Defining Ca2+/Calmodulin-dependent Protein Kinase Cascades in Transcriptional Regulation

calmodulin Ca2+/calmodulin-dependent protein kinase CaMKI/IV kinase calcineurin cAMP response element-binding protein CREB-binding protein cAMP response element dominant negative retinoic acid-related orphan receptor ligand binding domain Calcium is a well established regulator of transcription. Modulation of responses to this ubiquitous second messenger can occur by superposition of coincident Ca2+-independent signals, but there is also growing evidence that the strength, frequency, source, and location of the Ca2+ signal are determinants for specific transcriptional results. These complex variations must be translated into changes in protein function that preserve and process the information conveyed by the original signal. The Ca2+ receptor calmodulin (CaM)1 is involved in many of these changes through its effects on a variety of CaM-binding proteins (1Chin D. Means A.R. Trends Cell Biol. 2000; 10: 322-328Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar). Among these, the multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) are notable for their effects on components of transcription complexes, directly connecting Ca2+ with changes in gene expression. The highly homologous CaMKI and CaMKIV are distinct from the multimeric CaMKII, although all have broad and overlapping substrate preferences, because their activation is greatly enhanced following phosphorylation catalyzed by “upstream” kinases in a manner analogous to the mitogen-activated protein kinase cascade. Based on an evolving understanding of CaMKI/IV regulation and cloning of the CaMKI/IV kinases (CaMKKs), a Ca2+/CaM-dependent protein kinase I/IV cascade (CaMK cascade) has been proposed (2Means A.R. Mol. Endocrinol. 2000; 14: 4-13Crossref PubMed Scopus (159) Google Scholar, 3Soderling T.R. Trends Biochem. Sci. 1999; 24: 232-236Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). This review will discuss the biochemical and physiologic basis for the existence of this cascade and its potential for mediating Ca2+ regulation of transcription. CaMKI and CaMKIV are closely related protein kinases with many similarities in mode of activation and substrate preferences in vitro but with different tissue distributions. The kinases are regulated by Ca2+/CaM binding, which relieves intramolecular steric inhibition of the active site by a C-terminal autoinhibitory domain (Fig. 1). A second autoinhibitory mechanism unique to CaMKIV is relaxed by the autophosphorylation of Ser-12 and Ser-13 (4Chatila T. Anderson K.A. Ho N. Means A.R. J. Biol. Chem. 1996; 271: 21542-21548Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In addition to deinhibition, CaMKI and CaMIV are activated 10–50-fold bytrans phosphorylation on a single Thr residue in the activation loop. Once activated, CaMKIV acquires Ca2+/CaM independence, whereas CaMKI remains Ca2+/CaM-dependent (5Haribabu B. Hook S.S. Selbert M.A. Goldstein E.G. Tomhave E.D. Edelman A.M. Snyderman R. Means A.R. EMBO J. 1995; 14: 3679-3686Crossref PubMed Scopus (167) Google Scholar). Dephosphorylation and inactivation of activated CaMKI/IV can be catalyzed in vitro by PP1, PP2A, calcineurin (CaN), and the CaMK phosphatase, but the relevant phosphatase(s) in vivo is still unclear (6Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar,7Kitani T. Ishida A. Okuno S. Takeuchi M. Kameshita I. Fujisawa H. J. Biochem. (Tokyo). 1999; 125: 1022-1028Crossref PubMed Scopus (51) Google Scholar). CaMKI is ubiquitously expressed, whereas CaMKIV has a more limited distribution, although both enzymes are strongly expressed in the brain. Recognition of the ability of kinases in brain extract to phosphorylate and activate CaMKI/IV led to the cloning of two upstream kinases, CaMKKα and CaMKKβ (8Tokumitsu H. Enslen H. Soderling T.R. J. Biol. Chem. 1995; 270: 19320-19324Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 9Anderson K.A. Means R.L. Huang Q.H. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Fremeau R.T. Means A.R. J. Biol. Chem. 1998; 273: 31880-31889Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). In addition to the brain, where both CaMKKs are highly expressed, CaMKKα mRNA is found in thymus and spleen, whereas CaMKKβ is present at lower levels in all tissues that express CaMKIV. Although derived from distinct genes, rat CaMKKs are 80% similar, and either CaMKK can phosphorylate and activate CaMKI and CaMKIV in vitro. Both CaMKKs bind and are positively regulated by Ca2+/CaM in vitro, and although their CaM binding site is different from the other CaMKs, the autoinhibitory mechanism functions in a similar manner to that of other Ca2+/CaM-dependent kinases (10Tokumitsu H. Muramatsu M. Ikura M. Kobayashi R. J. Biol. Chem. 2000; 275: 20090-20095Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Importantly, Ca2+/CaM binding to CaMKI/IV is a prerequisite to phosphorylation by the CaMKKs (5Haribabu B. Hook S.S. Selbert M.A. Goldstein E.G. Tomhave E.D. Edelman A.M. Snyderman R. Means A.R. EMBO J. 1995; 14: 3679-3686Crossref PubMed Scopus (167) Google Scholar). Thus, in theory Ca2+/CaM could regulate CaMKI/IV activity on many levels. The substrate preferences of CaMKI and CaMKIV are similar and intersect with CaMKII. In vitro, all three can phosphorylate synapsin I, cAMP response element-binding protein (CREB), and activating transcription factor 1 (11Schulman H. Braun A. Carafoli E. Klee C. Calcium as a Cellular Regulator. Oxford University Press, New York1999: 311-343Google Scholar, 12Anderson K.A. Kane C.D. Biometals. 1998; 11: 331-343Crossref PubMed Scopus (44) Google Scholar). Their minimum consensus sequence Hyd-X-R-X-X-(S/T) (where Hyd is any hydrophobic amino acid), determined through peptide studies, provides only a rough template common to many protein kinases (11Schulman H. Braun A. Carafoli E. Klee C. Calcium as a Cellular Regulator. Oxford University Press, New York1999: 311-343Google Scholar). Additional specificity is provided by residues adjacent to the phosphorylation site of the substrate, producing differences in substrate preference among these kinases. The differences can have important transcriptional implications; for example, although all three can phosphorylate CREB on the activating site Ser-133, only CaMKII phosphorylates an additional inhibitory site, Ser-142 (13Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (649) Google Scholar). Interestingly, the presence of two additional basic amino acids 6 and 7 residues N-terminal to the phosphorylation site in certain peptide substrates allows phosphorylation by CaMKI/IV equally well with or without activation by a CaMKK (14Hook S.S. Kemp B.E. Means A.R. J. Biol. Chem. 1999; 274: 20215-20222Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This “activation independence” has not yet been demonstrated toward protein substrates. Nonetheless, because CaMKI/IV requires Ca2+/CaM for deinhibition in addition to activation loop phosphorylation, substrates of this type would not be phosphorylated by CaMKI/IV until a Ca2+ signal was initiated and so could represent Ca2+-dependent but activation-independent signaling targets. Subsequent activation by a CaMKK would increase the number of available substrates by enhancing CaMKI/IV activity toward a second set of substrates (Fig. 1). The first reconstruction of a CaMK cascade in cells used transient transfection experiments with CREB as the transcriptional target. CaMKI and CaMKIV phosphorylate CREB on its activating Ser-133 in vitro and stimulate Gal4-CREB-dependent transcription in response to a rise in intracellular Ca2+ when cells are cotransfected with Gal4-CREB and a Gal4 reporter gene (15Sun P. Lou L. Maurer R.A. J. Biol. Chem. 1996; 271: 3066-3073Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Additional cotransfection with a CaMKK increases reporter activity more than 10-fold (8Tokumitsu H. Enslen H. Soderling T.R. J. Biol. Chem. 1995; 270: 19320-19324Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 9Anderson K.A. Means R.L. Huang Q.H. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Fremeau R.T. Means A.R. J. Biol. Chem. 1998; 273: 31880-31889Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Mutation of the CaMKIV activation loop T to A abolishes CaMKK enhancement. To be a signaling cascade, kinase activation loop phosphorylation must be dependent upon induction of CaMKK activity. Phosphate incorporation into endogenous CaMKIV in Jurkat cells is induced rapidly following T-cell receptor stimulation and is blocked by chelation of extracellular Ca2+. This is accompanied by 8–14-fold increases in immunoprecipitated CaMKIV activity that is refractory to further activation by exogenous CaMKK and can be reversed by in vitro treatment with PP2A (16Park I.K. Soderling T.R. J. Biol. Chem. 1995; 270: 30464-30469Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Recombinant CaMKIV transfected into BJAB cells, which lack endogenous CaMKIV, demonstrates similar activation following anti-IgM stimulation that is abrogated by mutating the activation loop T to A (4Chatila T. Anderson K.A. Ho N. Means A.R. J. Biol. Chem. 1996; 271: 21542-21548Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Likewise, CaMKI phosphorylation is induced in PC12 cells, coincident with increased CaMKI activity and reduced activation of CaMKI by exogenous CaMKK (17Aletta J.M. Selbert M.A. Nairn A.C. Edelman A.M. J. Biol. Chem. 1996; 271: 20930-20934Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Collectively, these experiments provide strong evidence for an inducible, Ca2+-dependent activation of CaMKI and CaMKIV in intact cells via activation loop phosphorylation. However, the fact that activation loop phosphorylation requires Ca2+ does not confirm that the physiologic activator is itself Ca2+-dependent, because Ca2+/CaM binding to CaMKI or CaMKIV is required before these enzymes can be phosphorylated by the known CaMKKs. The issue of subcellular localization is a confounding one for any model of the CaMK cascade regulating transcription. There are several lines of evidence that CaMKKα and -β are both cytoplasmic. In contrast to an early study using a polyclonal antibody, immunohistochemistry of rat brain slices using monoclonal antibodies able to distinguish between the α and β isoforms found exclusively cytoplasmic immunoreactivity for both (18Sakagami H. Umemiya M. Saito S. Kondo H. Eur. J. Neurosci. 2000; 12: 89-99Crossref PubMed Scopus (54) Google Scholar). Furthermore, green fluorescent protein-tagged CaMKKα and -β are cytoplasmic in NG108 cells even after depolarizing stimulation (18Sakagami H. Umemiya M. Saito S. Kondo H. Eur. J. Neurosci. 2000; 12: 89-99Crossref PubMed Scopus (54) Google Scholar). This holds true for overexpressed CaMKKβ in Jurkat and BJAB cells. 2E. E. Corcoran and A. R. Means, unpublished data. Similarly, CaMKI appears to be excluded from the nucleus in brain slices as well as in cells overexpressing the protein, yet CaMKI immunoreactivity has recently been observed to translocate to the nuclei of hippocampal neurons during long term potentiation (19Picciotto M.R. Zoli M. Bertuzzi G. Nairn A.C. Synapse. 1995; 20: 75-84Crossref PubMed Scopus (97) Google Scholar,20Ahmed B.Y. Yamaguchi F. Tsumura T. Gotoh T. Sugimoto K. Tai Y. Konishi R. Kobayashi R. Tokuda M. Neurosci. Lett. 2000; 290: 149-153Crossref PubMed Scopus (16) Google Scholar). 3S. S. Hook and A. R. Means, unpublished data. In contrast, CaMKIV is predominantly nuclear but can also be found in neuronal soma and dendritic processes, where it could interact with a cytoplasmic CaMKK (21Nakamura Y. Okuno S. Sato F. Fujisawa H. Neuroscience. 1995; 68: 181-194Crossref PubMed Scopus (82) Google Scholar). The CaMK cascade then is well positioned to affect cytoplasmic events, but it is more difficult to explain its effects on transcription. There could be changes in subcellular localization, an unidentified nuclear CaMKIV activator, or cytosolic CaMK cascade targets that modulate nuclear events. Whether full-length CaMKI enters the nucleus or affects transcription through a cytoplasmic intermediate has not been well studied, but it can stimulate transcription of reporters in transient transfection assays (15Sun P. Lou L. Maurer R.A. J. Biol. Chem. 1996; 271: 3066-3073Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Consequently, although the biochemistry is unambiguous and transient transfections appear to reconstruct a cascade, the mechanism for transcriptional regulation by a CaMK cascade in cells is more complicated than expected. Several other pathways may influence or be influenced by the CaMK cascade. CaMKI and protein kinase A can phosphorylate CaMKKs on multiple inhibitory sites in vitro, and forskolin stimulation reduces CaMKI/IV activation in several cell lines (22Matsushita M. Nairn A.C. J. Biol. Chem. 1999; 274: 10086-10093Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Likewise, nuclear localization of CaMKIIαB and δB isoforms is inhibited by phosphorylation of their nuclear localization sequence by CaMKI or CaMKIV, presumably counteracting direct CaMKII effects on transcription (23Heist E.K. Srinivasan M. Schulman H. J. Biol. Chem. 1998; 273: 19763-19771Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In addition,in vitro experiments indicate CaMKK can phosphorylate and activate both AMP kinase and protein kinase B, and the AMP kinase kinase can phosphorylate and activate CaMKI, although all of these effects are far less substantial than those of the accepted activators AMP kinase kinase, phosphoinositide-dependent kinase 1, and CaMKK, respectively (24Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 25Yano S. Tokumitsu H. Soderling T.R. Nature. 1998; 396: 584-587Crossref PubMed Scopus (536) Google Scholar, 26Okuno S. Kitani T. Matsuzaki H. Konishi H. Kikkawa U. Fujisawa H. J. Biochem. (Tokyo). 2000; 127: 965-970Crossref PubMed Scopus (21) Google Scholar). Finally, Ras-independent activation of the mitogen-activated protein kinases ERK2, p38, and JNK1 was observed in cells transfected with constitutively active CaMKIV and further enhanced by cotransfection with CaMKKα (27Enslen H. Tokumitsu H. Stork P.J. Davis R.J. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10803-10808Crossref PubMed Scopus (261) Google Scholar). The physiologic significance of interacting signal cascades is still unclear, but these examples do serve as a reminder of the intricacy of signal processing. The evolutionary conservation of CaMKI/IV and CaMKK suggests a fundamental biological role. From Aspergillus nidulans toCaenorhabditis elegans to mammals, the cascade members are highly conserved and biochemically interchangeable in gross assays of cascade function in vitro (28Eto K. Takahashi N. Kimura Y. Masuho Y. Arai K. Muramatsu M.A. Tokumitsu H. J. Biol. Chem. 1999; 274: 22556-22562Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 29Joseph J.D. Means A.R. J. Biol. Chem. 2000; 275: 38230-38238Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). have also been in C.D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (30) Google Scholar, I. Mol. Biol. 1999; 10: PubMed Scopus Google and H. M. S. Y. C. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the CaMK cascade in will it is a physiologic and biological functions it However, to the biological significance of the cascade, targets must be CREB is a as it can be activated by the CaMK cascade in transfection experiments but many kinases can be induced to phosphorylate CREB Biochem. 1999; 68: PubMed Scopus Google Scholar, H. K. PubMed Scopus Google Scholar). that the physiologic on the example, of dominant negative CaMKIV in CREB phosphorylation on Ser-133, to effects that are of from K.A. M. Means A.R. Mol. Endocrinol. 11: PubMed Scopus Google Scholar). in experiments with from Ser-133 phosphorylation is but not and appears A. Anderson and A. R. Means, unpublished data. The for these is that the CREB phosphorylation not only by CaMKIV but also by other CREB kinases. In contrast, CREB phosphorylation is reduced in both and hippocampal neurons of CaMKIV but is in the K.A. Means A.R. 2000; PubMed Scopus Google Scholar, N. F. F. S. A. M. J. Neurosci. 2000; 20: PubMed Google Scholar, L. Means A.R. J. Neurosci. 2000; 20: PubMed Google Scholar). These that CaMKIV functions as a CREB kinase in but not all of CREB phosphorylation in the of CaMKKs of signal regulation could be in through regulated of CaMKIV with other between CaMKIV and PP2A has been demonstrated by and immunoprecipitated CaMKIV is with PP2A, although the is not true because of the of PP2A Anderson K.A. Means A.R. B.E. 1998; PubMed Scopus Google Scholar). of PP2A with or increases activation of Gal4-CREB which could from of CaMKIV, In contrast to the of CaMKIV with a potential to CaMKIV and CaMKK have been that the signaling complex may not the known its significance is not biochemical of extract that a of CaMKIV is with the nuclear Means A.R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). CaMKIV to in a signaling complex could be an important factor in its effects on transcription. the of CREB phosphorylation in yet when a transcription factor that can be regulated by CaMKIV in transfected cells will be regulated by CaMKIV in vivo and that specificity is CaMKIV has been with the regulation of many transcription other than response and activating transcription factor 1, and may have multiple of regulating transcription (Fig. K.A. Kane C.D. Biometals. 1998; 11: 331-343Crossref PubMed Scopus (44) Google Scholar). CREB transcriptional activation through its binding to the protein which many transcription to components of the transcriptional of CREB on Ser-133 increases its for but CREB Ser-133 phosphorylation is to transcription without other to itself is of or transfection with the CREB that is constitutively phosphorylated reporter transcription without additional J. M. M.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, K. H. M. Mol. Biol. 2000; 20: PubMed Scopus Google Scholar). experiments that induction of transcription by of cells requires nuclear Ca2+ but not increased CREB Ser-133 phosphorylation S. H. 1998; PubMed Scopus Google Scholar). reporter gene transcription by a is enhanced by constitutively active CaMKIV. in are which has led to the that CaMKIV by direct phosphorylation, but this remains to be CaMKIV stimulation of transcription by the orphan receptor may also be related to effects on A CaMKIV on was because of the similarities in between CaMKIV and In transfection CaMKIV a increase in transcription of a reporter C.D. Means A.R. EMBO J. 2000; PubMed Scopus Google Scholar). The ligand binding domain is not a substrate for CaMKIV in vitro, but binding that with endogenous the CaMKIV Although the mechanism remains to be these and the CaMKIV effects on a for CaMKIV in or transcriptional CaMKIV has also been in the Ca2+-dependent regulation of transcription (Fig. In and Ca2+ to activation with activation of p38, which phosphorylates on multiple sites in vitro those in its activation domain A. F. M. 1999; PubMed Scopus Google Scholar, M. New L. Y. H. F. R.J. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). has also been to be a substrate for CaMKIV in vitro F. Ho N. R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of CaMKI/IV and activate reporter but when There is evidence for two for the of of its nuclear and it to with to activate reporter and of which its binding 2000; PubMed Scopus Google Scholar). The can bind both and and may be involved in a complex on the may itself be a of a Ca2+-dependent signal EMBO J. 2000; PubMed Scopus Google Scholar). Furthermore, as a also and transcriptional activity in L. R. 1999; PubMed Scopus Google Scholar). binding to is with and is by Ca2+/CaM binding to L. R. 1999; PubMed Scopus Google Scholar, 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). also with and 1 and its from may a complex with an activating complex 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In a and is by but is by Ca2+/CaM binding to J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of with can also be inhibited by cotransfection with a constitutively active of which may provide an mechanism for CaMKI/IV of transcription J. R.L. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Ca2+ regulation of is both and from transcriptional from many of CaMKIV and transcription is a of CaMKK effects and between CaMKI and CaMKIV. the kinase and CaMKI, and and so be used to a for a specific CaMK H. H. 1996; PubMed Scopus (82) Google Scholar). these at with those used for CaMK so from the of these be J. D. N. J. 1999; 290: Google Scholar). of the kinases to constitutively active are also used to study but because this of the kinase functions are these proteins could be or example, the activating also the domain and both changes its activation biochemistry and nuclear (2Means A.R. Mol. Endocrinol. 2000; 14: 4-13Crossref PubMed Scopus (159) Google Scholar, H. Braun A. Carafoli E. Klee C. Calcium as a Cellular Regulator. Oxford University Press, New York1999: 311-343Google Scholar). CaMKI and CaMKIV, functions of the C-terminal are not as well but may also affect subcellular and substrate that these but has not yet been is to in the autoinhibitory for CaMKII, CaMKIV, and CaMKKs that activity without (10Tokumitsu H. Muramatsu M. Ikura M. Kobayashi R. J. Biol. Chem. 2000; 275: 20090-20095Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, S. Soderling T.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google H. J. H. J. Soderling T.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Finally, because understanding of the cascade is still so it is not to that transcriptional effects to CaMKIV are also regulated by the CaMKK without directly that These be in in the of experiments to one of the in a CaMK cascade. The evidence for a CaMK cascade regulating Ca2+-dependent transcription in cells is but still CaMKI and CaMKIV are substrates of the CaMKKs and are activated by activation loop phosphorylation that following stimulation of intact cells. The tissue of the CaMKKs those of CaMKI and CaMKIV, but the of subcellular localization still to be Whether the CaMKKs so far are the only kinases of activating CaMKI and CaMKIV is this cascade is does it function as a signal or as an phosphorylation by CaMKKs provides a mechanism for However, CaMKII, these kinases have activation that on a Ca2+ signal for multiple CaMKII, this complex activation biochemistry has been to stimulation P. Schulman H. 1998; PubMed Scopus Google but evidence yet for the CaMK cascade. The from peptide experiments that CaMKI/IV different substrate before and after activation for signal of the cascade also interact with a variety of other and the CaMKKs may have substrates other than both and are for a CaMK cascade. The regulation of these kinases provides many for Ca2+ K. E. J. D. C. D. K. A. and I. R. for and of the and S. S. Hook and K. A. Anderson for to discuss unpublished data.