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Rad and Rad-related GTPases Interact with Calmodulin and Calmodulin-dependent Protein Kinase II

1997; Elsevier BV; Volume: 272; Issue: 18 Linguagem: Inglês

10.1074/jbc.272.18.11832

1083-351X

Julie S. Moyers, Philip J. Bilan, Jianhua Zhu, C. Ronald Kahn,

Cell death mechanisms and regulation

Members of the Rad family of GTPases (including Rad, Gem, and Kir) possess several unique features of unknown function in comparison to other Ras-like proteins, with major N-terminal and C-terminal extensions, a lack of typical prenylation motifs, and several non-conservative changes in the sequence of the GTP binding domain. Here we show that Rad and Gem bind to calmodulin (CaM)-Sepharose in vitro in a calcium-dependent manner and that Rad can be co-immunoprecipitated with CaM in C2C12 cells. The interaction is influenced by the guanine nucleotide binding state of Rad with the GDP-bound form exhibiting 5-fold better binding to CaM than the GTP-bound protein. In addition, the dominant negative mutant of Rad (S105N) which binds GDP, but not GTP, exhibits enhanced binding to CaM in vivo when expressed in C2C12 cells. Peptide competition studies and expression of deletion mutants of Rad localize the binding site for CaM to residues 278–297 at the C terminus of Rad. This domain contains a motif characteristic of a calmodulin-binding region, consisting of numerous basic and hydrophobic residues. In addition, we have identified a second potential regulatory domain in the extended N terminus of Rad which, when removed, decreases Rad protein expression but increases the binding of Rad to CaM. The ability of Rad mutants to bind CaM correlates with their localization in cytoskeletal fractions of C2C12 cells. Immunoprecipitates of calmodulin-dependent protein kinase II, the cellular effector of Ca2+-calmodulin, also contain Rad, and in vitro both Rad and Gem can serve as substrates for this kinase. Thus, the Rad family of GTP-binding proteins possess unique characteristics of binding CaM and calmodulin-dependent protein kinase II, suggesting a role for Rad-like GTPases in calcium activation of serine/threonine kinase cascades. Members of the Rad family of GTPases (including Rad, Gem, and Kir) possess several unique features of unknown function in comparison to other Ras-like proteins, with major N-terminal and C-terminal extensions, a lack of typical prenylation motifs, and several non-conservative changes in the sequence of the GTP binding domain. Here we show that Rad and Gem bind to calmodulin (CaM)-Sepharose in vitro in a calcium-dependent manner and that Rad can be co-immunoprecipitated with CaM in C2C12 cells. The interaction is influenced by the guanine nucleotide binding state of Rad with the GDP-bound form exhibiting 5-fold better binding to CaM than the GTP-bound protein. In addition, the dominant negative mutant of Rad (S105N) which binds GDP, but not GTP, exhibits enhanced binding to CaM in vivo when expressed in C2C12 cells. Peptide competition studies and expression of deletion mutants of Rad localize the binding site for CaM to residues 278–297 at the C terminus of Rad. This domain contains a motif characteristic of a calmodulin-binding region, consisting of numerous basic and hydrophobic residues. In addition, we have identified a second potential regulatory domain in the extended N terminus of Rad which, when removed, decreases Rad protein expression but increases the binding of Rad to CaM. The ability of Rad mutants to bind CaM correlates with their localization in cytoskeletal fractions of C2C12 cells. Immunoprecipitates of calmodulin-dependent protein kinase II, the cellular effector of Ca2+-calmodulin, also contain Rad, and in vitro both Rad and Gem can serve as substrates for this kinase. Thus, the Rad family of GTP-binding proteins possess unique characteristics of binding CaM and calmodulin-dependent protein kinase II, suggesting a role for Rad-like GTPases in calcium activation of serine/threonine kinase cascades. Rad is the prototypic member of a new class of Ras-like GTP-binding proteins that includes Gem and Kir (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). In humans, Rad is most highly expressed in the heart, lung, and skeletal muscle and expression is increased in the skeletal muscle of some type II diabetic humans (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar). Rad exhibits a unique magnesium dependence for guanine nucleotide binding and is regulated by a Rad-specific GTPase-activating protein (GAP) 1The abbreviations used are: GAP, GTPase-activating protein; CaM, calmodulin; CaMKII, calmodulin-dependent protein kinase II; GTPγS, guanosine 5′-O-(2-thiodiphosphate); DTT, dithiothreitol; GST, glutathione S-transferase; MBP, myelin basic protein; Puro, puromycin; WT, wild type. 1The abbreviations used are: GAP, GTPase-activating protein; CaM, calmodulin; CaMKII, calmodulin-dependent protein kinase II; GTPγS, guanosine 5′-O-(2-thiodiphosphate); DTT, dithiothreitol; GST, glutathione S-transferase; MBP, myelin basic protein; Puro, puromycin; WT, wild type. (4Zhu J. Reynet C. Caldwell J.S. Kahn C.R. J. Biol. Chem. 1995; 270: 4805-4812Google Scholar). In cultured muscle and fat cells, Rad overexpression attenuates insulin-stimulated glucose uptake without altering expression or insulin-stimulated translocation of the Glut4 glucose transporter (5Moyers J.S. Bilan P.J. Reynet C.R. Kahn C.R. J. Biol. Chem. 1996; 271: 23111-23116Google Scholar). By expression library screening, Rad has been shown to interact with skeletal muscle β-tropomyosin, suggesting that Rad may participate in regulation of the cytoskeleton (6Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Google Scholar). The Gem gene product is expressed in the G1 phase in mitogen-activated T lymphocytes and shares approximately 60% amino acid identity with Rad (2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar), whereas kir was isolated from a pre-B-cell library and is overexpressed in cells expressingBCR/ABL or v-abl (3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). Murine kir andgem are 98.4% identical in nucleotide sequence and encode the same or very highly related proteins, referred to here as Kir/Gem (3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). When expressed in Saccharomyces cerevisiae,kir induces invasive pseudohyphal growth and may function upstream of the STE20 kinase (7Dorin D. Cohen L. Del-Villar K. Poullet P. Mohr R. Whiteway M. Witte O. Tamanoi F. Oncogene. 1995; 11: 2267-2271Google Scholar).rad and kir/gem encode GTP-binding proteins with several structural features that are distinct from other GTPases (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). The N terminus of Rad is extended by 88 amino acids, and Kir/Gem is extended by 72 amino acids in comparison to Ras, and the C terminus of each is extended by 31 amino acids. Although Rad and Kir/Gem share 100% identity in the final 11 amino acids, they lack a CAAX-like prenylation site present in other Ras-like molecules (8Casey P.J. Curr. Opin. Cell Biol. 1994; 6: 219-225Google Scholar, 9Glomset J.A. Farnsworth C.C. Annu. Rev. Cell Biol. 1994; 10: 181-205Google Scholar). Rad and Kir/Gem differ from each other and from other Ras-like proteins in the putative effector (G2) domain, suggesting that they interact with distinct GAPs or effector molecules. They also contain residues in the G3 consensus sequence for guanine nucleotide binding which are divergent from Ras (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar).Members of the Ras family of GTP-binding proteins participate in a number of cellular functions including proliferation (10Downward J. Bioessays. 1992; 14: 177-184Google Scholar), vesicular transport (11Baldini G. Hohl T. Lin H.Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5049-5052Google Scholar, 12Cormont M. Tanti J.-F. Zahraoui A. Van Obberghen E. Tavitian A. Le Marchand-Brustel Y. J. Biol. Chem. 1993; 268: 19491-19497Google Scholar), and cytoskeletal arrangement (13Ridley A.J. Hall A. Cell. 1992; 70: 389-399Google Scholar, 14Nishiyama T. Sasaki T. Takaishi K. Kato M. Yaku H. Araki K. Matsuura Y. Takai Y. Mol. Cell. Biol. 1994; 14: 2447-2456Google Scholar). Rac and cdc42 have been shown to interact with phosphatidylinositol 3-kinase and to participate in signaling leading to activation of the Jun kinases (15Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Google Scholar, 16Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Google Scholar). While the exact function of Rad and Kir/Gem is unknown, it has recently been reported in abstract that peptides based on the C terminus of Rad and Kir/Gem can bind to calmodulin (CaM)in vitro (17Berchtold, M. W., and Fischer, R. (1996) Keystone Symposia on Molecular and Cellular Biology, Tamarron, CO, Jan. 5–11, 1996, E7, 304 (abstr.), Keystone Symposia, Silverthorne, CO.Google Scholar). In this study, we show that the full-length Rad protein binds CaM in vitro and in vivo in a Ca2+-dependent manner and that the C-terminal residues 278–297 of human Rad are critical for this interaction. The binding of Rad and CaM is influenced by the guanine nucleotide bound state of Rad. We also demonstrate that Rad is present in complex with the cellular target of CaM, calmodulin-dependent protein kinase II (CaMKII), which can phosphorylate both Rad and Gem in vitro. These findings suggest that the Rad family of Ras-like proteins may participate in Ca2+-triggered signaling events involving CaM and the CaMKII serine/threonine kinase cascade.DISCUSSIONRad and Kir/Gem are members of a novel class of Ras-related GTP-binding proteins that contain unique and extended N and C termini as compared with other Ras-like proteins (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). In this study we have shown that these extended domains are involved in binding of CaM. Thus, Rad and Gem bind CaM-Sepharose in a Ca2+-dependent manner, and in cells, Rad co-immunoprecipitates with CaM in a manner that is disrupted by EGTA treatment. Therefore, the Rad family of proteins joins the growing list of CaM-binding proteins, including CaMKII, myosin light chain kinase, phosphofructokinase, plasma membrane Ca2+-ATPase, neuromodulin, and, more recently, IQGAP1 and Ras-guanine nucleotide releasing factor (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar, 23Bagchi I.C. Huang Q. Means A.R. J. Biol. Chem. 1992; 267: 3024-3029Google Scholar, 24Alexander K.A. Wakim B.T. Doyle G.S. Walsh K.A. Storm D.R. J. Biol. Chem. 1988; 263: 7544-7549Google Scholar, 25Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Google Scholar, 26Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Google Scholar). Berchtold and Fischer (17Berchtold, M. W., and Fischer, R. (1996) Keystone Symposia on Molecular and Cellular Biology, Tamarron, CO, Jan. 5–11, 1996, E7, 304 (abstr.), Keystone Symposia, Silverthorne, CO.Google Scholar) have shown that a peptide corresponding to the final C-terminal 30 amino acids of Kir/Gem binds to CaM in vitro, and in the current study we find that a synthetic peptide based on the final 30 amino acids of Rad (residues 278–308) competes for Rad binding to CaM-Sepharose. Further evidence that the CaM-binding domain of Rad is indeed located in the C-terminal extended region involved deletion mutants. Thus, while deletion of residues 297–308 from full-length Rad did not disrupt CaM binding, deletion of residues 278–308 abolished CaM binding completely. Based on these mutants, the specific residues critical for the CaM interaction lie in the 19-amino acid region encompassing residues 278–297 of human Rad. Modeling of this region as a helical wheel confirms the distribution of charged and hydrophobic residues typical of CaM-binding protein. It is likely that the corresponding region of Gem, which shares 79% homology to Rad and is also rich in charged and hydrophobic residues, mediates its binding to CaM. We have shown previously that in C2C12 cells, GDP-bound Rad binds skeletal muscle β-tropomyosin following Ca2+ ionophore treatment (6Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Google Scholar). Although the region of Rad that mediates this interaction has not been determined, addition of a 5-fold molar excess of purified tropomyosin did not affect the interaction of Rad and CaM, suggesting that the binding regions reside in different locations in Rad (not shown).The Rad-CaM interaction appears to be dependent on the guanine nucleotide-bound state of Rad since GDP-loaded purified Rad and the Rad dominant negative mutant (S105N) expressed in cells exhibit increased binding to CaM in comparison to GTPγS-loaded Rad and WT Rad in cells. We speculated that CaM may thus serve to sequester Rad in its inactive GDP-bound form, serving as a "switching off" mechanism; however, we failed to show an affect of CaM binding on the guanine nucleotide binding state of Rad, suggesting that this may not be the case. Alternatively, in the presence of Ca2+, Rad may serve to sequester CaM. Additionally, we have previously shown that treatment of C2C12 cells with Ca2+ ionophore, A23187, results in a rapid degradation of Rad protein (6Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Google Scholar). It is possible that Ca2+-CaM serves a role in the switching off of Rad by facilitating the degradation of Rad by Ca2+-activated proteases. CaM does not catalyze the inactivation of Rad by GTP hydrolysis, since CaM alone does not significantly affect Rad intrinsic GTPase activity nor Rad-GAP-stimulated GTP hydrolysis.In addition to binding CaM, Rad exists in complex with CaMKII, the serine/threonine kinase which is a cellular target of CaM. Deletion of the CaM-binding domain of Rad, treatment of immune complexes with EGTA, and competition studies with the CaM-binding domain peptide indicate that Rad interacts with CaMKII independent of its association with Ca2+-CaM and that different domains of Rad are involved in these interactions. In addition, Rad and Gem serve as in vitro substrates for CaMKII. Although the significance of this phosphorylation is not yet known, it is possible that CaMKII modulates the function of Rad or its binding to CaM in a feedback mechanism. Two consensus sites for CaMKII phosphorylation (serines 273 and 299) reside near the region of CaM binding (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar) and could potentially modulate the Rad-CaM interaction by introducing a negative charge in the binding region.It has been noted that several CaM-binding proteins, including CaMKII and myosin light chain kinase, contain a "CaM-like binding site" within the sequence of the molecule (i.e. rich in hydrophobic/anionic residues) which is proposed to act as an internal inhibitor of CaM binding by interacting with the hydrophobic/cationic CaM-binding site within the protein (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar). Our mutagenesis studies suggest that Rad may have such a region in that deletion of the N-terminal 88 amino acids of Rad resulted in a molecule that exhibits enhanced binding to CaM-Sepharose. The region of Rad spanning residues 68–88 contains a number of hydrophobic and negatively charged residues, making it a potential auto-inhibitory domain (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar). Thus, the unique N- and C-terminal regions of Rad may co-regulate CaM interaction. It is possible, of course, that deletion of the N terminus results in a more generalized alteration in conformation, exposing the CaM-binding site or altering the guanine nucleotide binding characteristics of this protein. In addition, accurate quantitation of the CaM binding efficiency of the N-terminal deletion mutant (Rad N88) is difficult since so little of the protein is detectable in the soluble lysate samples.Unlike Ras, which is localized to the plasma membrane via prenylation of its C-terminal CAAX-like motif, Rad is localized mainly to the cytoplasm, with portions of the protein associated with cytoskeletal2 and membrane fractions (5Moyers J.S. Bilan P.J. Reynet C.R. Kahn C.R. J. Biol. Chem. 1996; 271: 23111-23116Google Scholar). Although Rad lacks a CAAX-like C-terminal motif, deletion of the C-terminal residues 278–308 displaced Rad from the cytoskeleton, membrane skeleton, and soluble membrane fractions to the cytosol, whereas deletion of residues 297–308 did not. Thus, the critical residues for Rad localization to membrane and cytoskeletal components correspond to the CaM-binding domain of Rad, residues 278–297. It is also possible that CaM binding serves to localize Rad. Consistent with this, deletion of the N terminus of Rad, which enhances CaM binding, correlates with displacement of Rad from the cytosol to the cytoskeleton, membrane skeleton, and soluble membranes. Alternatively, in addition to an intact N terminus, Rad localization may require residues near, but distinct from, those C-terminal residues required for CaM interaction.In summary, we have shown that the Ras-like GTPases, Rad and Gem, possess the unique quality of binding Ca2+-CaM and have localized the site of CaM binding to the C-terminal residues 278–297 of human Rad. The interaction of Rad and CaM is dependent on the guanine nucleotide bound state of Rad, and deletion mutations that affect binding result in redistribution of Rad in the cell. In addition, Rad is found in complex with CaMKII, and Rad and Gem serve asin vitro substrates for this kinase, suggesting that the Rad-like GTPases participate in Ca2+-activated signaling cascades leading to the activation of serine kinases. Rad is the prototypic member of a new class of Ras-like GTP-binding proteins that includes Gem and Kir (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). In humans, Rad is most highly expressed in the heart, lung, and skeletal muscle and expression is increased in the skeletal muscle of some type II diabetic humans (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar). Rad exhibits a unique magnesium dependence for guanine nucleotide binding and is regulated by a Rad-specific GTPase-activating protein (GAP) 1The abbreviations used are: GAP, GTPase-activating protein; CaM, calmodulin; CaMKII, calmodulin-dependent protein kinase II; GTPγS, guanosine 5′-O-(2-thiodiphosphate); DTT, dithiothreitol; GST, glutathione S-transferase; MBP, myelin basic protein; Puro, puromycin; WT, wild type. 1The abbreviations used are: GAP, GTPase-activating protein; CaM, calmodulin; CaMKII, calmodulin-dependent protein kinase II; GTPγS, guanosine 5′-O-(2-thiodiphosphate); DTT, dithiothreitol; GST, glutathione S-transferase; MBP, myelin basic protein; Puro, puromycin; WT, wild type. (4Zhu J. Reynet C. Caldwell J.S. Kahn C.R. J. Biol. Chem. 1995; 270: 4805-4812Google Scholar). In cultured muscle and fat cells, Rad overexpression attenuates insulin-stimulated glucose uptake without altering expression or insulin-stimulated translocation of the Glut4 glucose transporter (5Moyers J.S. Bilan P.J. Reynet C.R. Kahn C.R. J. Biol. Chem. 1996; 271: 23111-23116Google Scholar). By expression library screening, Rad has been shown to interact with skeletal muscle β-tropomyosin, suggesting that Rad may participate in regulation of the cytoskeleton (6Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Google Scholar). The Gem gene product is expressed in the G1 phase in mitogen-activated T lymphocytes and shares approximately 60% amino acid identity with Rad (2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar), whereas kir was isolated from a pre-B-cell library and is overexpressed in cells expressingBCR/ABL or v-abl (3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). Murine kir andgem are 98.4% identical in nucleotide sequence and encode the same or very highly related proteins, referred to here as Kir/Gem (3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). When expressed in Saccharomyces cerevisiae,kir induces invasive pseudohyphal growth and may function upstream of the STE20 kinase (7Dorin D. Cohen L. Del-Villar K. Poullet P. Mohr R. Whiteway M. Witte O. Tamanoi F. Oncogene. 1995; 11: 2267-2271Google Scholar). rad and kir/gem encode GTP-binding proteins with several structural features that are distinct from other GTPases (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). The N terminus of Rad is extended by 88 amino acids, and Kir/Gem is extended by 72 amino acids in comparison to Ras, and the C terminus of each is extended by 31 amino acids. Although Rad and Kir/Gem share 100% identity in the final 11 amino acids, they lack a CAAX-like prenylation site present in other Ras-like molecules (8Casey P.J. Curr. Opin. Cell Biol. 1994; 6: 219-225Google Scholar, 9Glomset J.A. Farnsworth C.C. Annu. Rev. Cell Biol. 1994; 10: 181-205Google Scholar). Rad and Kir/Gem differ from each other and from other Ras-like proteins in the putative effector (G2) domain, suggesting that they interact with distinct GAPs or effector molecules. They also contain residues in the G3 consensus sequence for guanine nucleotide binding which are divergent from Ras (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). Members of the Ras family of GTP-binding proteins participate in a number of cellular functions including proliferation (10Downward J. Bioessays. 1992; 14: 177-184Google Scholar), vesicular transport (11Baldini G. Hohl T. Lin H.Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5049-5052Google Scholar, 12Cormont M. Tanti J.-F. Zahraoui A. Van Obberghen E. Tavitian A. Le Marchand-Brustel Y. J. Biol. Chem. 1993; 268: 19491-19497Google Scholar), and cytoskeletal arrangement (13Ridley A.J. Hall A. Cell. 1992; 70: 389-399Google Scholar, 14Nishiyama T. Sasaki T. Takaishi K. Kato M. Yaku H. Araki K. Matsuura Y. Takai Y. Mol. Cell. Biol. 1994; 14: 2447-2456Google Scholar). Rac and cdc42 have been shown to interact with phosphatidylinositol 3-kinase and to participate in signaling leading to activation of the Jun kinases (15Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Google Scholar, 16Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Google Scholar). While the exact function of Rad and Kir/Gem is unknown, it has recently been reported in abstract that peptides based on the C terminus of Rad and Kir/Gem can bind to calmodulin (CaM)in vitro (17Berchtold, M. W., and Fischer, R. (1996) Keystone Symposia on Molecular and Cellular Biology, Tamarron, CO, Jan. 5–11, 1996, E7, 304 (abstr.), Keystone Symposia, Silverthorne, CO.Google Scholar). In this study, we show that the full-length Rad protein binds CaM in vitro and in vivo in a Ca2+-dependent manner and that the C-terminal residues 278–297 of human Rad are critical for this interaction. The binding of Rad and CaM is influenced by the guanine nucleotide bound state of Rad. We also demonstrate that Rad is present in complex with the cellular target of CaM, calmodulin-dependent protein kinase II (CaMKII), which can phosphorylate both Rad and Gem in vitro. These findings suggest that the Rad family of Ras-like proteins may participate in Ca2+-triggered signaling events involving CaM and the CaMKII serine/threonine kinase cascade. DISCUSSIONRad and Kir/Gem are members of a novel class of Ras-related GTP-binding proteins that contain unique and extended N and C termini as compared with other Ras-like proteins (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). In this study we have shown that these extended domains are involved in binding of CaM. Thus, Rad and Gem bind CaM-Sepharose in a Ca2+-dependent manner, and in cells, Rad co-immunoprecipitates with CaM in a manner that is disrupted by EGTA treatment. Therefore, the Rad family of proteins joins the growing list of CaM-binding proteins, including CaMKII, myosin light chain kinase, phosphofructokinase, plasma membrane Ca2+-ATPase, neuromodulin, and, more recently, IQGAP1 and Ras-guanine nucleotide releasing factor (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar, 23Bagchi I.C. Huang Q. Means A.R. J. Biol. Chem. 1992; 267: 3024-3029Google Scholar, 24Alexander K.A. Wakim B.T. Doyle G.S. Walsh K.A. Storm D.R. J. Biol. Chem. 1988; 263: 7544-7549Google Scholar, 25Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Google Scholar, 26Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Google Scholar). Berchtold and Fischer (17Berchtold, M. W., and Fischer, R. (1996) Keystone Symposia on Molecular and Cellular Biology, Tamarron, CO, Jan. 5–11, 1996, E7, 304 (abstr.), Keystone Symposia, Silverthorne, CO.Google Scholar) have shown that a peptide corresponding to the final C-terminal 30 amino acids of Kir/Gem binds to CaM in vitro, and in the current study we find that a synthetic peptide based on the final 30 amino acids of Rad (residues 278–308) competes for Rad binding to CaM-Sepharose. Further evidence that the CaM-binding domain of Rad is indeed located in the C-terminal extended region involved deletion mutants. Thus, while deletion of residues 297–308 from full-length Rad did not disrupt CaM binding, deletion of residues 278–308 abolished CaM binding completely. Based on these mutants, the specific residues critical for the CaM interaction lie in the 19-amino acid region encompassing residues 278–297 of human Rad. Modeling of this region as a helical wheel confirms the distribution of charged and hydrophobic residues typical of CaM-binding protein. It is likely that the corresponding region of Gem, which shares 79% homology to Rad and is also rich in charged and hydrophobic residues, mediates its binding to CaM. We have shown previously that in C2C12 cells, GDP-bound Rad binds skeletal muscle β-tropomyosin following Ca2+ ionophore treatment (6Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Google Scholar). Although the region of Rad that mediates this interaction has not been determined, addition of a 5-fold molar excess of purified tropomyosin did not affect the interaction of Rad and CaM, suggesting that the binding regions reside in different locations in Rad (not shown).The Rad-CaM interaction appears to be dependent on the guanine nucleotide-bound state of Rad since GDP-loaded purified Rad and the Rad dominant negative mutant (S105N) expressed in cells exhibit increased binding to CaM in comparison to GTPγS-loaded Rad and WT Rad in cells. We speculated that CaM may thus serve to sequester Rad in its inactive GDP-bound form, serving as a "switching off" mechanism; however, we failed to show an affect of CaM binding on the guanine nucleotide binding state of Rad, suggesting that this may not be the case. Alternatively, in the presence of Ca2+, Rad may serve to sequester CaM. Additionally, we have previously shown that treatment of C2C12 cells with Ca2+ ionophore, A23187, results in a rapid degradation of Rad protein (6Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Google Scholar). It is possible that Ca2+-CaM serves a role in the switching off of Rad by facilitating the degradation of Rad by Ca2+-activated proteases. CaM does not catalyze the inactivation of Rad by GTP hydrolysis, since CaM alone does not significantly affect Rad intrinsic GTPase activity nor Rad-GAP-stimulated GTP hydrolysis.In addition to binding CaM, Rad exists in complex with CaMKII, the serine/threonine kinase which is a cellular target of CaM. Deletion of the CaM-binding domain of Rad, treatment of immune complexes with EGTA, and competition studies with the CaM-binding domain peptide indicate that Rad interacts with CaMKII independent of its association with Ca2+-CaM and that different domains of Rad are involved in these interactions. In addition, Rad and Gem serve as in vitro substrates for CaMKII. Although the significance of this phosphorylation is not yet known, it is possible that CaMKII modulates the function of Rad or its binding to CaM in a feedback mechanism. Two consensus sites for CaMKII phosphorylation (serines 273 and 299) reside near the region of CaM binding (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar) and could potentially modulate the Rad-CaM interaction by introducing a negative charge in the binding region.It has been noted that several CaM-binding proteins, including CaMKII and myosin light chain kinase, contain a "CaM-like binding site" within the sequence of the molecule (i.e. rich in hydrophobic/anionic residues) which is proposed to act as an internal inhibitor of CaM binding by interacting with the hydrophobic/cationic CaM-binding site within the protein (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar). Our mutagenesis studies suggest that Rad may have such a region in that deletion of the N-terminal 88 amino acids of Rad resulted in a molecule that exhibits enhanced binding to CaM-Sepharose. The region of Rad spanning residues 68–88 contains a number of hydrophobic and negatively charged residues, making it a potential auto-inhibitory domain (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar). Thus, the unique N- and C-terminal regions of Rad may co-regulate CaM interaction. It is possible, of course, that deletion of the N terminus results in a more generalized alteration in conformation, exposing the CaM-binding site or altering the guanine nucleotide binding characteristics of this protein. In addition, accurate quantitation of the CaM binding efficiency of the N-terminal deletion mutant (Rad N88) is difficult since so little of the protein is detectable in the soluble lysate samples.Unlike Ras, which is localized to the plasma membrane via prenylation of its C-terminal CAAX-like motif, Rad is localized mainly to the cytoplasm, with portions of the protein associated with cytoskeletal2 and membrane fractions (5Moyers J.S. Bilan P.J. Reynet C.R. Kahn C.R. J. Biol. Chem. 1996; 271: 23111-23116Google Scholar). Although Rad lacks a CAAX-like C-terminal motif, deletion of the C-terminal residues 278–308 displaced Rad from the cytoskeleton, membrane skeleton, and soluble membrane fractions to the cytosol, whereas deletion of residues 297–308 did not. Thus, the critical residues for Rad localization to membrane and cytoskeletal components correspond to the CaM-binding domain of Rad, residues 278–297. It is also possible that CaM binding serves to localize Rad. Consistent with this, deletion of the N terminus of Rad, which enhances CaM binding, correlates with displacement of Rad from the cytosol to the cytoskeleton, membrane skeleton, and soluble membranes. Alternatively, in addition to an intact N terminus, Rad localization may require residues near, but distinct from, those C-terminal residues required for CaM interaction.In summary, we have shown that the Ras-like GTPases, Rad and Gem, possess the unique quality of binding Ca2+-CaM and have localized the site of CaM binding to the C-terminal residues 278–297 of human Rad. The interaction of Rad and CaM is dependent on the guanine nucleotide bound state of Rad, and deletion mutations that affect binding result in redistribution of Rad in the cell. In addition, Rad is found in complex with CaMKII, and Rad and Gem serve asin vitro substrates for this kinase, suggesting that the Rad-like GTPases participate in Ca2+-activated signaling cascades leading to the activation of serine kinases. Rad and Kir/Gem are members of a novel class of Ras-related GTP-binding proteins that contain unique and extended N and C termini as compared with other Ras-like proteins (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar, 2Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Google Scholar, 3Cohen L. Mohr R. Chen Y.-Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. Witte O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Google Scholar). In this study we have shown that these extended domains are involved in binding of CaM. Thus, Rad and Gem bind CaM-Sepharose in a Ca2+-dependent manner, and in cells, Rad co-immunoprecipitates with CaM in a manner that is disrupted by EGTA treatment. Therefore, the Rad family of proteins joins the growing list of CaM-binding proteins, including CaMKII, myosin light chain kinase, phosphofructokinase, plasma membrane Ca2+-ATPase, neuromodulin, and, more recently, IQGAP1 and Ras-guanine nucleotide releasing factor (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar, 23Bagchi I.C. Huang Q. Means A.R. J. Biol. Chem. 1992; 267: 3024-3029Google Scholar, 24Alexander K.A. Wakim B.T. Doyle G.S. Walsh K.A. Storm D.R. J. Biol. Chem. 1988; 263: 7544-7549Google Scholar, 25Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Google Scholar, 26Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Google Scholar). Berchtold and Fischer (17Berchtold, M. W., and Fischer, R. (1996) Keystone Symposia on Molecular and Cellular Biology, Tamarron, CO, Jan. 5–11, 1996, E7, 304 (abstr.), Keystone Symposia, Silverthorne, CO.Google Scholar) have shown that a peptide corresponding to the final C-terminal 30 amino acids of Kir/Gem binds to CaM in vitro, and in the current study we find that a synthetic peptide based on the final 30 amino acids of Rad (residues 278–308) competes for Rad binding to CaM-Sepharose. Further evidence that the CaM-binding domain of Rad is indeed located in the C-terminal extended region involved deletion mutants. Thus, while deletion of residues 297–308 from full-length Rad did not disrupt CaM binding, deletion of residues 278–308 abolished CaM binding completely. Based on these mutants, the specific residues critical for the CaM interaction lie in the 19-amino acid region encompassing residues 278–297 of human Rad. Modeling of this region as a helical wheel confirms the distribution of charged and hydrophobic residues typical of CaM-binding protein. It is likely that the corresponding region of Gem, which shares 79% homology to Rad and is also rich in charged and hydrophobic residues, mediates its binding to CaM. We have shown previously that in C2C12 cells, GDP-bound Rad binds skeletal muscle β-tropomyosin following Ca2+ ionophore treatment (6Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Google Scholar). Although the region of Rad that mediates this interaction has not been determined, addition of a 5-fold molar excess of purified tropomyosin did not affect the interaction of Rad and CaM, suggesting that the binding regions reside in different locations in Rad (not shown). The Rad-CaM interaction appears to be dependent on the guanine nucleotide-bound state of Rad since GDP-loaded purified Rad and the Rad dominant negative mutant (S105N) expressed in cells exhibit increased binding to CaM in comparison to GTPγS-loaded Rad and WT Rad in cells. We speculated that CaM may thus serve to sequester Rad in its inactive GDP-bound form, serving as a "switching off" mechanism; however, we failed to show an affect of CaM binding on the guanine nucleotide binding state of Rad, suggesting that this may not be the case. Alternatively, in the presence of Ca2+, Rad may serve to sequester CaM. Additionally, we have previously shown that treatment of C2C12 cells with Ca2+ ionophore, A23187, results in a rapid degradation of Rad protein (6Zhu J. Bilan P.J. Moyers J.S. Antonetti D.A. Kahn C.R. J. Biol. Chem. 1996; 271: 768-773Google Scholar). It is possible that Ca2+-CaM serves a role in the switching off of Rad by facilitating the degradation of Rad by Ca2+-activated proteases. CaM does not catalyze the inactivation of Rad by GTP hydrolysis, since CaM alone does not significantly affect Rad intrinsic GTPase activity nor Rad-GAP-stimulated GTP hydrolysis. In addition to binding CaM, Rad exists in complex with CaMKII, the serine/threonine kinase which is a cellular target of CaM. Deletion of the CaM-binding domain of Rad, treatment of immune complexes with EGTA, and competition studies with the CaM-binding domain peptide indicate that Rad interacts with CaMKII independent of its association with Ca2+-CaM and that different domains of Rad are involved in these interactions. In addition, Rad and Gem serve as in vitro substrates for CaMKII. Although the significance of this phosphorylation is not yet known, it is possible that CaMKII modulates the function of Rad or its binding to CaM in a feedback mechanism. Two consensus sites for CaMKII phosphorylation (serines 273 and 299) reside near the region of CaM binding (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Google Scholar) and could potentially modulate the Rad-CaM interaction by introducing a negative charge in the binding region. It has been noted that several CaM-binding proteins, including CaMKII and myosin light chain kinase, contain a "CaM-like binding site" within the sequence of the molecule (i.e. rich in hydrophobic/anionic residues) which is proposed to act as an internal inhibitor of CaM binding by interacting with the hydrophobic/cationic CaM-binding site within the protein (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar). Our mutagenesis studies suggest that Rad may have such a region in that deletion of the N-terminal 88 amino acids of Rad resulted in a molecule that exhibits enhanced binding to CaM-Sepharose. The region of Rad spanning residues 68–88 contains a number of hydrophobic and negatively charged residues, making it a potential auto-inhibitory domain (22Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Google Scholar). Thus, the unique N- and C-terminal regions of Rad may co-regulate CaM interaction. It is possible, of course, that deletion of the N terminus results in a more generalized alteration in conformation, exposing the CaM-binding site or altering the guanine nucleotide binding characteristics of this protein. In addition, accurate quantitation of the CaM binding efficiency of the N-terminal deletion mutant (Rad N88) is difficult since so little of the protein is detectable in the soluble lysate samples. Unlike Ras, which is localized to the plasma membrane via prenylation of its C-terminal CAAX-like motif, Rad is localized mainly to the cytoplasm, with portions of the protein associated with cytoskeletal2 and membrane fractions (5Moyers J.S. Bilan P.J. Reynet C.R. Kahn C.R. J. Biol. Chem. 1996; 271: 23111-23116Google Scholar). Although Rad lacks a CAAX-like C-terminal motif, deletion of the C-terminal residues 278–308 displaced Rad from the cytoskeleton, membrane skeleton, and soluble membrane fractions to the cytosol, whereas deletion of residues 297–308 did not. Thus, the critical residues for Rad localization to membrane and cytoskeletal components correspond to the CaM-binding domain of Rad, residues 278–297. It is also possible that CaM binding serves to localize Rad. Consistent with this, deletion of the N terminus of Rad, which enhances CaM binding, correlates with displacement of Rad from the cytosol to the cytoskeleton, membrane skeleton, and soluble membranes. Alternatively, in addition to an intact N terminus, Rad localization may require residues near, but distinct from, those C-terminal residues required for CaM interaction. In summary, we have shown that the Ras-like GTPases, Rad and Gem, possess the unique quality of binding Ca2+-CaM and have localized the site of CaM binding to the C-terminal residues 278–297 of human Rad. The interaction of Rad and CaM is dependent on the guanine nucleotide bound state of Rad, and deletion mutations that affect binding result in redistribution of Rad in the cell. In addition, Rad is found in complex with CaMKII, and Rad and Gem serve asin vitro substrates for this kinase, suggesting that the Rad-like GTPases participate in Ca2+-activated signaling cascades leading to the activation of serine kinases. We thank Dr. Renee Emkey for valuable discussions on this work. We also thank Dr. Kathleen Kelly of National Institutes of Health for the gifts of the GST-Gem construct and the anti-Gem antibodies.