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• W1980961092 abstract "Isoforms of calcium/calmodulin-dependent protein kinase II from Drosophila (R1-R6 and R3A) showed differential activation by two series of mutant calmodulins, B1K-B4K and B1Q-B4Q. These mutant calmodulins were generated by changing a glutamic acid in each of the four calcium binding sites to either glutamine or lysine, altering their calcium binding properties. All mutations produced activation defects, with the binding site 4 and B1Q mutants the most severe. Activation differed substantially between isoforms. R4, R5, and R6 were the least sensitive to mutations in calmodulin, while R1, R3, and R3A were the most sensitive. Activation of R1 and R2 by B4K and activation of R3 and R3A by B2K and B2Q produced significant (6-fold and almost 3-fold, respectively) differences in Kact between isoforms that differ structurally by a single amino acid. These differences could not be accounted for by differential binding, as all isoforms showed almost identical binding patterns with the mutants. High binding affinity did not always correlate with ability to increase enzyme activity, implying that activation occurs in at least two steps. The isoform-specific differences seen in this study reflect a role for the COOH-terminal variable region in activation of CaM kinase II. Isoforms of calcium/calmodulin-dependent protein kinase II from Drosophila (R1-R6 and R3A) showed differential activation by two series of mutant calmodulins, B1K-B4K and B1Q-B4Q. These mutant calmodulins were generated by changing a glutamic acid in each of the four calcium binding sites to either glutamine or lysine, altering their calcium binding properties. All mutations produced activation defects, with the binding site 4 and B1Q mutants the most severe. Activation differed substantially between isoforms. R4, R5, and R6 were the least sensitive to mutations in calmodulin, while R1, R3, and R3A were the most sensitive. Activation of R1 and R2 by B4K and activation of R3 and R3A by B2K and B2Q produced significant (6-fold and almost 3-fold, respectively) differences in Kact between isoforms that differ structurally by a single amino acid. These differences could not be accounted for by differential binding, as all isoforms showed almost identical binding patterns with the mutants. High binding affinity did not always correlate with ability to increase enzyme activity, implying that activation occurs in at least two steps. The isoform-specific differences seen in this study reflect a role for the COOH-terminal variable region in activation of CaM kinase II. INTRODUCTIONCalcium/calmodulin-dependent protein kinase II (CaM kinase II) 1The abbreviations used are: CaM kinase IIcalcium/calmodulin-dependent protein kinase IICaMcalmodulinWTwild typesmMLCKsmooth muscle myosin light chain kinaseskMLCKskeletal muscle light chain kinaseTBSTTris-buffered saline with TweenPIPES1,4-piperazinediethanesulfonic acid. is a family of related proteins with molecular masses in the range of 50-65 kDa (for review see Ref. 1Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (657) Google Scholar). Homologs have been found in both vertebrates and invertebrates. In rat, where CaM kinase II has been most intensively studied, diversity is generated both by multiple genes coding for separate α, β, γ, and δ isozymes, and by alternative splicing of these genes (2Bennett M.K. Kennedy M.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1794-1798Crossref PubMed Scopus (214) Google Scholar, 3Benson D.L. Isakson P.J. Gall C.M. Jones E.G. J. Neurosci. 1991; 11: 31-47Crossref PubMed Google Scholar, 4Nghiem P. Saati S.M. Martens C.L. Gardner P. Schulman H. J. Biol. Chem. 1993; 268: 5471-5479Abstract Full Text PDF PubMed Google Scholar, 5Schworer C.M. Rothblum L.I. Thekkumkara T.J. Singer S.A. J. Biol. Chem. 1993; 268: 14443-14449Abstract Full Text PDF PubMed Google Scholar). The major variability between isozymes and between alternatively spliced forms of a single isozyme occurs between the calmodulin (CaM) binding site and the association domain, where 11-41-amino acid “inserts” are found. The function of the variable region in the rat kinase is largely unknown, although early experiments have suggested that the affinity of α and β for CaM differ (6Miller S.G. Kennedy M.B. J. Biol. Chem. 1985; 260: 9039-9046Abstract Full Text PDF PubMed Google Scholar) and a recent report demonstrates a nuclear localization signal in the δB isoform (7Srinivasan M. Edman C.F. Schulman H. J. Cell Biol. 1994; 126: 839-852Crossref PubMed Scopus (233) Google Scholar).In Drosophila, where CaM kinase II is encoded by a single gene (8Cho K.O. Wall J.B. Pugh P.C. Ito M. Mueller S.A. Kennedy M.B. Neuron. 1991; 7: 439-450Abstract Full Text PDF PubMed Scopus (61) Google Scholar), up to 18 isoforms are generated by alternative splicing to form a variable region between the CaM binding and association domains (9Griffith L.C. Greenspan R.J. J. Neurochem. 1993; 61: 1534-1537Crossref PubMed Scopus (45) Google Scholar). Table I shows the organization of sequences found in the variable regions of the seven Drosophila CaM kinase II isoforms used in this study. The variable domain is made up of sequences from two independent exons termed insert 1 and insert 2. In addition to these amino acid cassettes, a glutamic acid and/or an alanine can be added to the ends of the domain by use of alternative splice acceptor sites. There is no sequence homology between the Drosophila and rat variable regions.TABLE IVariable regions of Drosophila CaM kinase II The structures of the variable regions of the isoforms of Drosophila CaM kinase II are shown. Splicing occurs in a region between the CaM binding domain and the association domain (9Griffith L.C. Greenspan R.J. J. Neurochem. 1993; 61: 1534-1537Crossref PubMed Scopus (45) Google Scholar). Variable size amino acid insertions occur at this site by alternative splicing of two independent exons (insert 1 and insert 2; for sequences see Ref. 9Griffith L.C. Greenspan R.J. J. Neurochem. 1993; 61: 1534-1537Crossref PubMed Scopus (45) Google Scholar). Additional variation is induced by the existence of multiple acceptor sites at the 5′ end of the insert 1 exon (leading to addition of a glutamic acid, E, at the beginning of the variable region) and multiple splice acceptor sites for the 3′ end of the insert exons (leading to addition of an alanine, A, to the end of the variable region).R1Insert 2R2Insert 2 + AR3No insertR3AAR4E + insert 1R5E + insert 1 + insert 2 + AR6Insert 1 + insert 2 Open table in a new tab An examination of the catalytic, assembly, and autophosphorylation properties of Drosophila isoforms R1-R6 demonstrated that these proteins are indeed CaM kinase II homologs (10GuptaRoy B. Griffith L.C. J. Neurochem. 1996; 66: 1282-1288Crossref PubMed Scopus (20) Google Scholar). The only catalytic properties that showed any significant difference between isoforms were the Km for peptide substrate and the Kact for CaM, suggesting that the variable domain may influence the interaction between CaM and the catalytic domain. To investigate the function of CaM kinase II variable domains in activation by CaM, we have studied the interaction of this set of isoforms with a set of mutant CaMs with altered calcium binding and conformational properties. The use of the Drosophila isoforms allows unambiguous correlation of functional differences with the variable domain, since the proteins are otherwise identical. While the mammalian CaM kinase II isozymes show high homology outside this domain, they are not identical.The production of the active Ca2+/CaM complex has been studied extensively. Calcium binding to the four “EF” hands of CaM produces a conformational change required for activating target enzymes (for review, see Ref. 11Cohen P. Klee C.B. Calmodulin. Elsevier, New York1988: 35Google Scholar). The crystal structure of calmodulin has shown that there are two calcium binding sites in both the amino and carboxyl termini of CaM, which form two globular domains separated by a central linker region (12Babu Y.S. Sack J.S. Greenhough T.J. Bugg C.E. Means A.R. Cook W.J. Nature. 1985; 315: 37-40Crossref PubMed Scopus (796) Google Scholar, 13Taylor D. Sack J.S. Maune J.F. Beckingham K. Quicho R. J. Biol. Chem. 1991; 266: 21375-21380Abstract Full Text PDF PubMed Google Scholar). Most of the detectable conformational changes of CaM are completed upon occupation of two of the four calcium binding sites. Several studies indicate that the carboxyl-terminal sites (3 and 4) bind calcium with higher affinity than the amino-terminal sites (1 and 2) and therefore binding in the carboxyl domain is the primary contributor to calcium-induced structural changes (14Yazawa M. Ikura M. Hikichi Ying L. Yagi K. J. Biol. Chem. 1987; 262: 10951-10954Abstract Full Text PDF PubMed Google Scholar, 15Yoshida M. Minowa O. Yagi K. J. Biochem. (Tokyo). 1983; 94: 1925-1933Crossref PubMed Scopus (41) Google Scholar). Site-directed mutants of Drosophila CaM have been generated, which incapacitate each calcium binding site individually (16Maune J.F. Klee C.B. Beckingham K. J. Biol. Chem. 1992; 267: 5286-5295Abstract Full Text PDF PubMed Google Scholar). In each of these mutants, a conserved glutamic acid residue, which plays a critical role in calcium binding, at position 12 of one of the calcium binding loops has been mutated to either glutamine (Q) or lysine (K). Thus the B1Q mutant carries the glutamine mutation in binding site 1, while B1K has lysine at site 1, and so on. Calcium binding properties and conformational changes of these mutants and their ability to activate four target enzymes (skeletal myosin light chain kinase (skMLCK), smooth muscle myosin light chain kinase (smMLCK), type I adenylyl cyclase, and Ca2+-ATPase) have been examined (16Maune J.F. Klee C.B. Beckingham K. J. Biol. Chem. 1992; 267: 5286-5295Abstract Full Text PDF PubMed Google Scholar, 17Maune J.F. Beckingham K. Martin S.R. Bayley P.M. Biochemistry. 1992; 31: 7779-7786Crossref PubMed Scopus (70) Google Scholar, 18Beckingham K. J. Biol. Chem. 1991; 266: 6027-6030Abstract Full Text PDF PubMed Google Scholar). These studies have shown that, in both series of mutants, calcium binding at the mutated site is effectively eliminated over the calcium concentration range at which CaM functions and some component of the calcium-induced conformational change is also eliminated.We have used these CaM mutants to study the activation properties of seven isoforms of Drosophila CaM kinase II. In this report we show that, although the isoforms of CaM kinase II have similar biochemical properties (10GuptaRoy B. Griffith L.C. J. Neurochem. 1996; 66: 1282-1288Crossref PubMed Scopus (20) Google Scholar), they exhibit differential activation by mutant CaMs. The data support a role for the COOH-terminal variable regions of CaM kinase II in the mechanism of activation of this enzyme and demonstrate that stimulation of CaM kinase II catalytic activity by calmodulin is a multistep process, with separable binding and activation steps.DISCUSSIONThe data presented in this study provide information on the activation of CaM kinase II by calmodulin which can be interpreted at two levels. First, taken as a whole, the data are informative about the requirements of CaM kinase II for structural and functional properties of CaM, and about the mechanism of activation of CaM kinase II. This information comes from studying the rank orders of activation and binding and drawing conclusions based on the general properties of the population of isoforms. Second, the data provide evidence for distinct mechanisms of activation for the alternatively spliced isoforms of Drosophila CaM kinase II. These conclusions are based on examining absolute, rather than relative levels of activation, and from analyses of cases where specific isoforms behave differently than the population as a whole.Role of Calmodulin's Calcium Binding Sites in Activation of CaM Kinase IIStudies of the BK and BQ series of Drosophila CaM mutants have established that all of these mutants have greatly decreased calcium binding at the mutated site and some defect in calcium-induced conformational changes. In a previous study (20Gao Z.H. Krebs J. VanBerkum M.F.A. Tang W.J. Maune J.F. Means A.R. Stull J.T. Beckingham K. J. Biol. Chem. 1993; 268: 20096-20140Abstract Full Text PDF PubMed Google Scholar), three target enzymes of CaM, skMLCK, smMLCK, and type I adenylyl cyclase showed similar patterns of activation by the BK and BQ series CaM mutants. With the exception of poor activation of skMLCK by B1Q, mutations at site 4 were found to affect activation most severely followed by mutations at site 2 and site 3, and finally site 1. In earlier studies of UV circular dichroism (17Maune J.F. Beckingham K. Martin S.R. Bayley P.M. Biochemistry. 1992; 31: 7779-7786Crossref PubMed Scopus (70) Google Scholar), gel electrophoresis, and calcium binding (16Maune J.F. Klee C.B. Beckingham K. J. Biol. Chem. 1992; 267: 5286-5295Abstract Full Text PDF PubMed Google Scholar), a similar ranking of the sites emerged.For the CaM kinase II isoforms, if one ranks the Kact values for each isoform for all the mutant CaMs and the binding effectiveness of each mutant for each isoform (Table IV), one finds that the same order is followed as for the other effector enzymes with two notable exceptions. First, the B1Q mutant is almost as detrimental for activation as mutations in binding site 4 (as was seen with skMLCK). B1Q also performs poorly in the binding assay. This is in contrast to B1K, which is both a good activator and able to compete effectively in the binding assay.Activation of CaM Kinase II Is a Multistep ProcessAlthough for most of the mutant CaMs, there is good correspondence between their ability to bind CaM kinase II and to activate the enzyme, the site 2 mutants are interesting in that they do not show this correlation. B2K does not bind well to any of the isoforms, yet it can activate some isoforms very well. Conversely, although B2Q binds CaM kinase II better than B2K, it is unable to activate the same isoforms to a similar extent. This is clearly seen in a comparison of the rank order of activation by B2K and B2Q with the rank order of CaM binding (Table IV). Dissociation of binding and activation is also seen with the R4 isoform, which binds to B1K, B2Q, and B3K better than any of the other isoforms, yet does not show enhanced activation by these CaMs (TABLE II., TABLE III). This lack of correlation between binding and increased enzyme activity implies that activation of CaM kinase II consists of at least two steps: binding of CaM to the target and subsequent stimulation of enzyme activity by the CaM/target complex. In this context, complexes formed between the CaM kinase II isoforms studied here and mutants B2K and B2Q could be very informative with regard to the mechanism of activation of this enzyme.Role of the Variable Domain in ActivationThe general properties of Drosophila CaM kinase II can be inferred from the rank orders of Kact and CaM binding for the mutant CaMs. While consensus emerges from such an analysis, there are indications that there are also isoform-specific effects for each of the mutant CaMs as measured by Kact. There are also some cases where a particular isoform diverges from the general rank order for activation.Analysis of the Effects of Isoform Variability on Activation by Binding Site 1 MutantsActivation of the seven isoforms of Drosophila CaM kinase II tested in this study was adversely affected by all of the mutant CaMs in the K and Q series. The CaM with the most normal activation kinetics, B1K, still showed an increase of 1.5-14-fold in Kact, depending on the isoform assayed. Structural modeling studies have indicated that at site 1, the mutation to lysine may be neomorphic and elicit some of the conformational changes normally induced by calcium (16Maune J.F. Klee C.B. Beckingham K. J. Biol. Chem. 1992; 267: 5286-5295Abstract Full Text PDF PubMed Google Scholar). This meshes well with the activation data from four CaM target enzymes studied earlier, where it was shown to have a Kact only 1.5-2-fold greater than WT CaM (20Gao Z.H. Krebs J. VanBerkum M.F.A. Tang W.J. Maune J.F. Means A.R. Stull J.T. Beckingham K. J. Biol. Chem. 1993; 268: 20096-20140Abstract Full Text PDF PubMed Google Scholar). Thus the characteristics of B1K make it the mutant whose activation properties are most comparable to WT CaM.All the isoforms of CaM kinase II studied here showed nearly maximal stimulation by this mutant CaM at micromolar concentrations. The Kact values, however, differed from those seen with WT CaM and showed wider variation than did the other CaM effector enzymes tested previously. Since Kact and Vmax for WT CaM was virtually identical for all the isoforms (with the exception of R3A, which in other studies with WT bovine CaM had a significantly higher Kact value than other isoforms; Ref. 10GuptaRoy B. Griffith L.C. J. Neurochem. 1996; 66: 1282-1288Crossref PubMed Scopus (20) Google Scholar), it seems likely that the variations in activation by B1K are representative of some level of variable region-specific differences among the isoforms.The B1Q mutation has much more drastic effects on activation and binding than does the B1K mutation. Kact values range from 42- to 162-fold WT. Interestingly, although B1Q is one of the worst activators of Drosophila CaM kinase II, it is the least able to discriminate between isoforms. The difference between Kact values for the best and worst isoform is less than 4-fold. For all of the other mutants, the difference between best and worst is on the order of 10-20-fold. Since the B1K mutant may be neomorphic (17Maune J.F. Beckingham K. Martin S.R. Bayley P.M. Biochemistry. 1992; 31: 7779-7786Crossref PubMed Scopus (70) Google Scholar), the role of binding site 1 is probably best understood by examining the data for the B1Q mutant. This suggests that calcium binding at site 1 and/or the conformational changes dependent on site 1 do not interact significantly with the variable domain of CaM kinase II. The defect in activation for CaM kinase II may be a direct result of the decreased ability of the B1Q mutant to bind, and may be independent of variable domain influence.Analysis of the Effects of Isoform Variability on Activation by Binding Site 2 MutantsFor binding site 2, enzyme activity data shows clear effects of isoform variability. B2K Kact values range from 1- to 10-fold WT, while for B2Q they range from 3- to 54-fold WT. For B2K and B2Q, three classes of activation patterns emerge. The first of these is R5 and R6 (which have both insert 1 and insert 2), which are relatively unaffected by both the mutations; the second class is R1, R2, R3, and R4 (having either insert 1 or insert 2 or no insert), which are more severely affected by B2Q and relatively unaffected by B2K; and the third is R3A (no insert, additional alanine), which is poorly activated by both the mutants. Thus mutations in binding site 2 not only separate binding and activation (see above), they can also discriminate between isoform variable regions.Analysis of the Effects of Isoform Variability on Activation by Binding Site 3 MutantsBinding site 3 mutants are fairly uniformly detrimental to CaM kinase II activation. The only notable differences among isoforms is that R5 and R6 (which both contain insert 1 and insert 2) are relatively less affected than the other isoforms (which have single or no inserts).Analysis of the Effects of Isoform Variability on Activation by Binding Site 4 MutantsThe binding site 4 mutants are overall the worst activators of CaM kinase II, with Kact values ranging from 13- to 145-fold WT for B4Q and from 30- to 295-fold WT for B4K. The most dramatic discrimination between isoforms is the difference seen in activation of R1 and R2, both of which contain insert 2, with R2 having an extra alanine. R1 and R2 are activated equivalently by B4Q, but activation by B4K is quite different, with the calculated Kact of R1 being 6-fold higher than the Kact of R2. A difference of this magnitude implies that the addition of an alanine residue to the end of the variable domain has significant structural consequences, and that the variable domain is an active participant in stimulation of enzyme activity by the CaM/target complex.Importance of Isoform-specific InteractionsThe interaction of CaM kinase II with CaM is unusual. Most Ca2+/CaM effectors interact with CaM with subnanomolar-nanomolar affinities. CaM kinase II in its unphosphorylated state shows dissociation constants in the 25-100 nM range (1Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (657) Google Scholar, 21Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1202Crossref PubMed Scopus (505) Google Scholar). This contrasts with data for peptides based on the rat CaM kinase II CaM binding domain, which have a KD of 0.1-0.3 nM (1Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (657) Google Scholar). For the native rat cytoskeletal CaM kinase II, the binding constant for 125I-CaM was found to be >10-fold less than Kact (22LeVine III, H. Sahyoun N.E. Cuatrecasas P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 287-291Crossref PubMed Scopus (42) Google Scholar). In addition, autophosphorylation can modulate the affinity of CaM kinase II for CaM, increasing affinity by 3 orders of magnitude (21Meyer T. Hanson P.I. Stryer L. Schulman H. Science. 1992; 256: 1199-1202Crossref PubMed Scopus (505) Google Scholar). There is evidence that regions of skMLCK that are outside the CaM binding region interact with the first domain of CaM (23Zot H.G. Aden R. Samy S. Puett D. J. Biol. Chem. 1990; 265: 14796-14801Abstract Full Text PDF PubMed Google Scholar). Mutations that affect smMLCK activation but not binding have also been characterized (24Su Z. Fan D. George S.E. J. Biol. Chem. 1994; 269: 16761-16765Abstract Full Text PDF PubMed Google Scholar). Determinants other than the core CaM binding sequence are therefore very important to the binding of Ca2+/CaM by CaM kinase II. Using site-directed CaM mutants, we have shown here that isoforms of CaM kinase II that are structurally different show differences in their mechanisms of activation.As indicated by the above analyses, the correlation between the structure of the isoform and its activation is not uniform for different binding site mutants or for different mutations at the same binding site. If one examines these relative sensitivities, however, several generalizations can still be made. First, R1, R3, and R3A are usually the most sensitive to CaM mutations. Second, R4, R5, and R6 are usually most insensitive. R2 is quite variable, sometimes among the most sensitive, sometimes among the least. The differential sensitivity of the isoforms to mutations in the high or low affinity calcium binding sites could be indicative of selectivity in the pattern of isoforms activated at different subsaturating intracellular calcium concentrations. This diversity in activation of the Drosophila isoforms by mutant CaMs indicates that sequence variability in this region may regulate activation of CaM kinase II. Examination of the distribution of the kinase isoforms by mRNA in situ hybridization to adult tissue has revealed no remarkable differences (9Griffith L.C. Greenspan R.J. J. Neurochem. 1993; 61: 1534-1537Crossref PubMed Scopus (45) Google Scholar). The functional in vivo consequences of the regulatory diversity we have demonstrated remain to be determined. INTRODUCTIONCalcium/calmodulin-dependent protein kinase II (CaM kinase II) 1The abbreviations used are: CaM kinase IIcalcium/calmodulin-dependent protein kinase IICaMcalmodulinWTwild typesmMLCKsmooth muscle myosin light chain kinaseskMLCKskeletal muscle light chain kinaseTBSTTris-buffered saline with TweenPIPES1,4-piperazinediethanesulfonic acid. is a family of related proteins with molecular masses in the range of 50-65 kDa (for review see Ref. 1Hanson P.I. Schulman H. Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (657) Google Scholar). Homologs have been found in both vertebrates and invertebrates. In rat, where CaM kinase II has been most intensively studied, diversity is generated both by multiple genes coding for separate α, β, γ, and δ isozymes, and by alternative splicing of these genes (2Bennett M.K. Kennedy M.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1794-1798Crossref PubMed Scopus (214) Google Scholar, 3Benson D.L. Isakson P.J. Gall C.M. Jones E.G. J. Neurosci. 1991; 11: 31-47Crossref PubMed Google Scholar, 4Nghiem P. Saati S.M. Martens C.L. Gardner P. Schulman H. J. Biol. Chem. 1993; 268: 5471-5479Abstract Full Text PDF PubMed Google Scholar, 5Schworer C.M. Rothblum L.I. Thekkumkara T.J. Singer S.A. J. Biol. Chem. 1993; 268: 14443-14449Abstract Full Text PDF PubMed Google Scholar). The major variability between isozymes and between alternatively spliced forms of a single isozyme occurs between the calmodulin (CaM) binding site and the association domain, where 11-41-amino acid “inserts” are found. The function of the variable region in the rat kinase is largely unknown, although early experiments have suggested that the affinity of α and β for CaM differ (6Miller S.G. Kennedy M.B. J. Biol. Chem. 1985; 260: 9039-9046Abstract Full Text PDF PubMed Google Scholar) and a recent report demonstrates a nuclear localization signal in the δB isoform (7Srinivasan M. Edman C.F. Schulman H. J. Cell Biol. 1994; 126: 839-852Crossref PubMed Scopus (233) Google Scholar).In Drosophila, where CaM kinase II is encoded by a single gene (8Cho K.O. Wall J.B. Pugh P.C. Ito M. Mueller S.A. Kennedy M.B. Neuron. 1991; 7: 439-450Abstract Full Text PDF PubMed Scopus (61) Google Scholar), up to 18 isoforms are generated by alternative splicing to form a variable region between the CaM binding and association domains (9Griffith L.C. Greenspan R.J. J. Neurochem. 1993; 61: 1534-1537Crossref PubMed Scopus (45) Google Scholar). Table I shows the organization of sequences found in the variable regions of the seven Drosophila CaM kinase II isoforms used in this study. The variable domain is made up of sequences from two independent exons termed insert 1 and insert 2. In addition to these amino acid cassettes, a glutamic acid and/or an alanine can be added to the ends of the domain by use of alternative splice acceptor sites. There is no sequence homology between the Drosophila and rat variable regions.TABLE IVariable regions of Drosophila CaM kinase II The structures of the variable regions of the isoforms of Drosophila CaM kinase II are shown. Splicing occurs in a region between the CaM binding domain and the association domain (9Griffith L.C. Greenspan R.J. J. Neurochem. 1993; 61: 1534-1537Crossref PubMed Scopus (45) Google Scholar). Variable size amino acid insertions occur at this site by alternative splicing of two independent exons (insert 1 and insert 2; for sequences see Ref. 9Griffith L.C. Greenspan R.J. J. Neurochem. 1993; 61: 1534-1537Crossref PubMed Scopus (45) Google Scholar). Additional variation is induced by the existence of multiple acceptor sites at the 5′ end of the insert 1 exon (leading to addition of a glutamic acid, E, at the beginning of the variable region) and multiple splice acceptor sites for the 3′ end of the insert exons (leading to addition of an alanine, A, to the end of the variable region).R1Insert 2R2Insert 2 + AR3No insertR3AAR4E + insert 1R5E + insert 1 + insert 2 + AR6Insert 1 + insert 2 Open table in a new tab An examination of the catalytic, assembly, and autophosphorylation properties of Drosophila isoforms R1-R6 demonstrated that these proteins are indeed CaM kinase II homologs (10GuptaRoy B. Griffith L.C. J. Neurochem. 1996; 66: 1282-1288Crossref PubMed Scopus (20) Google Scholar). The only catalytic properties that showed any significant difference between isoforms were the Km for peptide substrate and the Kact for CaM, suggesting that the variable domain may influence the interaction between CaM and the catalytic domain. To investigate the function of CaM kinase II variable domains in activation by CaM, we have studied the interaction of this set of isoforms with a set of mutant CaMs with altered calcium binding and conformational properties. The use of the Drosophila isoforms allows unambiguous correlation of functional differences with the variable domain, since the proteins are otherwise identical. While the mammalian CaM kinase II isozymes show high homology outside this domain, they are not identical.The production of the active Ca2+/CaM complex has been studied extensively. Calcium binding to the four “EF” hands of CaM produces a conformational change required for activating target enzymes (for review, see Ref. 11Cohen P. Klee C.B. Calmodulin. Elsevier, New York1988: 35Google Scholar). The crystal structure of calmodulin has shown that there are two calcium binding sites in both the amino and carboxyl termini of CaM, which form two globular domains separated by a central linker region (12Babu Y.S. Sack J.S. Greenhough T.J. Bugg C.E. Means A.R. Cook W.J. Nature. 1985; 315: 37-40Crossref PubMed Scopus (796) Google Scholar, 13Taylor D. Sack J.S. Maune J.F. Beckingham K. Quicho R. J. Biol. Chem. 1991; 266: 21375-21380Abstract Full Text PDF PubMed Google Scholar). Most of the detectable conformational changes of CaM are completed upon occupation of two of the four calcium binding sites. Several studies indicate that the carboxyl-terminal sites (3 and 4) bind calcium with higher affinity than the amino-terminal sites (1 and 2) and therefore binding in the carboxyl domain is the primary contributor to calcium-induced st" @default.
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• W1980961092 title "Functional Diversity of Alternatively Spliced Isoforms of Drosophila Ca2+/Calmodulin-dependent Protein Kinase II" @default.
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