI, SCHULMANH: Neuronal Ca 2 +/Calmodulin-Dependent Protein Kinases. Annu Rev Biochem 1992, 61:559-601.
4.
SCHULMANH, HANSON PI, MEYER T: Decoding Calcium Sig-
nals by Multifunctional CaM Kinase. Cell Calcium 1992, 13:401-411. 5.
NGHIEM P, SAATI SM, MARTENS CL, GARDNER P, SCHULMAN H: Cloning and Analysis of Two New Isoforms of Multifunctional Ca2+/Calmodulin-Dependent Protein Kinase: Expression in Multiple H u m a n Tissues. J Biol Chem 1993, 258:5471-5479. T cells, but not B cells, contain two new ~/-CaM kinasedike isoforms that have the regulatory features of a-CaM kinase, •
6.
Wu K, HUANG Y, ADLER J, BLACKIB: On t h e Identity of the
•
Major Postsynaptic Density Protein. Proc Natl Acad Sci USA 1992, 89:3015-3019.
14. •
HANSONPI, SCHULMANH: Inhibitory Autophosphorylation of Multifunctional Ca2+/Calmodulin-Dependent Protein Kinase Analyzed by Site-Directed Mutagenesis. J Biol Chem 1992, 267: 17216-17224. Analysis of Ca 2 + independent autophosphorylation sites that block rebinding of cahnodulin. 15. ••
MEYERT, HANSONPI, STRYERL, SCHULMANH: Calmodulin Trapping by Calcium-Calmodulin-Dependent Protein Kinase. Science 1992, 256:1199-1202. Establishes a novel erect of autophosphorylation, namely the trapping of calmodulin on the Mnase. 16. ••
IKURAM, CLOREGM, GRONENBORNAM, ZHU G, KLEE CB, BAX A: Solution Structure of a Calmodulin-Target Peptide C o m p l e x by Multidimensional NMR. Science 1992, 256:632q538. First structural view of calmodulin wrapping around its peptide-binding site by the use of multidimensional nuclear magnetic resonance. 17. ••
MEADORWE, MEANSAR, QUIOCHO FA: Target Enzyme Recognition by Calmodulin: 2.4A Structure of a CalmodulinPeptide Complex. Science 1992, 257:1251-1255. First crystal structure of calmodulin wrapping around its peptide-binding site. 18.
PATTON BL, MILLER SG, KENNEDY MB: Activation of Type I! Calcium/Calmodulin-Dependent Protein Kinase by Caa+/Calmodulin is Inhibited by Autophosphorylation of
The multifunctional Ca2+/calmodulin-dependent protein kinases Schulman 253 Threonine within the Calmodulin-Binding Domain. J Biol Chem 1990, 265:11204-11212. 19. •
MACNICOLM, SCHULMANH: Multiple Ca 2+ Signaling Pathways Converge on CaM Kinase in PC12 Cells. FEBS Lett 1992, 304:237-240. CaM kinase is activated in situ by hormones that elevate Ca 2+ by either Ca2 + influx or intracellular release.
32.
MCCARRONJG, MCGEOWN JG, REARDON S, IKEBE M, FAY FS, WALSHJV JR: Calcium-Dependent Enhancement of Calcium Current in Smooth Muscle by Calmodulin-Dependent Protein Kinase II. Nature 1992, 357:74-77. CaM kinase-stimulated Ca 2+ current in smooth muscle is demonstrated by inhibitory peptides. •
MAcNICOLM, SCHULMANH: Cross-Talk between Protein Kinase C and Multifunctionai Ca2+/Calmodulin-Dependent Protein Kinase. J Biol Chem 1992, 267:12197-12201. Calmodulin may be limiting in cells because release of bound calmod ulin by PKC enhances CaM kinase activation.
TANSEYMG, WORD RA, HIDAKAH, SINGER HA, SCHWORERCM, KAMM RE, STULLJT: Phosphorylation of Myosin Light Chain Kinase by the Multifunctional Calmodulin-Dependent Protein Kinase II in Smooth Muscle Cells. J Biol Chem 1992, 267:12511-12516. CaM kinase turns out to be the kinase responsible for de-sensitization of MLCK in smooth muscle.
21.
34.
20. ,
KI.EE CB: Concerted Regulation of Protein Phosphorylation and Dephosphorylation by Calmodulin. Neurochem Res 1991, 16:1059-1065.
22. ••
SILVAAJ, PAYLORR, WEHNERJM, TONEGAWAS: Impaired Spatial Learning in ¢t-Calcium-Calmodulin Kinase II Mutant Mice. Science 1992, 257:206-211. Mice deficient in ~x-CaMkinase perform poorly in certain spatial learning tasks. 23. ..
SILVA AJ, STEVENSCF, TONEGAWAS, WANG Y: Deficient Hippocampal Long-Term Potentiation in ~-Calcium--Calmodulin Kinase II Mutant Mice. Science 1992, 257:201-206. This is the first gene targeted knockout of a major multifunctional ldnase, and it supports a role for a CAM kinase in the regulation of synaptic plasticity. 24.
MAUNOWR, SCHULMANH, TSIEN RW: Inhibition of Postsynaptic PKC or CaMKII Blocks Induction but not Expression of LTP. Science 1989, 245:862-866.
25. .
MACKLERSA, BROOKSBP, EBERWINEJH: Stimulus-lnduced Coordinate Changes in mRNA Abundance in Single Postsynaptic Hippocampal CA1 Neurons. Neuron 1992, 9:539-548. Stimuli that elicit LTP modulate expression of many genes, including induction of CaM kinase mRNA. 26. ••
WEGNERM, CAO Z, ROSENFELDMG: Calcium-Regulated Phosphorylation within the Leucine Zipper of C/EBP~. Science 1992, 256:370-373. Establishes that CaM kinase can mediate transcriptional regulation via phosphorylation of a trans-acting factor. 27.
SHENGM, GREENBERG ME: The Regulation and Function of c-los and Other Immediate Early Genes in the Nervous System. Neuron 1990, 4:477-485.
28.
SHENGM, THOMPSONMA, GREENBERGME: CREB: A Ca2+-Reg ulated Transcription Factor Phosphorylated by CalmodulinDependent Kinases. Science 1991, 252:142~1430.
29.
DASH PK, KARL KA, COUCOS MA, PRYWES R, KANDEL ER: cAMP Response Element-Binding Protein is Activated by Ca2+/Calmodulin - - as well as caMP-Dependent Protein Kinase. Proc Natl Acad Sci USA 1991, 88:5061-5065.
PLANKS-SILVAMD, MEANS AR: Expression of a Constitutive form of Calcium/Calmodulin Dependent Protein Kinase II Leads to Arrest of the Cell Cycle in G2. EMBO J 1992, 11:507-517. Describes how CaM kinase may mediate one of the effects of calmodufin on the cell cycle.
33. •
TSUNODA Y, FUNASAIG~. M, MODLIN IM, HIDAKA H, FOX LM, GOLDENRING JR: An Inhibitor of Ca2+/Calmodulin Dependent Protein Kinasc II KN-62 Inhibits CholinergicStimulated Parietal Cell Secretion. Am J Physiol 1992, 262:G118--G122. CaM kinase activation is necessary for secretion of acid from parietal cells. •
35. •
COUNTAWAYJL, NAIRNAC, DAVISRJ: Mechanism of Desensitization of the Epidermal Growth Factor Receptor ProteinTyrosine Kinase. J Biol Chem 1992, 267:1129-1140. Desensitization of the epidermal growth factor receptor at a site distinct from the PKC site is mediated by CaM kinase. 36.
37. ••
DEREMERMF, SAEU RJ, EDELMAN AM: Ca2+-Calmodulin-De pendent Protein Kinases Ia and Ib from Rat Brain. I. Identification, Purification, and Structural Comparisons. J Biol Chem 1992, 267:13460-13465. Identification and characterization of a new Ca2+/calmodulin dependent protein kinase. 38. •
DEREMERMF, SAELI RJ, BRAUTIGAN DL, EDELMAN AM: Ca 2+Calmodulin-Dependent Protein Kinases la and lb from Rat Brain. II. Enzymatic Characteristics and Regulation of Activities by Phosphorylation and Dephosphorylation. J Biol Chem 1992, 267:13466-13471. CaM kinase Ia is activated by autophosphorylation whereas CAM kinase Ib is probably regulated by another kinase. 39.
MEANS AR, CRUZALEGUI F, LEMAGUERESSE B, NEEDLEMAN DS, SLAUGHTER GR, ONO T: A Novel Ca2+/Calmodulin Dependent Protein Kinase and a Male Germ Cell-Specific Calmodulin-Binding Protein are Derived from the Same Gene. Mol Cell Biol 1991, 11:3960-3971.
40.
OHMSTEDE C A, BLAND MM, MERRILL BM, SAHYOUN N: Relationship of Genes Encoding Ca2+/Calmodulin-Dependent Protein Kinase Gr and Calspermin: a Gene within a Gene. Proc Natl Acad Sci USA 1991, 88:5784 5788.
41.
JENSENRE, OHMSTEDE CA, FISHERKS, OLINJK, SAHYOUNN: Acquisition and Loss of a Neuronal Ca 2 +/Calmodulin-Dependent Protein Kinase during Neuronal Differentiation. Proc Natl Acad Sci USA 1991, 88:4050-4053.
30. •
31. •
SCHUMANNMA, GARDNER P, RAFFIN TA: Recombinant Human Tumor Necrosis Factor ¢t Induces Calcium Oscillation and Calcium-Activated Chloride Current in Human Neutrophlls. The Role of Calcium/Calmodulin-Dependent Protein Kinase. J Biol Chem 1993, 268:2134-2140. Extends the function of CaM kinase to the regulation of chloride current in neutrophils.
NAmNAC, GREENGARDP: Purification and Characterization of Ca2+/Calmodulin-Dependent Protein Kinase I from Bovine Brain. J Biol Chem 1987, 262:7273 7281.
42. .
M~YANOO, KAMESHITA I, FUJISAWAH: Purification and Characterization of a Brain-Specific Multifunctional CalmodulinDependent Protein Kinase from Rat Cerebellum. J Biol Chem 1992, 267:1198-1203. Analysis of substrate specificity and other characteristics of cerebellar CAM kinase W.
H Schulman, Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332, USA.
Ca2+ mediates the effect of many hormones, neurotransmitters and growth factors on contractility and motility, carbohydrate metabolism, cell cycle, gene expression and neuronal plasticity. Multifunctional Ca2+/calmodulin-dependent (CAM) kinase, CaM kinase la, CaM kinase Ib and CaM kinase IV are four of the kinases that mediate Ca2+-signaling pathways. Recent studies have clarified our understanding of their structure, regulation and function.
Current Opinion in Cell Biology 1993, 5:247-253 Introduction Physiologic elevation in intracellular free Ca 2 + initiates a host of cellular changes that include secretion, contraction, metabolism and gene expression. The broad scope of Ca 2 + activity necessitates a diversity in Ca 2 +-linked receptor and signal-transduction systems. Intracellular effects of Ca 2 + are transmitted by many effector systems that include ATPases, ion channels, proteases and phospholipases, in addition to protein kinases and phosphatases. Protein kinases that are dedicated to the regulation of a single important process as well as general or multifunctional kinases that transmit information from cell stimuli to multiple target substrates are found in the Ca2+-signaling system (Fig. 1). Multifunctional Ca2+/calmodulin-dependent (CAM) protein kinase, also referred to as calmodulin-dependent multiprotein kinase, CaM kinase II or type II CaM kinase, is a general protein kinase. This review focuses on multifunctional Ca 2 +/calmodulin-dependent protein kinase kinase II, which I will refer to as multifunctional CaM kinase or simply as CaM kinase, as well as on CaM kinases Ia, Ib and 1V [calmodulin kinase-Gr (granule cell)], which have been far less characterized, but may also be capable of phosphorylating multiple substrates. In attempting to synthesize recent findings in a short review I have had to select a limited set of topics and have therefore omitted a number of potentially important contributions to the field. However, these have been well covered in several recent reviews with extensive citations [1-4].
Multifunctional Ca 2 +/calmodulin-dependent kinase Several considerations justify a special interest in multifunctional CaM kinase. It is the only clearly es tablished multifunctional Ca2+-stimulated kinase. Like cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC), two other major second messenger regulated kinases, CaM kinase is widespread in nature, responds to many hormones and neurotransmitters, and orchestrates their effects on almost every major cellular activity. Its regulation by autophosphorylation is extremely interesting and may be designed to potentiate its response to brief elevations in Ca 2 + and to decode the frequency by which the cell is stimulated.
Ca2+/calmodulin-dependent kinase structure CaM kinase represents a growing isozyme family derived from four genes, consisting of homomultimers or heteromultimers of 6-12 kinase subunits each. Three isoforms are neuronal-specific: a (54kDa), [3 (60kDa), and 13' (59 kDa). The Y (59 kDa) and 8 (60 kDa) isoforms s h o w a broader tissue distribution that includes brain. Two new isoforms, YB and To have recently been cloned from human Jurkat T cells and are distributed in human T cells, but not B cells, as well as in epithelial, skeletal muscle and neuronal tissue [5"]. A distinct 0t-like isoform may be the predominant protein in the post-synaptic specialization, termed post-synaptic density (PSD) [6"]. The 54 kDa subunit purified from PSD shows some differences in isoelectric point and immunogenicity from 'authentic' soluble aCaM kinase and contains a sequence of eight amino acids
Abbreviations CAM kinase~Ca2+/calmodulin-dependent kinase; LTP--Iong-term potentiation; MLCK~myosin light chain kinase; PKA--protein kinase A; PKC--protein kinase C; PSD---post-synapticdensity. (~) Current Biology Ltd ISSN 0955-0674
247
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;;;; j Fig. 1. Both dedicated and multifunctional CaM kinases mediate the actions of diverse signal transduction systems [voltage-sensitive Ca2+ channels, ligand-gated receptor/channels and phospholipase C (PLC)-Iinkedreceptors] that elevate intracellular Ca2+. (a) Dedicated kinases,such as myosin light chain kinase (MLCK),phosphorylasekinase and CaM kinase III, selectively phosphorylatethe unique substrates myosin light chain, phosphorylase and eukaryotic elongation factor II, respectively. (b) Multifunctional CaM kinases la, Ib, II and IV are activated by these signals and respond by phosphorylation of multiple substrates localized in the nucleus, cytoskeleton, membrane and cytosol. not found in any known CaM kinase isoform. The at- and ]3-isoforms in brain may serve some special functions as they are highly abundant in brain (2 % of all protein in the hippocampus); this is probably 50-fold higher than the level of Y- and 8-isoforms in either non-neuronal or neuronal tissue. Electron microscopic analysis of CaM kinase suggests a 'flower-with-petals' structure where the association domains of all subunits in a holo-enzyme assemble into a single globfilar hub from which radiate 8-12 smaller particles containing the catalytic and regulatory domains of the individual subunits [7°°]. It is not known which isoforms co-assemble and in which ratios, although the enzyme from brain is reported to contain homomultimers of either a- or 13-subunits [7°°], suggesting that heteromultimers may not exist, The primary differences between the otherwise highly homologous isoforms are 'inserts' of 11-39 amino acids between the regulatory and association domains. The inserts in 13- and fl'-isoforms correspond to distinct exons [8°]. The finding of new y-isoforms suggest that additional variants of ~, ~, 7 and 8 may be generated by alternative splicing [5°,9]. Functional differences between isoforms with almost identical catalytic domains may arise from targeting of isoforms to distinct substrates. Chemical crosslinking was used to demonstrate
a complex between synapsin I, the major CaM kinase substrate on synaptic vesicles, and the regulatory domain of ¢z- but not ]3-CAMkinase [10o]. This arrangement may add both specificity and speed to the phosphorylation that modulates synaptic release.
Regulation by Caa+/calmodulin and autophosphorylation In this section, I will summarize the regulatory motif governing the activity states of CaM kinase before detailing the evidence supporting it. An auto-inhibitory segment is positioned in the active site, sterically blocking access to its substrates. Ca2+/calmodulin displaces this segment by wrapping around it and thereby activating the enzyme. The kinase can then lock itself into activated states by auto-phosphorylation on a conserved threonine in the auto-inhibitory segment of all isoforms (Thr286 in ~). This markedly reduces the dissociation rate of calmodulin at either high or low Ca2 +. Even after calmodulin dissociates, disruption of the auto-inhibitory domain by phospho-Thr286 produces a partially active or autonomous kinase. An extended segment encompassing approximately amino acids 281-309 may be needed to sterically restrict access of both ATP and protein substrate to the
The multifunctional Ca 2 +/calmodulin-dependent protein kinases Schulman active site of CaM kinase [11,12.,13.]. Important interactions at the amino-terminal end, particularly at His282 and Thr286, may be responsible for positioning a downstream segment to interfere with ATP binding while the carboxyl-terminal end of the segment resides in the protein substrate binding site [12.]. Thr306 may be closest to the phosphotransferase acceptor site in the basal state as it is selectively, although weakly, autophosphorylated when kinase is incubated with high ATP without Ca 2 +/calmodulin [ 14.]. Molecular modeling of CaM kinase using coordinates derived from the crystal structure of the catalytic subunit of PKA also suggests that Thr286 does not occupy the peptide-binding site like a substrate as previously assumed [13"]. In fact, its autophosphorylation is found to occur by an intermolecular reaction when monomers made by truncation of the association domain are examined [15"]. Thr286 may be critical for proper folding or alignment of the amino acids that block the active site in the basal state. The solution and crystal structures of calmodulin bound to a target peptide [16.%17"] provide insights into activation of CaM kinase. Calmodulin, whose structure is usually elongated with two lobes at each end, was found to bend and wrap around its ¢~-helical binding peptide. In order to wrap around the calmodulin-binding domain on CaM kinase it would have to 'peel' this segment away from the active cleft, thereby de-inhibiting the kinase. When calmodulin is bound it blocks autophosphorylation of Thr305 and Thr306, which are in the calmodulinbinding domain. They become rapidly phosphorylated in a Ca 2 +-independent manner, however, after calmodulin dissociates; phosphorylation of either threonine blocks rebinding of calmodulin (Fig. 2) [14.,18]. Once calmodulin has bound, CaM kinase autophosphorylates on Thr286 and is thereby converted from an enzyme with one of the weakest affinities for calmodulin (45 nM) to an enzyme with one of the highest att~nities (60pM) [15..]. This is largely due to a reduced rate of dissociation of calmodulin. At high Ca 2 + levels, the dissociation rate of fluorescently labeled calmodulin is reduced by 1000-fold, from 0.4s to several hundred seconds. Even when Ca 2+ concentration is reduced below the level needed for activation of CaM kinase (e.g. 100 nM), fluorescently labeled calmodulin remains trapped for a minimum of 10 s. The low affinity of the non-phosphorylated kinase for calmodulin may mean that one or both lobes of calmodulin cannot fully wrap around the autoinhibitory domain. Autophosphorylation may 'loosen' the auto-inhibitory domain, allowing tighter binding. When calmodulin dissociates, the kinase continues to phosphorylate substrates in an autonomous or Ca 2 + -independent fashion because the phosphorylated auto-inhibitory domain is not an effective inhibitor of the active site. One obvious consequence of trapping and autonomy would be to extend the activity of the kinase for several seconds after Ca 2 + declines to sub-threshold levels. An equally important effect may arise during repetitive stimuli or oscillations that do not saturate the kinase with Ca2+/calmodulin and produce submaximal stimulation. Trapping may then enable the kinase to retain
some calmodulin during brief inter-spike intervals and recruit more calmodulin with each successive spike. Its activity or read-out may therefore be affected by the fiequency and number of oscillations.
Cellular regulation Conversion of CaM kinase to a Ca 2+-independent enz y m e in situ can be used to assess which signal transduction systems are subserved by it. In adrenal-like PC12 cells, multiple signals converge on CaM kinase, including ligand-gated Ca 2 + influx via receptors for ATP and acetylcholine (nicotinic) and IPymediated intracellular release of Ca 2 + via receptors for acetylcholine (muscarinic) and bradykinin [19"]. PKC may crosstalk with CaM kinase and other calmodulin-dependent processes by increasing the availability of free calmodulin [20"]. Activation of CaM kinase is enhanced under conditions in which PKC stimulation increases the level of unbound calmodulin. PKC may reduce the ability of a cell to buffer calmodulin by phosphorylating the calmodulin-binding domains of proteins such as neuromodulin (GAP-43), MARCKS and neurogranin (RC-3) [21].
Functional studies Gene knockout and memory The most exciting findings on CaM kinase during the period covered by this review involve its role in a form of neuronal plasticity referred to as long-term potentiation (LTP) and in a spatial-based form of learning by use of gene-targeted knockout [22"',23"]. Brief repetitive stimulation of a neuronal circuit is followed by an enhanced synaptic transmission through the circuit or LTP which lasts for hours when performed on an isolated slice of hippocampal tissue, or days and weeks when induced in vivo. Micro-injection of an auto-inhibitory peptide of CaM kinase was previously shown to block the induction of LTP [24]. Induction of LTP also increases transcription of a CaM kinase mRNA, which is not essential for the first few hours of LTP but may, along with transcriptional changes of other genes, be importam for its long-term maintenance [25"].
Mutant mice lacking ¢~-CaM kinase were generated by gene targeting of embryonic stem cells. Mice developed normally and no gross morphological changes in the brain were detected. The other major isoform, [3-CaM kinase, was unaffected. In most experiments, hippocampal slices from mutant animals displayed no LTP, supporting a role for CaM kinase in LTP. Surprisingly, in two experiments an entire population of synapses fully overcame the cx-CaM kinase deficit and exhibited a normal level of LTP. At individual synapses one can imagine that [3CaM kinase may compensate for the ¢~-CaMkinase deficit. However, a simple mechanism cannot explain how successful LTP can be induced in an all-or-none fashion in a large population of proximate synapses. The mutant mice were impaired in learning a Morris hidden platform task in which they find a hidden platform submerged in a round pool of water that has been made
249
250
Cell regulation
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Fig. 2. (a) The structure of multifunctional CaM kinase. The enzyme consists of 10 subunits; the catalytic and regulatory domains at the amino-terminal end of each subunit radiate from a central hub of association domains. The three phosphorylation sites in the aisoform are the autonomy site (Thr286) and two inhibitory sites (Thr305 and Thr306). The regulatory domain sequence is shown in single letter code. (b) Autophosphory[ation of CaM kinase. (i) Two kinase subunits in the inactive state. The auto-inhibitory segment blocks the active site of the enzyme. (ii) The Ca2+/calmodulin complex binds to the caimodulin-binding segment, including the inhibitory sites, which displaces the auto-inhibitory segment and activates the enzyme. Activation permits inter-subunit autophosphorylation of an autonomy site in one subunit (shaded arrow) which (iii) leaves that subunit in a highly active state by trapping bound calmodulin. (iv) After dissociation of calmodulin, the subunit is left in a partially active autonomous state. (v) Subsequent autophosphorylation of the inhibitory sites produces a calmoduiin-insensitive state in which activity is capped at the level of the autonomous activity until it is dephosphorylated. Phosphorylated sites are shaded. (Adapted from [4].)
opaque [23°°]. Normal mice are able to use an effective strategy involving multiple spatial relationships between the hidden platform and distal visual cues outside the pool. Mutant mice perform less well because they are unable to perform true spatial learning and use other, less
effective strategies, to eventually find the escape platform. The finding that mutant mice were defective in acquiring a form of 'real' memory strengthens the notion that LTP in the hippocampus is related to memory.
The multifunctional Ca2+/calmodulin-dependent protein kinases Schulman 251 Gene
expression
Multifunctional CaM kinase may be responsible for some Ca 2 +-induced gene expression that cannot be attributed to activation of PKC [26"*,27]. A DNA sequence corresponding to the binding site for C/EBP13, a m e m b e r of the bZip family of transcription factors, mediated the induction of a downstream reporter gene in response to elevated Ca2+ in a pituitary cell line. A reporter gene was induced 60-fold when co-transfected with DNA encoding a constitutive monomeric fragment of CaM kinase [a-CaM kinase(1-290)] and C/EBP13 [26--]. Ca 2+ influx stimulated C/EBPI3 phosphorylation, which was inhibited by KN-62, a cell-permeable inhibitor of CaM kinase. A serine residue in the leucine zipper dimerization domain was shown to be necessary for induction and to be phosphorylated by CaM kinase but not PKA. Thus, promoters with binding sites for C/EBP13, in addition to those with binding sites for CREB [28,29], may confer Ca2+-regu lated gene transcription in diverse cell systems via CaM kinase-mediated phosphorylation of the transcription factors.
Additional functions
New roles for CaM kinase in the cell cycle, smooth muscle contraction, secretion and growth factor desensitization have been demonstrated. Induction of a constitutive monomeric a-CaM kinase (1-291) stably transfected into a mammalian cell line led to a cessation of the cell cycle at the G2-M transition [30*]. The block occurred despite apparent activation of cdc2 kinase activity. Autoinhibitory peptides were utilized to demonstrate that CaM kinase mediates activation of a chloride current in neutrophils stimulated by tumor necrosis factor a [31"]. In smooth muscle, CaM kinase inhibitors blocked an important feedforward stimulation of Ca 2 + influx elicited by an initial rise in Ca 2+ [32"]. CaM kinase inhibits smooth muscle myosin light chain kinase (MLCK) by phosphorylation near its calmodulin-binding domain [33"]. Inhibitor KN-62 blocked phosphorylation of MLCK, thereby preventing the normal decrease in MLCK sensitivity to Ca 2+ . This suggests a role for CaM kinase in desensitizing MLCK following smooth muscle contraction. Secretagogue-stimulated acid secretion from parietal cells is blocked by KN-62, implicating a role for the enzyme in acid secretion [34"]. The kinase phosphorylates the epidermal growth factor receptor in vitro at a site necessary for desensitization of its tyrosine kinase activity and its down-regulation from the surface membrane [35"]. Phosphorylation is at the carboxyl-terminal end of the molecule and distinct from the site phosphorylated by PKC.
Other (possibly) multifunctional Ca2 +/calmodulin-dependent kinases Three other protein kinases, CaM kinases Ia, Ib and IV (CaM kinase-Gr) appear to have broad substrate speci-
ficity in vitro and may also serve to coordinate the phosphorylation of multiple substrates in response to elevated Ca 2 +. A final consensus about their physiological niche as dedicated or multifunctional kinases will have to await definitive demonstration of their action in situ.
Ca2 +/calmodulin-dependentkinases la and Ib CaM kinase I, a Ca2+/calmodulin-dependent kinase, is most abundant in brain but is also present in pancreas, heart and other rat tissues [36]. I n vitro substrates of the kinase are not numerous but include synapsin I and synapsin III at sites that are also phosphorylated by PICA. As a-CaM kinase is a large multimer of 20-30 nm that may have a difficult access to transcription factors in the nucleus, attention has begun to focus on CaM kinase I and IV because they are monomeric. Indeed, CaM kinase I is capable of phosphorylating CREB at a site shared by PKA and multifunctional CaM kinase [28]. An apparently similar activity has recently been resolved as two distinct monomeric protein kinases termed CaM kinases Ia (43kDa) and Ib (39kDa) [37",38"]. CaM kinase Ib is not simply a proteolytic fragment of CaM kinase Ia. Both are highly dependent on Ca2+/calmodulin for activity, although CaM kinase Ib is 20-fold more sensitive to calmodulin and autophosphorylates in its absence. The relationship between CaM kinase I used to phosphorylate CREB and CaM kinases Ia and Ib is uncertain. CaM kinase I most resembles CaM kinase Ib, but preparations of the enzyme may have included CaM kinase Ia as well. CaM kinase Ia exhibits two interesting regulatory features. First, Ca2+/calmodulin-stimulated autophosphorylation of the enzyme on threonine residues produces a timedependent increase in its maximal activity of up to 15fold, although the activity remains Ca2+/calmodulin-de pendent [38"]. Second, either the low or the high activity state of the enzyme requires a non-dialyzable 'activator', which is resolved from it on a calmodulin-atfinity column [37"]. Autophosphorylation of CaM kinase Ib does not affect its activity. However, dephosphorylation of the purified enzyme by protein phosphatase 2A reduces activity 10-fold [38"]. The dephosphorylated site(s) must be distinct from the autophosphorylation site as it cannot be reactivated by autophosphorylation. Thus, it appears to be subject to regulation by a distinct protein kinase.
Ca2+/calmodulin-dependent kinase IV (CaM kinase-Gr) CaM kinase 1V is a Ca2+/calmodulin-stimulated protein kinase derived from a gene that either produces the kinase in brain or is a calmodulin-binding protein termed calspermin in testes [39,40]. The catalytic domain of CaM kinase IV is 58 % homologous to multifunctional CaM kinase and lacking in calspermin. All of the carboxylterminal half of calspermin, a highly acidic domain, is present in CaM kinase IV. The acidic domain may regulate intracellular distribution of the kinase to nuclei and axons [41]. Its monomeric structure (53 kDa) and localization in nucleus as well as cytoplasm should enable it to participate in neuronal gene expression.
:252 Cell regulation Is CaM kinase IV a multifunctional kinase? In vitro, the kinase phosphorylates numerous substrates [42•]. However, it does so with a specific activity that is at best 5-10 % of that exhibited by multifunctional CaM kinase. Its localization is also more restricted. It is most enriched in the cerebellum, particularly in granule cells, but is also found in other brain regions [41]. If present, its level in non-neuronal tissues is only 1% of the level in cerebellum [42o]. Its phosphorylation of cerebellar protein in vitro suggests that it functions as a nmltifunctional kinase in restricted tissues.
Concluding remarks
The first indication that the major PSD protein may not be identical to wCaM kinase. 7. ••
KANASEKIT, IKEUCHI Y, SUGIURA H, YAMAUCHIT: Structural Features of Ca 2 +/Calmodulin-Dependent Protein Kinase II Revealed by Electron Microscopy. J Cell Biol 1991, 115:104~1060. Electron microscopy shows the multimeric structure of CaM kinase with 8-12 catalytic domain particles surrounding a core of association domains. 8.
KARTS U, MULLERU, GIIJ3ERT DJ, COPEIAND NG, JENVaNS NA, HARBERS K: Structure, Expression, and C h r o m o s o m e Location of the Gene for the [3 Subunit of Brain-Specific Ca2+/Calmodulin-Dependent Protein Kinase II Identified by Transgene Integration in an Embryonic Lethal Mouse Mutant. Mol Ceil Biol 1992, 12:3644-3652. Genomic cloning of [3-CaM kinase indicates that the variable domain of the [3- and [3'-isoforms are derived by alternative splicing. •
9.
We can now appreciate that multifunctional CaM kinase, CaM kinases Ia, Ib and 1V are integral components of Ca2+-based signal transduction systems that modulate many cell biological and physiological processes. Inhibitors must be refined to allow selective inhibition of these kinases. These, along with catalytic fragments and constitutive constructs of the enzymes will help to extend the list of functions mediated by them. In parallel, we must begin to examine how cells use the diversity of Ca2 + -dependent kinases to achieve specificity. These kinases and their multiple isoforms may be found to contain localization signals that target them to distinct intracellular sites. This would add specificity to the source of Ca 2 + that activates them and to the substrates that they effectively phosphorylate. It will be interesting to assess whether temporal aspects of Ca 2 + signals are also decoded by these kinases.
BULIEITRF, BENNETt MK, MOLLOY SS, HURLEYJB, KENNEDY MB: Conserved and Variable Regions in the Subunits of Brain Type II Ca 2 +/Calmodulin-Dependent Protein Kinase. Neuron 1988, 1:63-72.
10.
BENFENATI F, VALTORTA F, RUBENSTEIN JL, GORELICK FS, CZERNIK AJ: Synaptic Vesicle-Associated Ca2+/Calmodulin-Dependent Protein Kinase II is a Binding Protein for Synapsin I. Nature 1992, 359:417-420. First example of a CaM kinase that is physically associated with its sub strate. •
GREENGARD P,
11.
HAGrWARAT, OHSAKO S, YAMAUCHIT: Studies on the Regulatory Domain of Ca2+/Calmodulin-Dependent Protein Kinase II by Expression of Mutated cDNAs in Escherichia coll. J Biol Chem 1991, 266:16401-16408.
12. •
SMITH MK, COLBRAN RJ, BRICKEY DA, SODERLING TR: Functional Determinants in t h e Autoinhibitory Domain of Calcium/Calmodulin-Dependent Protein Kinase II. Role of His282 and Multiple Basic Residues. J Biol Chem 1992, 267:1761-1768. Detailed analysis of the auto-inhibitory domain reveals that it blocks the binding of both ATP and peptide. CRUZALEGUIFH, KAPILOFFMS, MOP,FIN J-P, KEMP BE, ROSENFELD MG, MEANS AR: Regulation of Intrasteric Inhibition of the Multifunctional Calcium/Calmodulin-Dependent Protein Kinase. Proc Natl Acad Sci USA 1993, 89:12127-12131. Describes site-directed mutagenesis and molecular modeling of the auto-inhibitory domain. 13.
•
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
DUNKLEYPR: Autophosphorylation of Neuronal Calcium/ Calmodulin-Stimulated Protein Kinase II. Mol Neurobiol 1991, 5:179-202.
2.
ROSTASJAP, DUNKLEYPR: Distribution of Multiple Forms of Calcium/Calmodulin Stimulated Protein Kinase II in Brain. J Neurochem 1992, 59:1191-1202.
3.
HANSONPI, SCHULMANH: Neuronal Ca 2 +/Calmodulin-Dependent Protein Kinases. Annu Rev Biochem 1992, 61:559-601.
4.
SCHULMANH, HANSON PI, MEYER T: Decoding Calcium Sig-
nals by Multifunctional CaM Kinase. Cell Calcium 1992, 13:401-411. 5.
NGHIEM P, SAATI SM, MARTENS CL, GARDNER P, SCHULMAN H: Cloning and Analysis of Two New Isoforms of Multifunctional Ca2+/Calmodulin-Dependent Protein Kinase: Expression in Multiple H u m a n Tissues. J Biol Chem 1993, 258:5471-5479. T cells, but not B cells, contain two new ~/-CaM kinasedike isoforms that have the regulatory features of a-CaM kinase, •
6.
Wu K, HUANG Y, ADLER J, BLACKIB: On t h e Identity of the
•
Major Postsynaptic Density Protein. Proc Natl Acad Sci USA 1992, 89:3015-3019.
14. •
HANSONPI, SCHULMANH: Inhibitory Autophosphorylation of Multifunctional Ca2+/Calmodulin-Dependent Protein Kinase Analyzed by Site-Directed Mutagenesis. J Biol Chem 1992, 267: 17216-17224. Analysis of Ca 2 + independent autophosphorylation sites that block rebinding of cahnodulin. 15. ••
MEYERT, HANSONPI, STRYERL, SCHULMANH: Calmodulin Trapping by Calcium-Calmodulin-Dependent Protein Kinase. Science 1992, 256:1199-1202. Establishes a novel erect of autophosphorylation, namely the trapping of calmodulin on the Mnase. 16. ••
IKURAM, CLOREGM, GRONENBORNAM, ZHU G, KLEE CB, BAX A: Solution Structure of a Calmodulin-Target Peptide C o m p l e x by Multidimensional NMR. Science 1992, 256:632q538. First structural view of calmodulin wrapping around its peptide-binding site by the use of multidimensional nuclear magnetic resonance. 17. ••
MEADORWE, MEANSAR, QUIOCHO FA: Target Enzyme Recognition by Calmodulin: 2.4A Structure of a CalmodulinPeptide Complex. Science 1992, 257:1251-1255. First crystal structure of calmodulin wrapping around its peptide-binding site. 18.
PATTON BL, MILLER SG, KENNEDY MB: Activation of Type I! Calcium/Calmodulin-Dependent Protein Kinase by Caa+/Calmodulin is Inhibited by Autophosphorylation of
The multifunctional Ca2+/calmodulin-dependent protein kinases Schulman 253 Threonine within the Calmodulin-Binding Domain. J Biol Chem 1990, 265:11204-11212. 19. •
MACNICOLM, SCHULMANH: Multiple Ca 2+ Signaling Pathways Converge on CaM Kinase in PC12 Cells. FEBS Lett 1992, 304:237-240. CaM kinase is activated in situ by hormones that elevate Ca 2+ by either Ca2 + influx or intracellular release.
32.
MCCARRONJG, MCGEOWN JG, REARDON S, IKEBE M, FAY FS, WALSHJV JR: Calcium-Dependent Enhancement of Calcium Current in Smooth Muscle by Calmodulin-Dependent Protein Kinase II. Nature 1992, 357:74-77. CaM kinase-stimulated Ca 2+ current in smooth muscle is demonstrated by inhibitory peptides. •
MAcNICOLM, SCHULMANH: Cross-Talk between Protein Kinase C and Multifunctionai Ca2+/Calmodulin-Dependent Protein Kinase. J Biol Chem 1992, 267:12197-12201. Calmodulin may be limiting in cells because release of bound calmod ulin by PKC enhances CaM kinase activation.
TANSEYMG, WORD RA, HIDAKAH, SINGER HA, SCHWORERCM, KAMM RE, STULLJT: Phosphorylation of Myosin Light Chain Kinase by the Multifunctional Calmodulin-Dependent Protein Kinase II in Smooth Muscle Cells. J Biol Chem 1992, 267:12511-12516. CaM kinase turns out to be the kinase responsible for de-sensitization of MLCK in smooth muscle.
21.
34.
20. ,
KI.EE CB: Concerted Regulation of Protein Phosphorylation and Dephosphorylation by Calmodulin. Neurochem Res 1991, 16:1059-1065.
22. ••
SILVAAJ, PAYLORR, WEHNERJM, TONEGAWAS: Impaired Spatial Learning in ¢t-Calcium-Calmodulin Kinase II Mutant Mice. Science 1992, 257:206-211. Mice deficient in ~x-CaMkinase perform poorly in certain spatial learning tasks. 23. ..
SILVA AJ, STEVENSCF, TONEGAWAS, WANG Y: Deficient Hippocampal Long-Term Potentiation in ~-Calcium--Calmodulin Kinase II Mutant Mice. Science 1992, 257:201-206. This is the first gene targeted knockout of a major multifunctional ldnase, and it supports a role for a CAM kinase in the regulation of synaptic plasticity. 24.
MAUNOWR, SCHULMANH, TSIEN RW: Inhibition of Postsynaptic PKC or CaMKII Blocks Induction but not Expression of LTP. Science 1989, 245:862-866.
25. .
MACKLERSA, BROOKSBP, EBERWINEJH: Stimulus-lnduced Coordinate Changes in mRNA Abundance in Single Postsynaptic Hippocampal CA1 Neurons. Neuron 1992, 9:539-548. Stimuli that elicit LTP modulate expression of many genes, including induction of CaM kinase mRNA. 26. ••
WEGNERM, CAO Z, ROSENFELDMG: Calcium-Regulated Phosphorylation within the Leucine Zipper of C/EBP~. Science 1992, 256:370-373. Establishes that CaM kinase can mediate transcriptional regulation via phosphorylation of a trans-acting factor. 27.
SHENGM, GREENBERG ME: The Regulation and Function of c-los and Other Immediate Early Genes in the Nervous System. Neuron 1990, 4:477-485.
28.
SHENGM, THOMPSONMA, GREENBERGME: CREB: A Ca2+-Reg ulated Transcription Factor Phosphorylated by CalmodulinDependent Kinases. Science 1991, 252:142~1430.
29.
DASH PK, KARL KA, COUCOS MA, PRYWES R, KANDEL ER: cAMP Response Element-Binding Protein is Activated by Ca2+/Calmodulin - - as well as caMP-Dependent Protein Kinase. Proc Natl Acad Sci USA 1991, 88:5061-5065.
PLANKS-SILVAMD, MEANS AR: Expression of a Constitutive form of Calcium/Calmodulin Dependent Protein Kinase II Leads to Arrest of the Cell Cycle in G2. EMBO J 1992, 11:507-517. Describes how CaM kinase may mediate one of the effects of calmodufin on the cell cycle.
33. •
TSUNODA Y, FUNASAIG~. M, MODLIN IM, HIDAKA H, FOX LM, GOLDENRING JR: An Inhibitor of Ca2+/Calmodulin Dependent Protein Kinasc II KN-62 Inhibits CholinergicStimulated Parietal Cell Secretion. Am J Physiol 1992, 262:G118--G122. CaM kinase activation is necessary for secretion of acid from parietal cells. •
35. •
COUNTAWAYJL, NAIRNAC, DAVISRJ: Mechanism of Desensitization of the Epidermal Growth Factor Receptor ProteinTyrosine Kinase. J Biol Chem 1992, 267:1129-1140. Desensitization of the epidermal growth factor receptor at a site distinct from the PKC site is mediated by CaM kinase. 36.
37. ••
DEREMERMF, SAEU RJ, EDELMAN AM: Ca2+-Calmodulin-De pendent Protein Kinases Ia and Ib from Rat Brain. I. Identification, Purification, and Structural Comparisons. J Biol Chem 1992, 267:13460-13465. Identification and characterization of a new Ca2+/calmodulin dependent protein kinase. 38. •
DEREMERMF, SAELI RJ, BRAUTIGAN DL, EDELMAN AM: Ca 2+Calmodulin-Dependent Protein Kinases la and lb from Rat Brain. II. Enzymatic Characteristics and Regulation of Activities by Phosphorylation and Dephosphorylation. J Biol Chem 1992, 267:13466-13471. CaM kinase Ia is activated by autophosphorylation whereas CAM kinase Ib is probably regulated by another kinase. 39.
MEANS AR, CRUZALEGUI F, LEMAGUERESSE B, NEEDLEMAN DS, SLAUGHTER GR, ONO T: A Novel Ca2+/Calmodulin Dependent Protein Kinase and a Male Germ Cell-Specific Calmodulin-Binding Protein are Derived from the Same Gene. Mol Cell Biol 1991, 11:3960-3971.
40.
OHMSTEDE C A, BLAND MM, MERRILL BM, SAHYOUN N: Relationship of Genes Encoding Ca2+/Calmodulin-Dependent Protein Kinase Gr and Calspermin: a Gene within a Gene. Proc Natl Acad Sci USA 1991, 88:5784 5788.
41.
JENSENRE, OHMSTEDE CA, FISHERKS, OLINJK, SAHYOUNN: Acquisition and Loss of a Neuronal Ca 2 +/Calmodulin-Dependent Protein Kinase during Neuronal Differentiation. Proc Natl Acad Sci USA 1991, 88:4050-4053.
30. •
31. •
SCHUMANNMA, GARDNER P, RAFFIN TA: Recombinant Human Tumor Necrosis Factor ¢t Induces Calcium Oscillation and Calcium-Activated Chloride Current in Human Neutrophlls. The Role of Calcium/Calmodulin-Dependent Protein Kinase. J Biol Chem 1993, 268:2134-2140. Extends the function of CaM kinase to the regulation of chloride current in neutrophils.
NAmNAC, GREENGARDP: Purification and Characterization of Ca2+/Calmodulin-Dependent Protein Kinase I from Bovine Brain. J Biol Chem 1987, 262:7273 7281.
42. .
M~YANOO, KAMESHITA I, FUJISAWAH: Purification and Characterization of a Brain-Specific Multifunctional CalmodulinDependent Protein Kinase from Rat Cerebellum. J Biol Chem 1992, 267:1198-1203. Analysis of substrate specificity and other characteristics of cerebellar CAM kinase W.
H Schulman, Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332, USA.