Activation of Calcium/Calmodulin-dependent Protein Kinase IV in Long Term Potentiation in the Rat Hippocampal CA1 Region

The importance of well characterized calcium/calmodulin-dependent protein kinase (CaMK) II in hippocampal long term potentiation (LTP) is widely well established; however, several CaMKs other than CaMKII are not yet clearly characterized and understood. Here we report the activation of CaMKIV, which is phosphorylated by CaMK kinase and localized predominantly in neuronal nuclei, and its functional role as a cyclic AMP-responsive element-binding protein (CREB) kinase in high frequency stimulation (HFS)-induced LTP in the rat hippocampal CA1 region. CaMKIV was transiently activated in neuronal nuclei after HFS, and the activation returned to the basal level within 30 min. Phosphorylation of CREB, which is a CaMKIV substrate, and expression of c-Fos protein, which is regulated by CREB, increased during LTP. This increase was inhibited mainly by CaMK inhibitors and also by an inhibitor for mitogen-activated protein kinase cascade, although to a lesser extent. Our results suggest that CaMKIV functions as a CREB kinase and controls CREB-regulated gene expression during HFS-induced LTP in the rat hippocampal CA1 region. The importance of well characterized calcium/calmodulin-dependent protein kinase (CaMK) II in hippocampal long term potentiation (LTP) is widely well established; however, several CaMKs other than CaMKII are not yet clearly characterized and understood. Here we report the activation of CaMKIV, which is phosphorylated by CaMK kinase and localized predominantly in neuronal nuclei, and its functional role as a cyclic AMP-responsive element-binding protein (CREB) kinase in high frequency stimulation (HFS)-induced LTP in the rat hippocampal CA1 region. CaMKIV was transiently activated in neuronal nuclei after HFS, and the activation returned to the basal level within 30 min. Phosphorylation of CREB, which is a CaMKIV substrate, and expression of c-Fos protein, which is regulated by CREB, increased during LTP. This increase was inhibited mainly by CaMK inhibitors and also by an inhibitor for mitogen-activated protein kinase cascade, although to a lesser extent. Our results suggest that CaMKIV functions as a CREB kinase and controls CREB-regulated gene expression during HFS-induced LTP in the rat hippocampal CA1 region. long term potentiation calcium/calmodulin-dependent protein kinase mitogen-activated protein kinase extracellular signal-regulated protein kinase MAPK/ERK kinase cyclic AMP-responsive element-binding protein high frequency stimulation fluorescein isothiocyanate excitatory postsynaptic potential field EPSP cyclic AMP-dependent protein kinase artificial cerebrospinal fluid Long term potentiation (LTP)1 in hippocampus (1Bliss T.V.P. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9604) Google Scholar, 2Malenka R.C. Nicoll R.A. Science. 1999; 285: 1870-1874Crossref PubMed Scopus (2250) Google Scholar) is thought to be a model for the molecular mechanism of learning and memory in the mammalian central nervous system. Among several kinds of molecules reported to be involved in LTP (3Sanes J.R. Lichtman J.W. Nat. Neurosci. 1999; 2: 597-604Crossref PubMed Scopus (281) Google Scholar), protein kinases and phosphatases are particularly important, because the protein phosphorylation and dephosphorylation are essential for the regulation of neuronal functions. We have been studying activation of calcium/calmodulin-dependent protein kinase II (CaMKII; Refs. 4Fukunaga K. Miyamoto E. Neurosci. Res. 2000; 38: 3-17Crossref PubMed Scopus (105) Google Scholar, 5Fukunaga K. Stoppini L. Miyamoto E. Muller D. J. Biol. Chem. 1993; 268: 7863-7867Abstract Full Text PDF PubMed Google Scholar, 6Fukunaga K. Muller D. Miyamoto E. J. Biol. 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Res. 2000; 38: 3-17Crossref PubMed Scopus (105) Google Scholar, 5Fukunaga K. Stoppini L. Miyamoto E. Muller D. J. Biol. Chem. 1993; 268: 7863-7867Abstract Full Text PDF PubMed Google Scholar, 6Fukunaga K. Muller D. Miyamoto E. J. Biol. Chem. 1995; 270: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 7Liu J. Fukunaga K. Yamamoto H. Nishi K. Miyamoto E. J. Neurosci. 1999; 19: 8292-8299Crossref PubMed Google Scholar) is particularly implicated in LTP induction, because its activation is accompanied by autophosphorylation (5Fukunaga K. Stoppini L. Miyamoto E. Muller D. J. Biol. Chem. 1993; 268: 7863-7867Abstract Full Text PDF PubMed Google Scholar, 6Fukunaga K. Muller D. Miyamoto E. J. Biol. Chem. 1995; 270: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 7Liu J. Fukunaga K. Yamamoto H. Nishi K. Miyamoto E. J. Neurosci. 1999; 19: 8292-8299Crossref PubMed Google Scholar) and the activity is essential for the induction of LTP (9Silva A.J. Stevens C.F. Tonegawa S. Wang Y. Science. 1992; 257: 206-211Crossref PubMed Scopus (1085) Google Scholar). Although the role of CaMKII in synaptic plasticity has been closely examined by many researchers (1Bliss T.V.P. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9604) Google Scholar, 2Malenka R.C. Nicoll R.A. Science. 1999; 285: 1870-1874Crossref PubMed Scopus (2250) Google Scholar, 3Sanes J.R. Lichtman J.W. Nat. Neurosci. 1999; 2: 597-604Crossref PubMed Scopus (281) Google Scholar, 4Fukunaga K. Miyamoto E. Neurosci. Res. 2000; 38: 3-17Crossref PubMed Scopus (105) Google Scholar), the roles of other CaMK subtypes expressed in neurons are still unknown. Neurons express at least five known CaMKs: CaMKI (10Nairn A.C. Greengard P. J. Biol. Chem. 1987; 262: 7273-7281Abstract Full Text PDF PubMed Google Scholar), CaMKII, CaMKIII (11Nairn A.C. Bhagat B. Palfrey H.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7939-7943Crossref PubMed Scopus (185) Google Scholar), CaMKIV (12Sikela J.M. Hahn W.E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3038-3042Crossref PubMed Scopus (59) Google Scholar, 13Ohmstede C.-A. Jensen K.F. Sahyoun N.E. J. Biol. Chem. 1989; 264: 5866-5877Abstract Full Text PDF PubMed Google Scholar), and CaMK kinase (14Tokumitsu H. Brickey D.A. Glod J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar). The activation mechanisms of CaMKI and CaMKIV (14Tokumitsu H. Brickey D.A. Glod J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar, 15Soderling T.R. Trends Biochem. Sci. 1999; 24: 232-236Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) differ from that of CaMKII; specific threonine residues of CaMKI (Thr177; Ref. 16Haribabu 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) and IV (Thr196; Ref. 17Serbert M.A. Anderson K.A. Huang Q.-H. Goldstein E.G. Means A.R. Edelman A.M. J. Biol. Chem. 1995; 270: 17616-17621Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) are phosphorylated by CaMK kinase, and phosphorylation is essential for the activation of CaMKI and CaMKIV. CaMKIV, also called CaMK Gr, is expressed in the nuclei of neurons (18Miyano O. Kameshita I. Fujisawa H. J. Biol. Chem. 1992; 267: 1198-1203Abstract Full Text PDF PubMed Google Scholar, 19Jensen K.F. Ohmstede C.A. Fisher R.S. Sahyoun N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2850-2853Crossref PubMed Scopus (183) Google Scholar), although its expression is not only limited to the nuclei (19Jensen K.F. Ohmstede C.A. Fisher R.S. Sahyoun N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2850-2853Crossref PubMed Scopus (183) Google Scholar). We previously reported regulation of CaMKIV in cultured rat hippocampal neurons (20Kasahara J. Fukunaga K. Miyamoto E. J. Neurosci. Res. 2000; 59: 594-600Crossref PubMed Scopus (17) Google Scholar, 21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and showed that CaMKIV was activated by N-methyl-d-aspartate glutamate receptor stimulation or high K+-induced depolarization (20Kasahara J. Fukunaga K. Miyamoto E. J. Neurosci. Res. 2000; 59: 594-600Crossref PubMed Scopus (17) Google Scholar). Inactivation of CaMKIV was regulated primarily by protein phosphatases 2A (21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 22Park I.-K. Soderling T.R. J. Biol. Chem. 1995; 270: 30464-30469Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 23Westpharl R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (224) Google Scholar) and calcineurin (21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Because a major substrate for CaMKIV in the nucleus is cyclic AMP-responsive element-binding protein (CREB; Ref. 24Enslen H. Sun P. Brickey D. Soderling S.H. Klamo E. Soderling T.R. J. Biol. Chem. 1994; 269: 15520-15527Abstract Full Text PDF PubMed Google Scholar), CaMKIV is thought to regulate gene expression in a neuronal activity-dependent manner. In support of this hypothesis, suppression of CaMKIV expression by antisense oligonucleotides abolished high K+- or electrical stimulation-induced CREB phosphorylation in cultured rat hippocampal neurons (25Bito H. Deisseroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar), and CaMKIV-deficient mice showed the decrease in CREB phosphorylation (26Ho N. Liauw J.A. Blaeser F. Wei F. Hanissian S. Muglia L.M. Wozniak D.F. Nardi A. Arvin K.L. Holtzman D.M. Linden D.J. Zhuo M. Muglia L.J. Chatila T.A. J. Neurosci. 2000; 20: 6459-6472Crossref PubMed Google Scholar,27Riber T.J. Rodriguiz R.M. Khiroug L. Wetsel W.C. Augustine G.J. Means A.R. J. Neurosci. 2000; 20 (, 1–5): RC107Crossref PubMed Google Scholar). Introduction of a dominant negative CaMKIV or dominant negative CREB into cultured cerebellar Purkinje neurons resulted in defects in the late phase of long term depression in response to glutamate (28Ahn S. Ginty D.D. Linden D.J. Neuron. 1999; 23: 559-568Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). CaMKIV-deficient mice also showed decreased potentiation at a later stage of rat hippocampal CA1 LTP (26Ho N. Liauw J.A. Blaeser F. Wei F. Hanissian S. Muglia L.M. Wozniak D.F. Nardi A. Arvin K.L. Holtzman D.M. Linden D.J. Zhuo M. Muglia L.J. Chatila T.A. J. Neurosci. 2000; 20: 6459-6472Crossref PubMed Google Scholar). These reports suggest that CaMKIV functions as a CREB kinase and regulates CREB-dependent gene expression in neurons, which may in turn be linked to a protein synthesis-dependent long lasting synaptic plasticity. To understand the molecular mechanisms of synaptic plasticity and function of CaMKIV, it is essential to know how CaMKIV activity changes following application of conditioning stimuli such as high frequency stimulation (HFS) to induce LTP and how CREB phosphorylation is regulated by CaMKIV and other protein kinases under these conditions. In the present study, we examined CaMKIV activation CREB phosphorylation and stimulation of gene expression during HFS-induced LTP in the rat hippocampal CA1 region. The following and from the from and from from and from and from protein from from and from from from and from to a report J. Fukunaga K. Yamamoto H. Nishi K. Miyamoto E. J. Neurosci. 1999; 19: 8292-8299Crossref PubMed Google Scholar). The from and the hippocampal a in artificial cerebrospinal fluid at for at least a a was to an and at a of at 30 The field EPSP was by stimuli a the and from the of CA1 a In the of a was the in the and the from the of The was a and the of the was and for an and a The was to a of the was a of frequency and was a and the was for the in the the for the The following inhibitors for protein kinases in as a at the following and at and and and The in to at the following and and and was in at a of as a and in at a of The these and during the as in the the to a and the CA1 in and at the in of the 30 and by at for min. CaMKIV was an (21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and protein and CaMKIV activity was as a substrate, as reported previously (20Kasahara J. Fukunaga K. Miyamoto E. J. Neurosci. Res. 2000; 59: 594-600Crossref PubMed Scopus (17) Google Scholar, 21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The the and the at in of and by at for and and and for min. to and to a at for a in a for at and the at the (21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), (21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and and by a or an and the of the the at in and of from in and a The in and for at and the and by of CaMKIV phosphorylation was as The of the and of neurons in neurons, the that are in a the stimulation and in the CA1 The of the neurons in which the of the nuclei are than of the was which have in the nuclei, as The of hippocampal and was The are the was by the of was of to have We examined the changes of CaMKIV activity during LTP. of to the potentiation of which in the CA1 of at after CaMKIV activity was and after application and was increased within of and of to basal within 30 after This was by the (21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) to of CaMKIV phosphorylation at an essential for activation of CaMKIV by CaMK This is by in D. Phosphorylation of was increased after of and returned to the basal CaMKIV protein and 30 after HFS, although a increase of was after and is the previously reported M. M. Miyamoto O. F. R. O. Res. PubMed Scopus Google Scholar). We also examined the phosphorylation of mitogen-activated protein kinase signal-regulated protein kinase and CREB, and phosphorylation of was in Phosphorylation of was increased after of and returned to basal Because activated not changes in phosphorylation of examined in the present This is the reports J. Fukunaga K. Yamamoto H. Nishi K. Miyamoto E. J. Neurosci. 1999; 19: 8292-8299Crossref PubMed Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that activity of is transiently increased by in the rat hippocampal CA1 region. In CREB phosphorylation an increased of of 30 of and of after HFS, and the increased phosphorylation was during LTP Although the of and after HFS, a increase was 30 of and of after The protein of CREB not at after not To the of activated CaMKIV in neurons, we an the In was in the was not by the (21Kasahara J. Fukunaga K. Miyamoto E. J. Biol. Chem. 1999; 274: 9061-9067Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) that it was The in the of CaMKIV activation in the nuclei not was in the nuclei of neurons in and that was the was the that it was Although the in the present was it that the protein the may be called These results suggest that CaMKIV is activated in the nuclei of CA1 neurons by was also in the of neurons because its expression was not limited to nuclei (19Jensen K.F. Ohmstede C.A. Fisher R.S. Sahyoun N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2850-2853Crossref PubMed Scopus (183) Google Scholar). We expression of CaMKIV in the and of cultured neurons not Because it was reported that the major CREB kinase was CaMKIV in response to neuronal stimulation in cultured rat hippocampal neurons (25Bito H. Deisseroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar), we CaMKIV as a CREB kinase during LTP. To CaMKIV we a and a CaMK inhibitors have been previously to CaMKIV activity in cultured rat hippocampal neurons (20Kasahara J. Fukunaga K. Miyamoto E. J. Neurosci. Res. 2000; 59: 594-600Crossref PubMed Scopus (17) Google Scholar). The application of or inhibited induction of by CaMKII K. Muller D. Miyamoto E. J. Biol. Chem. 1995; 270: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 7Liu J. Fukunaga K. Yamamoto H. Nishi K. Miyamoto E. J. Neurosci. 1999; 19: 8292-8299Crossref PubMed Google Scholar). and CaMKIV phosphorylation phosphorylation and HFS-induced CREB phosphorylation however, inhibited by and 30 after an to LTP induction CREB phosphorylation and specific by These results a CaMKIV activation and CREB phosphorylation and are the that CaMKIV functions as a CREB kinase during HFS-induced LTP. We examined the of HFS-induced CREB The is thought to be involved in CREB phosphorylation the activation of kinase J. Ginty D.D. Science. 1996; PubMed Scopus (1085) Google Scholar). We previously reported that a high of an inhibited LTP induction J. Fukunaga K. Yamamoto H. Nishi K. Miyamoto E. J. Neurosci. 1999; 19: 8292-8299Crossref PubMed Google Scholar). the of this inhibitor was of CaMKII, and we that inhibited LTP of CaMKII than J. Fukunaga K. Yamamoto H. Nishi K. Miyamoto E. J. Neurosci. 1999; 19: 8292-8299Crossref PubMed Google Scholar). In this study, we examined the of M. A.J. D.A. R.A. F. R.A. J.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), which at a than the not the induction of HFS-induced LTP a other reports D. A. B. Neuron. 1999; 24: Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. T.J. J. Neurosci. 2000; 20: PubMed Google Scholar). inhibited phosphorylation CaMKIV phosphorylation To 20 and 30 was and of CaMKIV phosphorylation was not the other CREB phosphorylation was not inhibited after was inhibited 30 after These results suggest that not only CaMKIV also function as CREB kinases in HFS-induced LTP. be that CaMKIV functions as a CREB kinase at a stage of HFS-induced functions at a later during the We examined the of and inhibitors for HFS-induced CREB because and In and not HFS-induced LTP in the CA1 In the LTP was by inhibitors as previously reported Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), that these inhibitors under conditions. Because has been to have other kinases than we its CaMKIV and in CaMKIV and CREB phosphorylation was inhibited by by during LTP in the CA1 The results for CREB phosphorylation are in D. of the inhibitors inhibited CREB phosphorylation by LTP at and 30 after in the CA1 region. These results suggest that the is involved in HFS-induced LTP in CREB phosphorylation at an stage and are the report M. Zhuo M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). To CREB phosphorylation by gene we examined the expression of the c-Fos protein, of the for cyclic AMP-responsive gene expression H. Ginty D.D. Science. 1993; PubMed Scopus Google Scholar). The expression of the c-Fos protein was and after LTP induction and increased 30 of and of after LTP induction that the phosphorylated CREB c-Fos We c-Fos expression was regulated by CaMKIV during LTP. The increase in c-Fos expression at after was inhibited by the of or of hippocampal inhibited the HFS-induced c-Fos inhibited c-Fos expression The of by and was of CREB phosphorylation in and These results suggest that CaMKIV and function as CREB which in turn regulate gene expression in HFS-induced LTP. Here we that CaMKIV is activated accompanied phosphorylation of CREB phosphorylation and stimulation of c-Fos expression during LTP. To this is the report to that CaMKIV is activated and involved in stimulation of gene expression during LTP in the hippocampal CA1 region. The activation of CaMKIV in was and returned to the level in a This activation from that of CaMKII, which showed a long lasting activation during LTP (5Fukunaga K. Stoppini L. Miyamoto E. Muller D. J. Biol. Chem. 1993; 268: 7863-7867Abstract Full Text PDF PubMed Google Scholar, 6Fukunaga K. Muller D. Miyamoto E. J. Biol. Chem. 1995; 270: 6119-6124Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). 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