A Domain with Homology to Neuronal Calcium Sensors Is Required for Calcium-dependent Activation of Diacylglycerol Kinase α

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase α is an 82-kDa DGK isoform that is activated in vitro by Ca2+. The DGKα regulatory region includes tandem C1 protein kinase C homology domains and Ca2+-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca2+ regulation, we expressed a series of DGKα deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca2+-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca2+ to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGKα fragment that included both the RVH domain and EF hands. These observations suggested that Ca2+ elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGKα. As with Ca2+, this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca2+-induced DGKα activation. Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase α is an 82-kDa DGK isoform that is activated in vitro by Ca2+. The DGKα regulatory region includes tandem C1 protein kinase C homology domains and Ca2+-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca2+ regulation, we expressed a series of DGKα deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca2+-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca2+ to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGKα fragment that included both the RVH domain and EF hands. These observations suggested that Ca2+ elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGKα. As with Ca2+, this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca2+-induced DGKα activation. diacylglycerol N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate diacylglycerol kinase octadecyltrimethylammonium chloride phosphatidic acid protein kinase C phenylmethylsulfonyl fluoride phosphatidylserine N α-p-tosyl-l-lysine chloromethyl ketone polyacrylamide gel electrophoresis recoverin homology polymerase chain reaction dithiothreitol glutathione S-transferase Hydrolysis of phosphatidylinositol 4,5-bisphosphate is a common mechanism of stimulus transduction (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2368) Google Scholar). Diacylglycerol (DAG)1 released in this reaction activates protein kinase C (PKC) and is then rapidly metabolized back to phosphatidylinositol in a series of reactions initiated by a diacylglycerol kinase (DGK). As such, DGKs attenuate DAG-mediated PKC activation (2Bishop W.R. Ganong B.R. Bell R.M. J. Biol. Chem. 1986; 261: 6993-7000Abstract Full Text PDF PubMed Google Scholar). Recent studies indicate that DGKs are also activated by mechanisms independent of phosphoinositide turnover (3Flores I. Casaseca T. Martinez C. Kanoh H. Merida I. J. Biol. Chem. 1996; 271: 10334-10340Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 4Montgomery R.B. Moscatello D.K. Wong A.J. Stahl W.L. Biochem. Biophys. Res. Commun. 1997; 232: 111-116Crossref PubMed Scopus (9) Google Scholar). Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of sn-1,2-diacylglycerol to form phosphatidic acid (PA), which is also a lipid mediator (5Erickson R.W. Langel-Peveri P. Traynor-Kaplan A.E. Heyworth P.G. Curnutte J.T. J. Biol. Chem. 1999; 274: 22243-22250Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 6Sciorra V.A. Daniel L.W. J. Biol. Chem. 1996; 271: 14226-14232Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Several DGK isoforms have been cloned (7Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). All these sequences share a homologous catalytic domain and two or three C1 protein kinase C homology domains (7Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 8Kanoh H. Sakane F. Imai S. Wada I. Cell Signal. 1993; 5: 495-503Crossref PubMed Scopus (40) Google Scholar, 9Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (321) Google Scholar). Some DGKs contain EF hands, which are Ca2+-binding sites (7Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). These DGKs also have a domain at their N termini with homology to the recoverin family of neuronal calcium sensors (Fig. 1). We term this the recoverin homology (RVH) domain. In S-modulin, the frog orthologue of recoverin, this domain associates with the EF hands to mediate Ca2+-dependent inhibition of rhodopsin kinase (10Tachibanaki S. Nanda K. Sasaki K. Ozaki K. Kawamura S. J. Biol. Chem. 2000; 275: 3313-3319Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The varied structures of DGK regulatory domains suggest divergent mechanisms of regulation. Several studies have shown variation among DGKs with regard to activation by phospholipids, sphingosine, or Ca2+ (11Walsh J.P. Suen R. Glomset J.A. J. Biol. Chem. 1995; 270: 28647-28653Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12Yamada K. Sakane F. Biochim. Biophys. Acta. 1993; 1169: 211-216Crossref PubMed Scopus (31) Google Scholar, 13Kato M. Takenawa T. J. Biol. Chem. 1990; 265: 794-800Abstract Full Text PDF PubMed Google Scholar, 14Yada Y. Ozeki T. Kanoh H. Nozawa Y. J. Biol. Chem. 1990; 265: 19237-19243Abstract Full Text PDF PubMed Google Scholar, 15Sakane F. Yamada K. Kanoh H. FEBS Lett. 1989; 255: 409-413Crossref PubMed Scopus (99) Google Scholar). Kanoh and co-workers (16Sakane F. Yamada K. Imai S. Kanoh H. J. Biol. Chem. 1991; 266: 7096-7100Abstract Full Text PDF PubMed Google Scholar, 17Yamada K. Kanoh H. Biochem. J. 1988; 255: 601-608PubMed Google Scholar, 18Goto K. Watanabe M. Kondo H. Yuasa H. Sakane F. Kanoh H. Brain Res. Mol. Brain Res. 1992; 16: 75-87Crossref PubMed Scopus (106) Google Scholar) have studied DGKα, a Ca2+-activated isoform highly expressed in oligodendrocytes and thymocytes. They have shown that Ca2+binds the EF hand region of the enzyme and that deletion of the EF hands results in constitutive enzyme activation (19Sakane F. Imai S. Yamada K. Kanoh H. Biochem. Biophys. Res. Commun. 1991; 181: 1015-1021Crossref PubMed Scopus (42) Google Scholar, 20Sakane F. Kai M. Wada I. Imai S. Kanoh H. Biochem. J. 1996; 318: 583-590Crossref PubMed Scopus (68) Google Scholar). We have now examined a series of DGKα mutants in which the RVH and EF hand domains are sequentially deleted. Our results indicate that the N-terminal RVH domain is required for Ca2+ to activate this enzyme. In contrast to the constitutive activation seen with deletion of the EF hands, DGKs with deletions involving only the RVH domain expressed activity similar to that of wild-type enzyme in the absence of Ca2+. Sites within both the EF hands and RVH domain were protected from trypsin proteolysis by Ca2+, indicating that both domains participate in a Ca2+-induced conformational change. A cationic amphiphile, octadecyltrimethylammonium chloride, markedly stimulated DGKα activity in vitro. This effect, like Ca2+-dependent activation, was dependent on the RVH domain. The DGKα RVH domain does not itself bind Ca2+. However, it does appear to function together with the EF hands to couple Ca2+ binding to release of EF hand-mediated autoinhibition of DGKα. Restriction and DNA-modifying enzymes were from or A was from S. and were from phosphatidylserine and phosphatidic acid were from was by of the with C J.P. Bell R.M. 1992; PubMed Scopus Google Scholar). phenylmethylsulfonyl fluoride and were from and were from Protein were from was from was from was from and from chloride was from the by A was from was from for protein and electrophoresis were from was from and were from gel were from were by and were by F. Sakane and H. Kanoh (19Sakane F. Imai S. Yamada K. Kanoh H. Biochem. Biophys. Res. Commun. 1991; 181: 1015-1021Crossref PubMed Scopus (42) Google Scholar, 20Sakane F. Kai M. Wada I. Imai S. Kanoh H. Biochem. J. 1996; 318: 583-590Crossref PubMed Scopus (68) Google Scholar). To the DGKα and was DGK as a and sites were on the sequences and were to the and was from reactions were or the for for for for and a at for were with and cloned DGKα and the sequences were To of a was at C of DGKα. and were to a DGKα was at for for and for for a of with a at for The fragment was cloned This was with and and fragment was in of a fragment at the DGK C The was the DGKα COS-1 were in with of COS-1 was by the C. H. Mol. Biol. PubMed Scopus Google Scholar). were and by in and of by the were at for at to The were rapidly in a and at of the of from COS-1 DGKα or the mutants was to were to in for the was with in for were with and with in for in the were by to domains in the DGKα regulatory region were as in the sequences were by as a reactions were at for for and for for a of with a at for and which the RVH domain. and which the EF hands. and which the C1 domains. and were to and The were the and They were then and and All were in with The were expressed in were at in with to of and with for at or at were and in and by two a The were by at for The were then to by of the was the recombinant protein to the was with of protein at The was then and at for to the The was and were rapidly and at The DGK in of the and enzyme (11Walsh J.P. Suen R. Glomset J.A. J. Biol. Chem. 1995; 270: 28647-28653Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). In a an of in was a of in a To the were the of of and enzyme to a of of and were as J.P. Bell R.M. 1992; PubMed Scopus Google Scholar). were initiated by of were to for at and by the of of and of The was with of and in The of the was of this of of and phosphatidylserine were of In these the of was at to the was the of and which was as J.H. J. Chem. I. Google Scholar). of these the of was to The in these was were Ca2+ was to the of and the was at The and Ca2+ were to the of J.P. Bell R.M. J. Biol. Chem. 1986; 261: Full Text PDF PubMed Google Scholar). and or and of have been Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). All are of at that within in results are of two or independent with independent enzyme binding was by the of K. T. S. J. Biochem. PubMed Scopus Google Scholar). of recombinant or DGKα mutants were by and to The was of Ca2+ in of It was three with and and then for at in of the with The was then with and to The was with to protein To of the DGKα mutants for these COS-1 with DGKα or the mutants were with and The was with protein to was then together with protein and the was at The were by The was with the only and the were which includes the RVH and EF hand was expressed in and the as trypsin proteolysis was as by M. I. F. I. K. PubMed Scopus Google Scholar). The reaction in a of and or The reaction was initiated by of trypsin and at were at and by of N α-p-tosyl-l-lysine chloromethyl The were by To the of on the reaction was with and and the trypsin was The was together with the to the which activity is (11Walsh J.P. Suen R. Glomset J.A. J. Biol. Chem. 1995; 270: 28647-28653Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). and were also included in the from of the trypsin were by an of were with The were with a of in and The was The was the of a was as the at and was was with a of a of and a of were an of were the and both with a at an of and with at a of of were on the to the were the with the binding of recombinant domain EF hands, and hands to phenyl-Sepharose was by a of the of PubMed Scopus Google Scholar). of recombinant in a of was of phenyl-Sepharose in and or The were with of the and were of DGK from and been Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). stimulated the from and of the Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). for of DGK in have also been Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). Protein of enzyme were by the Biochem. PubMed Scopus Google Scholar). of and was as J.P. Bell R.M. 1992; PubMed Scopus Google Scholar). of in DGK were by of J. Biol. Chem. Full Text PDF PubMed Google Scholar). Protein sequences with homology to the DGKα N were by and J. Res. 1997; PubMed Scopus Google Scholar, F. F. Res. 1997; PubMed Scopus Google Scholar). of with homology to DGKs were Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). domains of Ca2+-activated DGKs contain These EF hands and tandem C1 PKC homology domains H. Sakane F. Imai S. Wada I. Cell Signal. 1993; 5: 495-503Crossref PubMed Scopus (40) Google Scholar, 9Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (321) Google Scholar). EF DGKs also contain a acid at their N termini K. M. Kondo H. Sci. S. PubMed Scopus Google Scholar, F. Kanoh H. J. Biochem. Cell Biol. 1997; PubMed Scopus Google Scholar). that this domain is related to the recoverin family of neuronal calcium sensors (Fig. 1). We to this region as the RVH domain. The homology DGKs and neuronal calcium sensors the EF hands (Fig. 1). To the of the RVH domain in DGKα regulation, a series of N-terminal deletion mutants was DGKα the of the RVH domain and DGKα the RVH DGKα both the RVH domain and the EF hands. A to the C termini and of protein These mutants were expressed in COS-1 cells. from COS-1 DGKα or the deletion mutants a in DGK activity as with with The were in were only from or The of COS-1 were for The of protein with the was by (Fig. of the that DGKα DGKα and DGKα were expressed at and the of wild-type DGKα. COS-1 an DGK expressed at this of the COS-1 and for the of DGKα and DGKα which RVH were not from wild-type enzyme However, deletion of the EF hands resulted in an in results were in both the and All of the were activated as the of in was increased from to As the and were also activated by and to To that the mutants DGK we also expressed the DGKs in S. All of the mutants expressed DGK activity in the and in the with on not As not an of activity in this that the DGKs are Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). These results indicate that the DGKs a functional catalytic domain. expressed by DGKα and DGKα with the C termini were to expressed by the indicating that the does not DGKα of trypsin proteolysis of DGK EF was as a and the as trypsin was both with and Ca2+ of the reaction were at and the were by with of the reaction are the of in Ca2+. the with the Ca2+ by indicate the of and of DGK α of DGKα mutants in this are EF and catalytic domains are of DGKα deletion mutants expressed in COS-1 cells. The of of COS-1 with the were to and to a were with as DGKα DGKα DGKα DGKα, of wild-type DGKα and deletion to were on of from COS-1 the DGK These were for DGK activity by the and by the with both and All are by the DGK of a of COS-1 in the with the of DGKα and its mutants in COS-1 were by of In the of the was in the with the was from as DGKα, DGKα DGKα DGKα not of the are All are of in within results were in two independent in a were on of from COS-1 the DGK These were for DGK activity by the and by the with both and All are by the DGK of a of COS-1 in the with the of DGKα and its mutants in COS-1 were by of In the of the was in the with the was from as DGKα, DGKα DGKα DGKα not of the are All are of in within results were in two independent We examined Ca2+ activation of DGKα and the deletion enzyme by Ca2+, to activity with However, of the DGKα and which the EF hands, Ca2+-dependent activation. To the that deletion of RVH domain the of EF hands, we DGKα and with and Ca2+ on these (Fig. Our results that deletion of RVH domain did not Ca2+ As deletion of the RVH domain also on Ca2+ binding to regulatory domain sequences expressed in Ca2+ binding at the EF hands, the RVH domains are also required for Ca2+-dependent activation. The of the and mutants were similar to that of wild-type DGKα in the absence of Ca2+. deletion of the EF hands increased the activity to a to that of Ca2+-activated wild-type enzyme (Fig. This that sequences in or near the EF hands the catalytic domain and that in DGKα, this of DGK α deletion mutants expressed in COS-1 cells. COS-1 with the DGKα were and with as The were by and to A Ca2+ was on the and by Ca2+ of DGKα and of DGKα and The indicate the of and and are also seen in the Binding of Ca2+ to recoverin elicits a of the region to the DGKα RVH domain to the EF hands R. T. M. 1997; PubMed Scopus Google Scholar). The loss of Ca2+-dependent activation in the mutants loss of a similar conformational change during DGKα activation. We expressed the RVH EF hands, and C1 domains of DGKα as recombinant in that and of which the EF hands, bind Ca2+ (Fig. The RVH domain did not bind Ca2+ and was not required for Ca2+ binding (Fig. To probe for a Ca2+-induced conformational we trypsin proteolysis of with and Ca2+. of the reaction were at and by (Fig. protected from proteolysis (Fig. In the absence of Ca2+ the was within in These results are not to an of Ca2+ on of Ca2+ protect trypsin from and its these in the for these M. M. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, T. 9: PubMed Scopus Google Scholar). Ca2+ were trypsin the we To sites to in the and were by of the in In the Ca2+ only on the of from sequences the N and However, cleavage at by Ca2+. markedly cleavage at sites from to the C In the Ca2+ the of which is with the that was on As was seen in the the of from cleavage at and were by Ca2+. However, increased the of three in this of includes both EF hands, and the EF hand of these from by Ca2+ that both EF hands of DGKα Ca2+, as by Yamada K. Sakane F. Kanoh H. Biochem. J. 1997; PubMed Scopus Google Scholar). Ca2+ cleavage of sites within the RVH domain in the the RVH domain and the EF hands and within the EF hands of DGKα hands was with trypsin as in the to of the from the and were by as The the as in the and and the in in a was with trypsin as in the to of the from the and were by as The the as in the and and the in conformational of EF hand neuronal calcium in binding to PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. 1995; PubMed Scopus Google Scholar). We examined the binding of EF to As shown in of EF to by the The in was and in Ca2+ it was and The EF hands to both with and Ca2+, and were not the for this not This of a loss of the RVH domain. these observations are with a Ca2+-induced conformational change involving both the RVH domain and the EF hands. We have shown that DGKα is markedly stimulated by the cationic amphiphile, (11Walsh J.P. Suen R. Glomset J.A. J. Biol. Chem. 1995; 270: 28647-28653Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This agent stimulated three Ca2+-dependent DGK from and to a similar not Several DGK an from and from and were only activated by (11Walsh J.P. Suen R. Glomset J.A. J. Biol. Chem. 1995; 270: 28647-28653Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). DGKα activity expressed in COS-1 was activated by was seen with the amphiphile, were required to the activation. In of COS-1 the DGK and of the mutants was to that seen with In the of of Ca2+ does not activate wild-type DGKα Y. Sakane F. Kanoh H. J.P. Biochem. 2000; PubMed Scopus (112) Google Scholar). These results suggest that and have two on a seen with isoforms and an seen only with Ca2+-activated This effect, like Ca2+-dependent activation, required the RVH domains. and appear to act RVH and EF hand domains to Ca2+-dependent activation of DGKα. We examined like Ca2+, from trypsin In the absence of Ca2+, the proteolysis of not In the of Ca2+ protected increased the was by of or in the binding and of on binding not these results suggest that and Ca2+-dependent DGKα activation by EF hand-mediated autoinhibition not with Ca2+ binding or the Ca2+-induced conformational change. of and of DGKα activity with deletion of the RVH domain independent of a for this domain in enzyme of DGKα mutants by and of of COS-1 with the were for DGK activity in with and or as DGK from COS-1 with only were to of is as of activity in with activity in are from which within in results were in two independent in a The of of COS-1 with the were for DGK activity in with and or as DGK from COS-1 with only were to of is as of activity in with activity in are from which within in results were in two independent Our results indicate that Ca2+-dependent diacylglycerol kinase α activation an N-terminal RVH domain with homology to the recoverin family of neuronal calcium sensors. Several within the N-terminal region of recoverin that to the DGK RVH domain to Ca2+-dependent inhibition of rhodopsin kinase (10Tachibanaki S. Nanda K. Sasaki K. Ozaki K. Kawamura S. J. Biol. Chem. 2000; 275: 3313-3319Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). calcium sensors from an EF hand protein have a Cell Res. 1999; PubMed Scopus Google Scholar). In contrast to the of and neuronal calcium sensors are structures R. T. M. 1997; PubMed Scopus Google S. 1993; Full Text PDF PubMed Scopus Google Scholar, M. K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biol. 1999; 6: PubMed Scopus Google Scholar). calcium sensors also have at the N and C termini and and that are not in (Fig. and R. T. M. 1997; PubMed Scopus Google Scholar, S. 1993; Full Text PDF PubMed Scopus Google Scholar). and are related to neuronal calcium sensors not as in the to the RVH domain (Fig. 1). with the N-terminal of a that of recoverin M. K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of the of DGKα with recoverin that of its are also in DGKs (Fig. 1). in the the N-terminal region of recoverin and its EF hands have in DGKs (Fig. from the Our that the DGKα EF hands bind phenyl-Sepharose the RVH EF hand of of these loss of the RVH domain. These suggest that the DGK N-terminal region calcium sensors are at their N In recoverin, the is a by (Fig. from the R. T. M. 1997; PubMed Scopus Google Scholar). These are also in DGKα and EF suggesting DGKs are not this is Binding of Ca2+ to recoverin results in a of the region to the DGKα RVH domain to EF hands and R. T. M. 1997; PubMed Scopus Google Scholar, S. T. Y. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The two domains which is in binding to recoverin also the and N-terminal R. T. M. 1997; PubMed Scopus Google Scholar, S. T. Y. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (21) Google Scholar). DGKs the for N-terminal with neuronal calcium sensors in the region to the N-terminal (Fig. In DGKα, Ca2+ protected from trypsin cleavage. The acid in recoverin is in a the and from the EF hands, and as a of the Ca2+-induced conformational In recoverin, sequences to the and are and it is binding of Ca2+ to that elicits the conformational change S. T. Y. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The two EF hands of DGKs to the and these suggest that Ca2+ activation of DGKα a conformational of the N-terminal region to that of with a conformational sites within both the RVH domain and the EF hands from trypsin cleavage. Deletion of the EF hands from DGKα resulted in a constitutively active enzyme, suggesting that Ca2+ activation an by this The conformational change of the RVH domain and EF hands a region of the EF hands in autoinhibition to Ca2+-induced of the enzyme. a cationic amphiphile, and a amphiphile, also activated DGKα. As with Ca2+-dependent activation, this required the of the RVH domain. However, did not protect the hands from trypsin indicating that it does not the conformational change as Ca2+. This that the RVH domain a in DGKα activation. results indicate that functional of the DGKα EF hands to its RVH domain is required to couple Ca2+ binding to release of the catalytic domain from EF hand-mediated autoinhibition. We and for and The of in is We also F. Sakane and H. Kanoh for DGK and for on the