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• W2100025585 abstract "Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is phosphorylated at Thr196 by Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), resulting in induction of both autonomous activity and a high level of Ca2+/CaM-dependent activity. We have shown that the kinetics of Thr196 phosphorylation of CaM-KIV by CaM-KK is well correlated with the generation of its autonomous activity, although Thr177 phosphorylation of CaM-KI does not induce its autonomous activity. The activities of CaM-KI chimera mutants fused with C-terminal regions (residues 296–469 and 296–350) of CaM-KIV are completely dependent on Ca2+/CaM, which is also the case for CaM-KI. Unlike wild-type CaM-KI, however, phosphorylation of Thr177 in the chimera mutants by CaM-KK resulted in generation of significant autonomous activities, indicating that the phosphorylation of Thr in the activation loop is sufficient to partially release the autoinhibitory region of CaM-KIV from the catalytic core. Indeed, the CaM-KIV peptide (residues 304–325) containing minimum autoinhibitory sequences (residues 314–321) suppressed the activity of non-phosphorylated CaM-KIV with an IC50 of ∼50 μm, and this suppression was competitive with respect to the peptide substrate; however, the CaM-KIV peptide was not capable of inhibiting Thr196-phosphorylated CaM-KIV. Taken together, these results indicated that the Thr196 phosphorylation of CaM-KIV by CaM-KK reduced the interaction of the catalytic core with the autoinhibitory region, resulting in generation of the autonomous activity. Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is phosphorylated at Thr196 by Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), resulting in induction of both autonomous activity and a high level of Ca2+/CaM-dependent activity. We have shown that the kinetics of Thr196 phosphorylation of CaM-KIV by CaM-KK is well correlated with the generation of its autonomous activity, although Thr177 phosphorylation of CaM-KI does not induce its autonomous activity. The activities of CaM-KI chimera mutants fused with C-terminal regions (residues 296–469 and 296–350) of CaM-KIV are completely dependent on Ca2+/CaM, which is also the case for CaM-KI. Unlike wild-type CaM-KI, however, phosphorylation of Thr177 in the chimera mutants by CaM-KK resulted in generation of significant autonomous activities, indicating that the phosphorylation of Thr in the activation loop is sufficient to partially release the autoinhibitory region of CaM-KIV from the catalytic core. Indeed, the CaM-KIV peptide (residues 304–325) containing minimum autoinhibitory sequences (residues 314–321) suppressed the activity of non-phosphorylated CaM-KIV with an IC50 of ∼50 μm, and this suppression was competitive with respect to the peptide substrate; however, the CaM-KIV peptide was not capable of inhibiting Thr196-phosphorylated CaM-KIV. Taken together, these results indicated that the Thr196 phosphorylation of CaM-KIV by CaM-KK reduced the interaction of the catalytic core with the autoinhibitory region, resulting in generation of the autonomous activity. Ca2+/calmodulin-dependent protein kinases (CaM-Ks) 1The abbreviations used are: CaM-K, Ca2+/CaM-dependent protein kinase; CaM, calmodulin; GST, glutathione S-transferase; DTT, dithiothreitol; CREB, cAMP-response element-binding protein. constitute a diverse group of enzymes, which are involved in many cellular responses mediated by an increase in the concentration of intracellular calcium (1Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (663) Google Scholar, 2Lukas T.J. Mirzoeva S. Watterson D.M. Van Eldik L.J. Watterson D.M. Calmodulin and Signal Transduction. Academic Press, New York1998: 65-168Crossref Scopus (26) Google Scholar, 3Soderling T.R. Stull J.T. Chem. Rev. 2001; 101: 2341-2352Crossref PubMed Scopus (167) Google Scholar). CaM-KIV, also known as CaM-kinase Gr, is one of the multifunctional CaM-Ks, and is predominantly localized in cell nuclei (4Ohmstede C.A. Jensen K.F. Sahyoun N.E. J. Biol. Chem. 1989; 264: 5866-5875Abstract Full Text PDF PubMed Google Scholar, 5Jensen 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, 6Miyano O. Kameshita I. Fujisawa H. J. Biol. Chem. 1992; 267: 1198-1203Abstract Full Text PDF PubMed Google Scholar). Consistent with the nuclear localization of CaM-KIV, an important role has been demonstrated for CaM-KIV in the regulation of Ca2+-dependent gene expression by the phosphorylation of transcription factors such as cAMP-responsive element-binding protein (CREB) (7Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1289) Google Scholar, 8Matthews R.P. Guthrie C.R. Wailes L.M. Zhao X. Means A.R. McKnight G.S. Mol. Cell. Biol. 1994; 14: 6107-6116Crossref PubMed Scopus (497) Google Scholar, 9Enslen 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, 10Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (649) Google Scholar, 11Bito H. Deisserroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar). A recent study of transgenic mice carrying dominant negative CaM-KIV alleles that confer defects in the phosphorylation of CREB indicates that these animals exhibit a disruption of late phase long term potentiation and that they are impaired in the consolidation/retention phase of hippocampus-dependent memory (12Kang H. Sun L.D. Atkins C.M. Soderling T.R. Wilson M.A. Tonegawa S. Cell. 2001; 106: 771-783Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Analysis of mice deficient in CaM-KIV has revealed that the CaM-KIV-mediated pathway plays an important role in the function and development of both the cerebellum and hippocampal CA1 neurons (13Ribar T.J. Rodriguiz R.M. Khiroug L. Wetsel W.C. Augustine G.J. Means A.R. J. Neurosci. 2000; 20: RC107Crossref PubMed Google Scholar, 14Ho 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), and is critical for male and female fertility (15Wu J.Y. Gonzalez-Robayna I.J. Richards J.S. Means A.R. Endocrinology. 2000; 141: 4777-4783Crossref PubMed Scopus (49) Google Scholar, 48Wu J.Y. Ribar T.J. Cummings D.E. Burton K.A. McKnight G.S. Means A.R. Nat. Genet. 2000; 25: 448-452Crossref PubMed Scopus (196) Google Scholar). Previous studies have demonstrated that full activation of CaM-KIV requires not only Ca2+/CaM binding but also phosphorylation of its activation loop Thr residue (Thr196) by an upstream protein kinase (CaM-KK) (9Enslen 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, 16Cruzalegui F.H. Means A.R. J. Biol. Chem. 1993; 268: 26171-26178Abstract Full Text PDF PubMed Google Scholar, 17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar, 18Tokumitsu H. Enslen H. Soderling T.R. J. Biol. Chem. 1995; 270: 19320-19324Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 19Selbert 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). Phosphorylation of Thr196 in CaM-KIV results in the induction of a large amount of Ca2+/CaM-dependent activity. In addition to the induction of total activity, Ca2+/CaM-independent activity (autonomous activity) is generated by activation with CaM-KK. This important regulatory mechanism would allow a transient elevation in intracellular Ca2+ levels to produce a prolonged CaM-KIV activation to regulate gene transcription via the phosphorylation of transcription factor(s). Autonomous activity of CaM-KIV has been observed with purified enzyme from rat brain (20Frangakis M.V. Ohmstede C.A. Sahyoun N. J. Biol. Chem. 1991; 266: 11309-11316Abstract Full Text PDF PubMed Google Scholar), immunoprecipitated enzyme from anti-TCR/CD3 monoclonal antibody-stimulated Jurkat cells (21Hanissian S.H. Frangakis M. Bland M.M. Jawahar S. Chatila T.A. J. Biol. Chem. 1993; 268: 20055-20063Abstract Full Text PDF PubMed 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), and ionomycin-treated, transfected COS-7 and HeLa cells (23Tokumitsu H. Soderling T.R. J. Biol. Chem. 1996; 271: 5617-5622Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 24Tokumitsu H. Inuzuka H. Ishikawa Y. Ikeda M. Saji I. Kobayashi R. J. Biol. Chem. 2002; 277: 15813-15818Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). A recent study suggested that the autonomous activity appears to be required for CaM-KIV to regulate CREB-mediated transcription, indicating that it might be physiologically relevant (25Anderson K.A. Noeldner P.K. Reece K. Wadzinski B.E. Means A.R. J. Biol. Chem. 2004; 279: 31708-31716Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We have shown that the induction of the total activity of CaM-KIV by CaM-KK phosphorylation was because of mainly decreasing Km for its substrate (17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar). However, this does not account for generation of the autonomous activity. At a minimum, Thr196 phosphorylation by CaM-KK is required for generation of Ca2+/CaM-independent activity of CaM-KIV, because a mutation of Thr196 by Ala has been shown to abolish the generation of autonomous activity (19Selbert 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, 23Tokumitsu H. Soderling T.R. J. Biol. Chem. 1996; 271: 5617-5622Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Therefore, it has not been resolved whether the Ca2+-independent activity is a direct consequence of Thr196 phosphorylation or whether it is the result of subsequent autophosphorylation. In contrast, it has been well characterized that CaM-KII, another member of multifunctional CaM-Ks, is converted to the Ca2+/CaM-independent form by autophosphorylation at Thr286 in its autoinhibitory region, which suppresses the autoinhibitory function of the enzyme (26Lou L.L. Lloyd S.J. Schulman H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9497-9501Crossref PubMed Scopus (136) Google Scholar, 27Lai Y. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4253-4257Crossref PubMed Scopus (216) Google Scholar, 28Miller S.G. Kennedy M.B. Cell. 1986; 44: 861-870Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 29Schworer C.M. Colbran R.J. Soderling T.R. J. Biol. Chem. 1986; 261: 8581-8584Abstract Full Text PDF PubMed Google Scholar, 30Colbran R.J. Smith M.K. Schworer C.M. Fong Y.L. Soderling T.R. J. Biol. Chem. 1989; 264: 4800-4804Abstract Full Text PDF PubMed Google Scholar, 31Yang E. Schulman H. J. Biol. Chem. 1999; 274: 26199-26208Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Thr286 autophosphorylation has been shown to be sensitive to the duration, magnitude, and frequency of the imposed calcium transient (32Hanson P.I. Meyer T. Stryer L. Schulman H. Neuron. 1994; 12: 943-956Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 33De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1086) Google Scholar) and also to be important in the regulation of synaptic plasticity and behavior in vivo (34Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Science. 1998; 279: 870-873Crossref PubMed Scopus (889) Google Scholar). Although there is an equivalent Thr (Thr308) in the putative autoinhibitory region of CaM-KIV, mutation of Thr308 to Ala did not impair the generation of autonomous activity, indicating that the Thr308 is not involved in the generation of autonomous activity (23Tokumitsu H. Soderling T.R. J. Biol. Chem. 1996; 271: 5617-5622Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 35Chatila 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, multiple autophosphorylation sites have been identified in either the N-terminal region of the catalytic domain or the C-terminal region of CaM-KIV (36McDonald O.B. Merrill B.M. Bland M.M. Taylor L.C. Sahyoun N. J. Biol. Chem. 1993; 268: 10054-10059Abstract Full Text PDF PubMed Google Scholar, 37Watanabe S. Okuno S. Kitani T. Fujisawa H. J. Biol. Chem. 1996; 271: 6903-6910Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 38Okuno S. Kitani T. Fujisawa H. J. Biochem. (Tokyo). 1995; 117: 686-690Crossref PubMed Scopus (27) Google Scholar), but none of them has been shown to be involved in its autonomous activity (35Chatila 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). Thus the precise mechanism of the autonomous activity of CaM-KIV generated by CaM-KK phosphorylation remains uncertain. In this report, we have examined the mechanism of generation of the autonomous activity of CaM-KIV by CaM-KK phosphorylation using Escherichia coli-expressed recombinant enzymes, including CaM-KI/CaM-KIV chimera mutants and the autoinhibitory peptide of CaM-KIV, and demonstrated that Thr196 phosphorylation reduced the affinity of its autoinhibitory region to the catalytic core and is likely sufficient to generate the autonomous activity of the enzyme. Materials—CaM-KKα cDNA (GenBank™ accession number L42810) was obtained from a rat brain cDNA library (18Tokumitsu H. Enslen H. Soderling T.R. J. Biol. Chem. 1995; 270: 19320-19324Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Recombinant CaM-KKα was expressed in E. coli and purified as described previously (39Tokumitsu H. Iwabu M. Ishikawa Y. Kobayashi R. Biochemistry. 2001; 40: 13925-13932Crossref PubMed Scopus (69) Google Scholar). Recombinant rat CaM was expressed in the E. coli strain BL21(DE3) using pET-CaM (kindly provided by Dr. Nobuhiro Hayashi, Fujita Health University, Toyoake, Japan) and purified by phenyl-Sepharose column chromatography (40Hayashi N. Matsubara M. Takasaki A. Titani K. Taniguchi H. Protein Expression Purif. 1998; 12: 25-28Crossref PubMed Scopus (134) Google Scholar). Anti-CaM-KIV antibody and anti-CaM-KI antibody were obtained from Signal Transduction Laboratories and Santa Cruz Biotechnology, Inc., respectively. Anti-phospho-CaM-KIV at Thr196 and anti-phospho-CaM-KI at Thr177 monoclonal antibodies were generated against the synthetic phosphopeptides corresponding to residues 189–203 of rat CaM-KIV (CEHQVLMKT(p)VCGTPGY) and corresponding to residues 169–184 of rat CaM-KI (CEDPGSVLST-(p)ACGTPGY), respectively. Peptides were conjugated with keyhole limpet hemocyanin via the N terminus cysteine and were injected into BALB/c mice as described previously (41Kimura K. Nozaki N. Saijo M. Kikuchi A. Ui M. Enomoto T. J. Biol. Chem. 1994; 269: 24523-24526Abstract Full Text PDF PubMed Google Scholar). Biotinylated CaM was purchased from Biomedical Technologies Inc. (Stoughton, MA). A synthetic peptide corresponding to residues 304–325 of mouse CaM-KIV (VHMDTAQKKLQEFNARRKLKAA) was obtained from Bio-Synthesis Inc. (Lewisville, TX) with >95% purity. All other chemicals were from standard commercial sources. Construction and Expression of CaM-KI and CaM-KIV—GST-fused CaM-KI was constructed by amplification of cDNA fragments using rat CaM-KI cDNA as a template, and a sense primer (5′-GCTCTAGAGATGCCAGGGGCAGTGGAAGGC-3′) and an antisense primer (5′-CCGCTCGAGTCAGTCCATGGCCCTAGAGC-3′). GST-fused CaM-KIVs were constructed by amplification of cDNA fragments using mouse CaM-KIV cDNA (17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar) as a template, and a sense primer (5′-GCTCTAGAGATGCTCAAAGTCACGGTGCCC-3′) and the following antisense primers: wild-type, 5′-CCGCTCGAGTTAGTACTCTGGCTGAATCGC-3′; CaM-KIV-(1–350), 5′-CCGCTCGAGTCACTTGTGGTTCTCTTGGAT-3′; CaM-KIV-(1–336), 5′-CCGCTCGAGTTATCCCAGCCGAGAAGAGGC-3′; CaM-KIV-(1–331), 5′-CCGCTCGAGTTAGGCCACCACAGCCTTCAC-3′; CaM-KIV-(1–327), 5′-CCGCTCGAGTCACTTCACCGCTGCCTTAAG-3′; CaM-KIV-(1–321), 5′-CCGCTCGAGTCACTTGCGCCGAGCATTG-3′; CaM-KIV-(1–313), 5′-CCGCTCGAGTCAAAGTTTCTTCTGAGCAG-3′; CaM-KIV-(1–309), 5′-CCGCTCGAGTCAAGCAGTGTCCATGTGAAC-3′. The PCR products were then ligated into the XbaI/XhoI site of the pGEX-PreS vector (39Tokumitsu H. Iwabu M. Ishikawa Y. Kobayashi R. Biochemistry. 2001; 40: 13925-13932Crossref PubMed Scopus (69) Google Scholar). GST-fused CaM-KI/IV469 and CaM-KI/IV350 were constructed by replacement of the StuI/XhoI-digested fragment of the wild-type pGEX-PreS-CaM-KI by a PCR fragment amplified using mouse CaM-KIV cDNA as a template, and sense (5′-CCTTGCAGCACCCATGGGTCACAGGTAAAG-3′) and antisense primers (wild-type and CaM-KIV-(1–350), respectively) as described above. The nucleotide sequences of CaM-KIV mutants and CaMKI/KIV chimera mutants, including the wild-type enzymes, were confirmed by an ABI377 automated sequencer. cDNAs carrying GST-fused enzymes were introduced into E. coli JM-109, and the expression of recombinant enzyme was induced by 1 mm isopropyl-β-d-thiogalactopyranoside. Purification of Recombinant Enzymes—An E. coli pellet containing recombinant enzyme was lysed with PBS followed by purification using glutathione-Sepharose (Amersham Biosciences) column chromatography as described in the manufacturer's protocol. Then further purification was carried out by using CaM-Sepharose column chromatography as described in the manufacturer's protocol for the enzymes that are capable of binding Ca2+/CaM (wild-type CaM-KI, CaM-KIV, CaM-KI/IV469, CaM-KI/IV350, CaM-KIV-(1–350), and CaM-KIV-(1–336)). These enzymes were eluted from the column with 150 mm NaCl, 50 mm Tris-HCl (pH 7.5), 1 mm dithiothreitol (DTT), 1 mm EDTA, and 2 mm EGTA (Buffer A). Recombinant enzymes which do not bind Ca2+/CaM (CaM-KIV-(1–331), -(1–327), -(1–321), -(1–313), and -(1–309)) were dialyzed against Buffer A. Then purified enzymes were digested with PreScission protease (10 unit, Amersham Biosciences) for 12 h at 5 °C to cleave GST followed by purification using glutathione-Sepharose column chromatography as described in the manufacturer's protocol. Pass-through fractions from the column were collected and concentrated using Microcon YM-30 (Millipore Corporation, Bedford, MA). After addition of equal volumes of 80% glycerol and 20% ethylene glycol solution, purified enzymes were stored at –30 °C. In Vitro Activation of CaM-KI and CaM-KIV by CaM-KK—Purified recombinant CaM-Ks (0.1 mg/ml) were incubated with CaM-KKα (3 μg/ml) at 30 °C for the indicated periods in a solution containing 50 mm HEPES (pH 7.5), 10 mm Mg(Ac)2, 1 mm DTT, 2 mm CaCl2, 10 μm CaM, and 200 μm ATP. The reaction was initiated by addition of ATP and terminated by 10–20-fold dilution with ice-cold 50 mm HEPES (pH 7.5), 2 mg/ml bovine serum albumin, 10% ethylene glycol, and 2 mm EDTA. Five μl of the diluted sample was then subjected to the protein kinase assay or Western blot analysis. In Vitro Assay for CaM-KI and CaM-KIV Activity—CaM-KI and CaM-KIV were assayed at 30 °C for 5–10 min in a solution (25 μl) containing 50 mm HEPES (pH 7.5), 10 mm Mg(Ac)2, 40 μm syntide-2, 1 mm DTT, and 200 μm [γ-32P]ATP (∼1000 cpm/pmol) in the presence of either 1 mm EGTA (autonomous activity) or 1 mm CaCl2, 4 μm CaM. The reaction was initiated by the addition of the enzyme and terminated by spotting aliquots (15 μl) onto phosphocellulose paper (Whatman P-81) followed by several washes with 75 mm phosphoric acid (42Roskowski R. Methods Enzymol. 1985; 99: 3-6Crossref Scopus (691) Google Scholar). Phosphate incorporation into syntide-2 was determined by liquid scintillation counting of the filters. Others—Western blotting was performed as described previously using horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Biosciences) or anti-goat IgG antibody as a secondary antibody and chemiluminescence reagent (PerkinElmer Life Sciences) for detection. CaM overlay was carried out using biotinylated CaM (0.5 μg/ml) in the presence of 1 mm CaCl2 as described previously (17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar) using chemiluminescence reagent (PerkinElmer Life Sciences) for detection. Protein concentration was estimated by staining with Coomassie Brilliant Blue (Bio-Rad) using bovine serum albumin as a standard (43Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217315) Google Scholar). Activation and Phosphorylation of CaM-KIV and CaM-KI by CaM-KK—It has been shown that CaM-KIV is phosphorylated at Thr196 by CaM-KK, resulting in induction of a high level of Ca2+/CaM-dependent activity as well as generation of Ca2+/CaM-independent activity (17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar, 18Tokumitsu H. Enslen H. Soderling T.R. J. Biol. Chem. 1995; 270: 19320-19324Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 19Selbert 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). Because CaM-KIV undergoes autophosphorylation of multiple residues subsequent to activation (36McDonald O.B. Merrill B.M. Bland M.M. Taylor L.C. Sahyoun N. J. Biol. Chem. 1993; 268: 10054-10059Abstract Full Text PDF PubMed Google Scholar, 37Watanabe S. Okuno S. Kitani T. Fujisawa H. J. Biol. Chem. 1996; 271: 6903-6910Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 38Okuno S. Kitani T. Fujisawa H. J. Biochem. (Tokyo). 1995; 117: 686-690Crossref PubMed Scopus (27) Google Scholar), the relationship between activation of the kinase activity and Thr196 phosphorylation has not been precisely determined in vitro. Therefore, we have attempted to perform time course experiments on CaM-KIV activation and phosphorylation of Thr196 using anti-phospho-Thr196 monoclonal antibody (Fig. 1, A and B). We have expressed GST-fused enzymes and then GST was cleaved by PreScission protease treatment followed by removing GST and GST-fused protease to avoid the effect of GST on the enzyme activities and regulation. Thus the recombinant enzymes used in this study contain five residues (Gly-Pro-Ile-Leu-Glu) fused with the first Met. As shown in Fig. 1A, both Ca2+/CaM-dependent and -independent activities (∼30% of total activity) of CaM-KIV were induced by CaM-KK treatment in the presence of Ca2+/CaM and Mg-ATP and were saturated for 20 min. This activation kinetics was well correlated with Thr196 phosphorylation under this condition (t½ = ∼5 min, Fig. 1B). It is noteworthy that we did not observe a significant time lag between generation of the autonomous activity and phosphorylation of Thr196. These characteristic features of the recombinant CaM-KIV used in this study, including generation of autonomous activity by activation and kinetic parameters of the enzyme (Km for ATP = 27 μm and Km for syntide-2 = 264 μm, Fig. 5C), were similar to the previous results obtained using enzymes expressed in Sf9 cells (16Cruzalegui F.H. Means A.R. J. Biol. Chem. 1993; 268: 26171-26178Abstract Full Text PDF PubMed Google Scholar, 17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar, 19Selbert 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). We also performed similar experiments using CaM-KI and the same concentration (3 μg/ml) of CaM-KK as used for CaM-KIV activation (Fig. 1C). Protein kinase activity assay and Western blot analysis using anti-phospho-Thr177 monoclonal antibody revealed that, in comparison to CaM-KIV, CaM-KI was more rapidly activated and phosphorylated at Thr177 by CaM-KK (t½ = <1 min) than CaM-KIV. However, significant autonomous activity of CaM-KI was not observed during the activation. These results indicated that the autoinhibitory mechanisms might differ between CaM-KIV and CaM-KI, at least for the phosphorylated form of enzymes, which in turn might affect the generation of the autonomous activity of CaM-KIV.Fig. 5Inhibition of CaM-KIV activity by CaM-KIV autoinhibitory peptide (residues 304–325).A, wild-type CaM-KIV was either treated with CaM-KK at 30 °C for 30 min as described in Fig. 1A (+) or left untreated (–), and then the enzymes (40 ng) were subjected to Western blotting analysis using anti-CaM-KIV antibody (upper panel), anti-phospho-Thr196 antibody (middle panel), or CaM overlay analysis in the presence of Ca2+ (lower panel). B, protein kinase activities of either the CaM-KK-treated (closed circle, 2 μg/ml) or untreated CaM-KIV wild-type (open circle, 2 μg/ml) were measured with various concentrations of the synthetic peptide corresponding to residues 304–325 of CaM-KIV (0–250 μm) in the presence of 1 mm CaCl, 4 μm CaM at 30 °C for 5 min (closed circle) or 10 min (open circle). 2Activities are expressed as a percentage of the value in the absence of the peptide. Results represent the duplicate experiments. C, kinetic analysis of CaM-KIV inhibition. CaM-KIV was assayed in either the presence (closed circle) or absence (open circle) of 100 μm CaM-KIV peptide (residues 304–325) as described in panel B except for concentrations of substrates. For titration of syntide-2 (left panel), 200 μm [γ-32P]ATP and 50–400 μm syntide-2 were used. For titration of ATP (right panel), 400 μm syntide-2 and 25–200 μm [γ-32P]ATP were used. The results were performed in duplicate for each point and are presented as double-reciprocal plots (Lineweaver-Burk).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Characterization of CaM-KI/CaM-KIV Chimera Mutants— To examine the role of the autoinhibitory mechanism of CaM-KIV in the generation of autonomous activity, we have constructed, expressed, and purified chimera mutants in which the CaM-KI catalytic domain was fused with various C-terminal portions of CaM-KIV (residues 296–469 and residues 296–350; Fig. 2, A and B, upper panel), because it has been shown that CaM-KI does not undergo autophosphorylation in the catalytic domain subsequent to the activation (44Sugita R. Mochizuki H. Ito T. Yokokura H. Kobayashi R. Hidaka H. Biochem. Biophys. Res. Commun. 1994; 203: 694-701Crossref PubMed Scopus (36) Google Scholar) and also does not generate autonomous activity in association with Thr177 phosphorylation (Fig. 1C). CaM-overlay analysis revealed that the chimera mutants possess the same degree of functional Ca2+/CaM binding ability as the wild-type CaM-KI (Fig. 2B, lower panel). Without activation by CaM-KK, CaM-KI/CaM-KIV469 and CaM-KI/CaM-KIV350 were inactive in the absence of Ca2+/CaM and exhibited kinase activity only in the presence of Ca2+/CaM, which results are indistinguishable from those for the wild-type CaM-KI (Fig. 2C). This indicates that the autoinhibitory region in the C-terminal of CaM-KIV (within residues 296–350) functionally suppresses the catalytic activity of CaM-KI, which is conformationally neutralized by Ca2+/CaM binding. When we treated these CaM-KI chimera mutants with CaM-KK in the presence of Ca2+/CaM and Mg-ATP for 5 min as described in Fig. 1C, both mutants including wild-type CaM-KI were phosphorylated at Thr177 (Fig. 2D, inset). However, unlike wild-type CaM-KI, which does not generate significant autonomous activity (Figs. 1C and 2D), CaM-KI/CaM-KIV469 and CaM-KI/CaM-KIV350 showed a high level of autonomous activity after their activation. This result strongly supports the idea that the autoinhibitory mechanism of CaM-KIV was partially disrupted by phosphorylation of the activation loop Thr residue, thereby resulting in generation of autonomous activity, because it has been shown that CaM-KI is phosphorylated at Thr177 by CaM-KK, but no other autophosphorylation in the catalytic domain has been observed (44Sugita R. Mochizuki H. Ito T. Yokokura H. Kobayashi R. Hidaka H. Biochem. Biophys. Res. Commun. 1994; 203: 694-701Crossref PubMed Scopus (36) Google Scholar). Mapping of the Autoinhibitory Sequence in CaM-KIV—To clarify the involvement of the autoinhibitory function in generation of the autonomous activity of CaM-KIV, we attempted to precisely map the autoinhibitory sequence in CaM-KIV. Previous studies have demonstrated that the truncation at Leu313 (16Cruzalegui F.H. Means A.R. J. Biol. Chem. 1993; 268: 26171-26178Abstract Full Text PDF PubMed Google Scholar) and a block mutation (Phe316–Asn317 to Asp–Asp) are converted to the Ca2+/CaM-independent form of CaM-KIV (17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar), suggesting that the C-terminal region after Leu313 contains the autoinhibitory sequence of CaM-KIV. Because the precise location of the autoinhibitory sequence has not been determined, we expressed and purified a series of C-terminal truncation mutants to map a minimum autoinhibitory sequence (Fig. 3, A and B, insert upper panel). As shown in Fig. 3B, measurement of the protein kinase activities of these mutants in either the absence or presence of Ca2+/CaM revealed that truncation after Gly336 did not alter either the Ca2+/CaM dependence of the activity or the activity itself. The truncation mutants at Ala331, Lys327, and Lys321 are completely inactive in either the absence or presence of Ca2+/CaM, indicating that these mutants contain a functional autoinhibitory sequence. Semiquantitative analysis of the Ca2+/CaM binding of these mutants using the CaM overlay method in the presence of Ca2+ (Fig. 3B, insert lower panel) showed that the truncation mutant at Ala331 fails to bind Ca2+/CaM as well as other truncation mutants (1–327, 1–321, 1–313, and 1–309), suggesting that the residues between Ser332 and Gly336 are the C-terminal end of the Ca2+/CaM binding sequence. Thus the 1–331, 1–327, and 1–321 mutants were not activated by Ca2+/CaM. Further truncation at Leu313 generated the constitutively active enzyme to the same degree as did truncation at Ala309, consistent with a previous report (16Cruzalegui F.H. Means A.R. J. Biol. Chem. 1993; 268: 26171-26178Abstract Full Text PDF PubMed Google Scholar), and which suggested that a minimum autoinhibitory sequence is located between Gln314 and Lys321. This is also consistent with our previous observation that double Asp mutations at Phe316 and Asn317 impair the autoinhibitory function of CaM-KIV, resulting in generation of Ca2+/CaM-independent activity (17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar). We also examined whether CaM-KIV-(1–336) generates its autonomous activity by activation, because the mutant lacks the C-terminal portion of CaM-KIV (residues 337–469) but contains the functional regulatory domain, including the autoinhibitory and Ca2+/CaM binding sequences that completely suppress the catalytic activity of CaM-KIV in the absence of Ca2+/CaM (Fig. 3). During incubation with CaM-KK in the presence of Mg-ATP and Ca2+/CaM, Ca2+/CaM-independent activity was significantly increased in association with the Thr196 phosphorylation (Fig. 4), which was similar to the results for the wild-type CaM-KIV (Fig. 1, A and B). This result indicated that the C-terminal portion of CaM-KIV after Gly336 is unlikely to be involved in the generation of its autonomous activity. Inhibition of CaM-KIV Activity by CaM-KIV Autoinhibitory Peptide—Based on the mapping of the regulatory domain in CaM-KIV (Fig. 3), we were able to synthesize the CaM-KIV peptide (residues 304–325) containing the minimum autoinhibitory sequence (residues 314–321) to examine its inhibition of unphosphorylated and Thr196-phosphorylated CaM-KIV (Fig. 5A, upper and middle panels). We also confirmed by CaM overlay analysis that the Ca2+/CaM bindings of two enzymes were indistinguishable (Fig. 5A, lower panel). In the presence of Ca2+/CaM, the CaM-KIV peptide inhibited the total activity of unphosphorylated CaM-KIV with an IC50 value of ∼50 μm (Fig. 5B), which is ∼3-fold less potent than the inhibition by the CaM-KII autoinhibitory peptide (residues 281–302, T286A) (17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar). Furthermore, inhibition of CaM-KIV by the CaM-KIV peptide (residues 304–325) was not competitive with ATP but was competitive with the peptide substrate syntide-2 (Fig. 5C). This kinetic profile of enzyme inhibition was similar to that observed for the inhibition of CaM-KIV by the CaM-KII autoinhibitory peptide (residues 281–302, T286A) (17Tokumitsu H. Brickey D.A. Gold J. Hidaka H. Sikela J. Soderling T.R. J. Biol. Chem. 1994; 269: 28640-28647Abstract Full Text PDF PubMed Google Scholar). When we assayed the same concentration (2 μg/ml) of phosphorylated CaM-KIV by CaM-KK for 30 min (Fig. 1A), the CaM-KIV peptide at a concentration of up to 250 μm was unable to suppress the activity of activated CaM-KIV (Fig. 5B). We performed the same kinase assay as shown in Fig. 5B in the absence of the peptide substrate (syntide-2), but significant phosphate incorporation into the CaM-KIV peptide was not detected (data not shown). We also confirmed by mass spectrometry analysis that the autoinhibitory peptide was not phosphorylated at Thr308 by activated CaM-KIV (data not shown). This result indicated that the interaction between the catalytic core of CaM-KIV and the autoinhibitory peptide was abolished by Thr196 phosphorylation, resulting in a loss of the inhibitory potency of the peptide. The enzymatic activities of Ca2+/CaM-dependent protein kinases are thought to be strictly autoinhibited by intrasteric interaction between their own autoinhibitory segment and the catalytic core when the intracellular Ca2+ concentration is low enough to dissociate from CaM. Once the concentration rises upon stimulation of the cells, the Ca2+-CaM complex binds to the Ca2+/CaM binding segment of the kinases, which either overlaps or is adjacent to the autoinhibitory segment. Then Ca2+/CaM binding conformationally neutralize the autoinhibitory function, resulting in the generation of protein kinase activity. Two members of the CaM-K family, CaM-KII and CaM-KIV, generate Ca2+/CaM-independent activity in association with autophosphorylation at Thr286 and trans-phosphorylation at Thr196 by an upstream kinase (CaM-KK), respectively. Here we have shown that the phosphorylation of Thr196 in the activation loop by CaM-KK was likely sufficient to generate the autonomous activity of CaM-KIV because of: 1) the in vitro kinetics of the activation and Thr196 phosphorylation of CaM-KIV were well correlated; 2) CaM-KI chimera mutants containing the regulatory domain of CaM-KIV generated autonomous activities that were associated with Thr177 phosphorylation by CaM-KK, whereas the CaM-KI wild-type did not exhibit Ca2+/CaM-independent activity by Thr177 phosphorylation and also did not undergo autophosphorylation in the catalytic domain subsequent to the activation (44Sugita R. Mochizuki H. Ito T. Yokokura H. Kobayashi R. Hidaka H. Biochem. Biophys. Res. Commun. 1994; 203: 694-701Crossref PubMed Scopus (36) Google Scholar); 3) as shown in the experiment with a truncation mutant (CaM-KIV-(1–336)), the C-terminal of CaM-KIV (residues 337–469) is not directly involved in generation of autonomous activity; and 4) CaM-KIV autoinhibitory peptide (residues 304–325) failed to inhibit activated CaM-KIV without phosphorylation by CaM-KIV. Although multiple autophosphorylation sites in the N-terminal region of the catalytic domain of CaM-KIV have been identified (36McDonald O.B. Merrill B.M. Bland M.M. Taylor L.C. Sahyoun N. J. Biol. Chem. 1993; 268: 10054-10059Abstract Full Text PDF PubMed Google Scholar, 37Watanabe S. Okuno S. Kitani T. Fujisawa H. J. Biol. Chem. 1996; 271: 6903-6910Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 38Okuno S. Kitani T. Fujisawa H. J. Biochem. (Tokyo). 1995; 117: 686-690Crossref PubMed Scopus (27) Google Scholar), a previous study has shown that the deletion of six N-terminal Ser residues (Ser8, Ser10, Ser12, Ser13, Ser15, and Ser16 in the human enzyme) did not impair the generation of autonomous activity subsequent to the activation by CaM-KK (35Chatila 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). Collectively, these results suggest that the Thr196 phosphorylation is most likely sufficient to generate the autonomous activity of CaM-KIV. However, although it is unlikely, we cannot completely exclude the possibility that the autophosphorylation at unidentified residue(s) subsequent to phosphorylation at Thr196 by CaM-KK may be involved in the generation of autonomous activity. Interestingly, CaM-KI is also phosphorylated on Thr177 in the activation loop by CaM-KK, resulting in a large increase in Ca2+/CaM-dependent activity without generation of autonomous activity (45Haribabu B. Hook S.S. Selbert M.A. Goldstein E.G. Tomhave E.D. Edelman A.M. Synderman R. Means A.R. EMBO J. 1995; 14: 3679-3686Crossref PubMed Scopus (167) Google Scholar, 46Matsushita M. Nairn A.C. J. Biol. Chem. 1998; 273: 21473-21481Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). This result, together with our present data, suggests that the autoinhibitory mechanisms of CaM-KI and CaM-KIV are distinct, which is supported by the fact that the CaM-KI chimera mutants fused with the regulatory domain of CaM-KIV, generating Ca2+/CaM-independent activity by Thr177 phosphorylation, whereas the CaM-KIV autoinhibitory domain completely suppressed the catalytic activity of unphosphorylated chimera mutants in the absence of Ca2+/CaM. Therefore, the autoinhibitory segment of CaM-KIV no longer tightly binds to the Thr177-phosphorylated CaM-KI catalytic core in the absence of Ca2+/CaM, but that of CaM-KI does. Consistent with these findings, we directly demonstrated that the inhibitory function of the autoinhibitory peptide of CaM-KIV (residues 304–325), by preventing the substrate binding, was completely lost toward phosphorylated CaM-KIV on Thr196. This observation clearly indicates that the autonomous activity of CaM-KIV is generated by a reduction of the interaction between the catalytic core and the autoinhibitory region. Notably, the Ca2+/CaM-independent activity of activated CaM-KIV accounts for ∼30% of the total activity suggesting that the autoinhibitory region is partially but not fully released from the catalytic core. This may indicate that the intramolecular interaction of the catalytic core with the autoinhibitory region is not completely destroyed by Thr196 phosphorylation, as distinct from our results with the intermolecular inhibition by the autoinhibitory peptide. The crystal structure of the autoinhibited form of CaM-KI revealed that the regulatory segment of CaM-KI (residues 286–316) interacted with the catalytic core at some distance from Thr177 (47Goldberg J. Nairn A.C. Kuriyan J. Cell. 1996; 84: 875-887Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). This may account for the finding that the autoinhibitory mechanism of CaM-KI appeared not to be affected by Thr177 phosphorylation. We could therefore speculate that the autoinhibitory domain of CaM-KIV might be located closer to the activation loop in its autoinhibited form of unphosphorylated kinase to prevent the substrate binding. Subsequent to phosphorylation by CaM-KK, the interaction of the autoinhibitory segment with the catalytic core of CaM-KIV could be reduced by either Thr196 phosphorylation directly or by conformational changes of the catalytic domain mediated by Thr196 phosphorylation in the absence of Ca2+/CaM, resulting in the partial release of the autoinhibitory domain from the catalytic core. In contrast to the findings for CaM-KIV, it has been extensively characterized that direct autophosphorylation of the autoinhibitory region at Thr286 in CaM-KII suppresses the autoinhibitory function that is necessary to generate CaM-KII autonomous activity (26Lou L.L. Lloyd S.J. Schulman H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9497-9501Crossref PubMed Scopus (136) Google Scholar, 27Lai Y. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4253-4257Crossref PubMed Scopus (216) Google Scholar, 28Miller S.G. Kennedy M.B. Cell. 1986; 44: 861-870Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 29Schworer C.M. Colbran R.J. Soderling T.R. J. Biol. Chem. 1986; 261: 8581-8584Abstract Full Text PDF PubMed Google Scholar, 30Colbran R.J. Smith M.K. Schworer C.M. Fong Y.L. Soderling T.R. J. Biol. Chem. 1989; 264: 4800-4804Abstract Full Text PDF PubMed Google Scholar, 31Yang E. Schulman H. J. Biol. Chem. 1999; 274: 26199-26208Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Therefore, closely related multifunctional CaM-Ks appear to show different modes of regulating the autoinhibitory mechanism in association with the activation. Because the three-dimensional structure of CaM-KIV has not been determined, future structural studies will be needed to clarify the mechanism of the enzymatic regulation of CaM-KIV in greater detail, and to reveal the diverse range of autoinhibitory mechanisms among the CaM-K family. We thank Drs. Y. Shigeri, Y. Tatsu, and N. Yumoto (National Institute of Advanced Industrial Science and Technology, Osaka, Japan) for synthesis of the phosphopeptides and Dr. Y. Watanabe (Kagawa University) for helpful discussion. We also thank Y. Ishikawa (Kagawa University) for preparing recombinant enzymes and N. Ishikawa and F. Naruse (Kagawa University) for excellent technical assistance." @default.
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• W2100025585 title "Mechanism of the Generation of Autonomous Activity of Ca2+/Calmodulin-dependent Protein Kinase IV" @default.
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