Identification of Targets for Calcium Signaling through the Copine Family of Proteins

Abstract

We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of “target” proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways. The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins. In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil. A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets. We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins. We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of “target” proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways. The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins. In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil. A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets. We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins. glutathione S-transferase metal-induced adhesion site mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Calcium regulates a number of intracellular activities in stimulated cells by associating with specific motifs in calcium-binding proteins. In some cases the calcium-binding motif is embedded in an enzyme or structural protein that is directly regulated by calcium. However, in the case of the important signaling protein calmodulin, calcium first binds to calmodulin and then the calcium-calmodulin complex binds to and regulates a wide variety of “target” proteins. Copines, proteins of unknown function first described inParamecium (1Creutz C.E. Tomsig J.L. Snyder S.L. Gautier M.C. Skouri F. Beisson J. Cohen J. J. Biol. Chem. 1998; 273: 1393-1402Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 2Tomsig J.L. Creutz C.E. Cell. Mol. Life Sci. 2002; 59: 1467-1477Crossref PubMed Scopus (96) Google Scholar), are expressed from multigene families in plants, animals, and protozoa. They bind phospholipid membranes through the action of two “C2 domains” in the N-terminal portion that are activated by calcium. The C-terminal half of the copine molecule is distantly related to the “A domain” (3Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar) (or “I domain”) that enables the extracellular portion of integrins to bind extracellular matrix proteins. The binding of the integrin A domain to matrix proteins is dependent upon the presence of a bound magnesium or manganese ion through a mechanism that has been termed MIDAS for metal-induced adhesion site (3Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar). Two lines of evidence have led to the hypothesis that the copine A domain may also be involved in targeted protein-protein interactions. First, critical residues involved in the chelation of Mg2+ are conserved in the copine molecule (1Creutz C.E. Tomsig J.L. Snyder S.L. Gautier M.C. Skouri F. Beisson J. Cohen J. J. Biol. Chem. 1998; 273: 1393-1402Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and second, native copine I binds Mg2+ and Mn2+(4Tomsig J.L. Creutz C.E. Biochemistry. 2000; 39: 16163-16175Crossref PubMed Scopus (63) Google Scholar). It is, however, unknown whether the copine A domain is in fact a protein-protein interaction motif. We report here, based on in vivo and in vitro evidence obtained by yeast two-hybrid screening and “pull-down” experiments using immobilized copine partners, that the copine A domain binds to a number of intracellular target proteins. We also demonstrate that full-length copine mediates the Ca2+-dependent association of target proteins with phospholipids, a phenomenon that could influence the activities and intracellular localization of the target proteins. The copines may thus comprise another pathway for calcium signaling to proteins involved in a wide range of biological activities including growth control, exocytosis, mitosis, apoptosis, gene transcription, and cytoskeletal organization.CONCLUSIONMultiple copines are expressed in a given organism (1Creutz C.E. Tomsig J.L. Snyder S.L. Gautier M.C. Skouri F. Beisson J. Cohen J. J. Biol. Chem. 1998; 273: 1393-1402Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 2Tomsig J.L. Creutz C.E. Cell. Mol. Life Sci. 2002; 59: 1467-1477Crossref PubMed Scopus (96) Google Scholar), thus multiple independent and branching pathways for calcium signaling may exist within cells based on this family of proteins. Because of their ability to associate with membranes the copines may be particularly important for regulating signaling processes on membrane surfaces. With the exception of collagen, all of the copine target domains identified here are known or predicted to be on intracellular proteins or on the intracellular portions of membrane proteins. Therefore, these targets should be accessible to copines in vivo. In many cases the targets are signaling molecules themselves, including calcium-binding proteins, kinases, a phosphatase, ubiquitin (or NEDD8)-conjugating enzymes, and transcription modulators. Mutation of a copine inArabidopsis has been shown to lead to alterations of growth patterns and apoptotic responses to stress (24Hua J. Grisafi P. Cheng S.H. Fink G.R. Genes Dev. 2001; 15: 2263-2272Crossref PubMed Scopus (118) Google Scholar, 25Jambunathan N. Siani J.M. McNellis T.W. Plant Cell. 2001; 13: 2225-2240Crossref PubMed Scopus (154) Google Scholar). Possibly this reflects alterations in calcium signaling networks based on copine-target interactions that underlie these processes. Calcium regulates a number of intracellular activities in stimulated cells by associating with specific motifs in calcium-binding proteins. In some cases the calcium-binding motif is embedded in an enzyme or structural protein that is directly regulated by calcium. However, in the case of the important signaling protein calmodulin, calcium first binds to calmodulin and then the calcium-calmodulin complex binds to and regulates a wide variety of “target” proteins. Copines, proteins of unknown function first described inParamecium (1Creutz C.E. Tomsig J.L. Snyder S.L. Gautier M.C. Skouri F. Beisson J. Cohen J. J. Biol. Chem. 1998; 273: 1393-1402Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 2Tomsig J.L. Creutz C.E. Cell. Mol. Life Sci. 2002; 59: 1467-1477Crossref PubMed Scopus (96) Google Scholar), are expressed from multigene families in plants, animals, and protozoa. They bind phospholipid membranes through the action of two “C2 domains” in the N-terminal portion that are activated by calcium. The C-terminal half of the copine molecule is distantly related to the “A domain” (3Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar) (or “I domain”) that enables the extracellular portion of integrins to bind extracellular matrix proteins. The binding of the integrin A domain to matrix proteins is dependent upon the presence of a bound magnesium or manganese ion through a mechanism that has been termed MIDAS for metal-induced adhesion site (3Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar). Two lines of evidence have led to the hypothesis that the copine A domain may also be involved in targeted protein-protein interactions. First, critical residues involved in the chelation of Mg2+ are conserved in the copine molecule (1Creutz C.E. Tomsig J.L. Snyder S.L. Gautier M.C. Skouri F. Beisson J. Cohen J. J. Biol. Chem. 1998; 273: 1393-1402Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and second, native copine I binds Mg2+ and Mn2+(4Tomsig J.L. Creutz C.E. Biochemistry. 2000; 39: 16163-16175Crossref PubMed Scopus (63) Google Scholar). It is, however, unknown whether the copine A domain is in fact a protein-protein interaction motif. We report here, based on in vivo and in vitro evidence obtained by yeast two-hybrid screening and “pull-down” experiments using immobilized copine partners, that the copine A domain binds to a number of intracellular target proteins. We also demonstrate that full-length copine mediates the Ca2+-dependent association of target proteins with phospholipids, a phenomenon that could influence the activities and intracellular localization of the target proteins. The copines may thus comprise another pathway for calcium signaling to proteins involved in a wide range of biological activities including growth control, exocytosis, mitosis, apoptosis, gene transcription, and cytoskeletal organization. CONCLUSIONMultiple copines are expressed in a given organism (1Creutz C.E. Tomsig J.L. Snyder S.L. Gautier M.C. Skouri F. Beisson J. Cohen J. J. Biol. Chem. 1998; 273: 1393-1402Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 2Tomsig J.L. Creutz C.E. Cell. Mol. Life Sci. 2002; 59: 1467-1477Crossref PubMed Scopus (96) Google Scholar), thus multiple independent and branching pathways for calcium signaling may exist within cells based on this family of proteins. Because of their ability to associate with membranes the copines may be particularly important for regulating signaling processes on membrane surfaces. With the exception of collagen, all of the copine target domains identified here are known or predicted to be on intracellular proteins or on the intracellular portions of membrane proteins. Therefore, these targets should be accessible to copines in vivo. In many cases the targets are signaling molecules themselves, including calcium-binding proteins, kinases, a phosphatase, ubiquitin (or NEDD8)-conjugating enzymes, and transcription modulators. Mutation of a copine inArabidopsis has been shown to lead to alterations of growth patterns and apoptotic responses to stress (24Hua J. Grisafi P. Cheng S.H. Fink G.R. Genes Dev. 2001; 15: 2263-2272Crossref PubMed Scopus (118) Google Scholar, 25Jambunathan N. Siani J.M. McNellis T.W. Plant Cell. 2001; 13: 2225-2240Crossref PubMed Scopus (154) Google Scholar). Possibly this reflects alterations in calcium signaling networks based on copine-target interactions that underlie these processes. Multiple copines are expressed in a given organism (1Creutz C.E. Tomsig J.L. Snyder S.L. Gautier M.C. Skouri F. Beisson J. Cohen J. J. Biol. Chem. 1998; 273: 1393-1402Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 2Tomsig J.L. Creutz C.E. Cell. Mol. Life Sci. 2002; 59: 1467-1477Crossref PubMed Scopus (96) Google Scholar), thus multiple independent and branching pathways for calcium signaling may exist within cells based on this family of proteins. Because of their ability to associate with membranes the copines may be particularly important for regulating signaling processes on membrane surfaces. With the exception of collagen, all of the copine target domains identified here are known or predicted to be on intracellular proteins or on the intracellular portions of membrane proteins. Therefore, these targets should be accessible to copines in vivo. In many cases the targets are signaling molecules themselves, including calcium-binding proteins, kinases, a phosphatase, ubiquitin (or NEDD8)-conjugating enzymes, and transcription modulators. Mutation of a copine inArabidopsis has been shown to lead to alterations of growth patterns and apoptotic responses to stress (24Hua J. Grisafi P. Cheng S.H. Fink G.R. Genes Dev. 2001; 15: 2263-2272Crossref PubMed Scopus (118) Google Scholar, 25Jambunathan N. Siani J.M. McNellis T.W. Plant Cell. 2001; 13: 2225-2240Crossref PubMed Scopus (154) Google Scholar). Possibly this reflects alterations in calcium signaling networks based on copine-target interactions that underlie these processes.