Calcium Signaling Regulates Translocation and Activation of Rac

Abstract

Rac is activated in response to various stimuli including growth factors and by adhesion to the extracellular matrix. However, how these stimuli ultimately result in Rac activation is poorly understood. The increase in intracellular calcium [Ca2+] i represents a ubiquitous second messenger system in cells, linking receptor activation to downstream signaling pathways. Here we show that elevation of [Ca2+] i , either artificially or by thrombin receptor activation, potently induces Rac activation. Lamellipodia formation induced by artificial elevation of [Ca2+] i is blocked by inhibition of Rac signaling, indicating that calcium-induced cytoskeletal changes are controlled by the activation of Rac. Calcium-dependent Rac activation was dependent on the activation of a conventional protein kinase C. Furthermore, both increased [Ca2+] i and protein kinase C activation induce phosphorylation of RhoGDIα and induce the translocation of cytosolic Rac to the plasma membrane. Intracellular calcium signaling may thus contribute to the intracellular localization and activation of Rac to regulate the cytoskeletal changes in response to receptor stimulation. Rac is activated in response to various stimuli including growth factors and by adhesion to the extracellular matrix. However, how these stimuli ultimately result in Rac activation is poorly understood. The increase in intracellular calcium [Ca2+] i represents a ubiquitous second messenger system in cells, linking receptor activation to downstream signaling pathways. Here we show that elevation of [Ca2+] i , either artificially or by thrombin receptor activation, potently induces Rac activation. Lamellipodia formation induced by artificial elevation of [Ca2+] i is blocked by inhibition of Rac signaling, indicating that calcium-induced cytoskeletal changes are controlled by the activation of Rac. Calcium-dependent Rac activation was dependent on the activation of a conventional protein kinase C. Furthermore, both increased [Ca2+] i and protein kinase C activation induce phosphorylation of RhoGDIα and induce the translocation of cytosolic Rac to the plasma membrane. Intracellular calcium signaling may thus contribute to the intracellular localization and activation of Rac to regulate the cytoskeletal changes in response to receptor stimulation. The Rho family of small GTPases, including Rho, Rac, and Cdc42 isoforms, regulates different aspects of cytoskeletal organization, which are coordinated in the process of cell migration (1Hall A. Nobes C.D. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2000; 355: 965-970Crossref PubMed Scopus (424) Google Scholar). Of these, Rac is involved in the protrusion of lamellipodia, which occur principally at the leading edge of migrating cells but also emerge from around newly adherent cells to mediate cell spreading (2Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. 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Rac is activated in response to various stimuli, including growth factors and adhesion to the extracellular matrix. However, how these stimuli ultimately result in Rac activation is poorly understood. The principal regulators of Rac activation are the guanine nucleotide exchange factors (GEFs) 1The abbreviations used are: GEF, guanine nucleotide exchange factor; PKC, protein kinase C; PDGF, platelet-derived growth factor; TRP, thrombin-related peptide; PMA, phorbol 12-myristate 13-acetate; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; EGFP, enhanced GFP; PLC, phospholipase C; PH, pleckstrin homology; PIPES, 1,4-piperazinediethanesulfonic acid; CRIB, Cdc42/Rac interactive binding; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester. and GTPase activating proteins. GEFs induce activation by exchanging GDP for GTP, whereas GTPase activating proteins enhance the intrinsic rate of hydrolysis of bound GTP to GDP, resulting in inactivation. In cells, Rac exists predominantly in its inactive GDP-bound form in a complex with RhoGDI (8Olofsson B. Cell. Signal. 1999; 11: 545-554Crossref PubMed Scopus (410) Google Scholar). RhoGDI binds and masks the hydrophobic C-terminal region of Rac, the same region that is responsible for targeting Rac to the plasma membrane (9Scheffzek K. Stephan I. Jensen O.N. Illenberger D. Gierschik P. Nat. Struct. Biol. 2000; 7: 122-126Crossref PubMed Scopus (182) Google Scholar). Thus RhoGDI maintains Rac in the cytoplasm and must dissociate to allow Rac to translocate to the membrane and interact with membrane-associated activators (10Stam J.C. Sander E.E. Michiels F. van Leeuwen F.N. Kain H.E. van der Kammen R.A. Collard J.G. J. Biol. 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Increased intracellular calcium [Ca2+] i represents a ubiquitous second messenger system in cells, linking receptor activation to downstream signaling pathways. Previous studies (15Price L.S. Norman J.C. Ridley A.J. Koffer A. Curr. Biol. 1995; 5: 68-73Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 16van Leeuwen F.N. van Delft S. Kain H.E. van der Kammen R.A. Collard J.G. Nat. Cell Biol. 1999; 1: 242-248Crossref PubMed Scopus (184) Google Scholar, 17Hirata K. Kikuchi A. Sasaki T. Kuroda S. Kaibuchi K. Matsuura Y. Seki H. Saida K. Takai Y. J. Biol. Chem. 1992; 267: 8719-8722Abstract Full Text PDF PubMed Google Scholar, 18O'Sullivan A.J. Brown A.M. Freeman H.N. Gomperts B.D. Mol. Biol. Cell. 1996; 7: 397-408Crossref PubMed Scopus (52) Google Scholar) have described relationships between intracellular calcium and the activation and function of Rho family GTPases in processes including muscle contraction and the exocytic response. Intracellular calcium is also required for thrombin- and collagen-induced Rac activation in platelets (19Soulet C. Gendreau S. Missy K. Benard V. Plantavid M. Payrastre B. FEBS Lett. 2001; 507: 253-258Crossref PubMed Scopus (58) Google Scholar). In neutrophils, however, chemoattractant-induced Rac activation is independent of intracellular calcium (20Geijsen N. van Delft S. Raaijmakers J.A. Lammers J.W. Collard J.G. Koenderman L. Coffer P.J. Blood. 1999; 94: 1121-1130Crossref PubMed Google Scholar), suggesting that the relationship between calcium and Rac signaling is dependent on the cell type and/or the growth factor receptor involved. Ras-GRF 1 and 2, exchange factors specific for both Ras and Rac (21Fan W.T. Koch C.A. de Hoog C.L. Fam N.P. Moran M.F. Curr. Biol. 1998; 8: 935-938Abstract Full Text Full Text PDF PubMed Google Scholar, 22Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (77) Google Scholar), harbor a calcium-calmodulin binding site (23Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (393) Google Scholar) whereas the Rac exchange factor, Tiam1, is phosphorylated by calcium-calmodulin-dependent protein kinase II, which leads to increased nucleotide exchange on Rac (24Fleming I.N. Elliott C.M. Buchanan F.G. Downes C.P. Exton J.H. J. Biol. Chem. 1999; 274: 12753-12758Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). These findings suggest that nucleotide exchange on Rac may be regulated by changes in intracellular calcium. Various studies have implicated protein kinase C (PKC) in the activation of Rac. In Swiss cells, phorbol induces membrane which is of Rac activation (2Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3076) Google Scholar), whereas is required for Rac activation in cells F.G. C.M. M. Exton J.H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). However, how Rac is Here we have the of intracellular calcium on Rac that intracellular calcium induce the membrane translocation and activation of Rac. an of of Rac by calcium calcium induces a of the the translocation of Rac to the plasma membrane be activated by membrane-associated or guanine nucleotide exchange the the acid was used and at the The the and was was as described K. J. Cell Biol. 1992; PubMed Scopus Google Scholar). BAPTA-AM, and from and from and from and B was from and of Cell by cells R.A. C.M. A. S. W.B. Google Scholar) in with in a at and with cells E.E. van Delft S. van der Kammen R.A. Collard J.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar) in in to a of cells at on and for cells by with a 1996; 7: PubMed Scopus Google Scholar, F. van der Kammen R.A. L. Collard J.G. 2000; PubMed Google Scholar). that was at the same as Rac was also as by Rac activation as a GTPase was as described E.E. van Delft S. van der Kammen R.A. Collard J.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar), with the that of a to the of was used to Rac. of cells with the and stimuli, cells and with a Cell by and with and in Rac was with Cell of and in cytosolic in cells by of the green and Cell 11: PubMed Scopus Google Scholar). for by in with of the of these in the of and in for to the was at and the was a and a at in a in and of phospholipase C activation and [Ca2+] i , cells with der J. R. P. T. K. J. Biol. 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Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). cells of for and with for with or and with or in and 1 and a RhoGDI was with and protein RhoGDI and RhoGDI from cell was by and with a Cell cells in with and with with in for to allow of cytosolic proteins. of Rac and of the cytosolic protein kinase and RhoGDIα was to as a was by The cells with and with by at for at and was to the on and and with Rac, and for of protein Intracellular and Rac the relationship between intracellular calcium signaling and Rac we cells with the thrombin receptor and the on intracellular calcium and on activation of Rac. of the thrombin receptor with in of calcium from intracellular of cells with induced a and increase in intracellular calcium [Ca2+] i Rac we used a modified Rac which a to a region of the of the Rac region binds to Rac is in its The as the protein used in studies E.E. van Delft S. T. van der Kammen R.A. Michiels F. Collard J.G. J. 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Cell Biol. 1998; PubMed Scopus Google Scholar). of cells that induced the translocation of from membrane to findings that thrombin receptor activation leads to the activation of induced translocation well in the or of However, of [Ca2+] i that blocked calcium that thrombin signaling, which ultimately leads to of intracellular is by these we that intracellular calcium is required for thrombin Rac activation and Increased to the of Rac activation on calcium signaling, we used of [Ca2+] of cells with which calcium from intracellular by from the by to a elevation of [Ca2+] i that of the of Rac activation that induced a activation of Rac that was at of cells with which induces a elevation of [Ca2+] i of of extracellular also induced activation of Rac C and we that the of calcium elevation to with that of Rac the elevation of [Ca2+] i induced by and with elevation of Rac whereas in [Ca2+] i and Rac activation. of Rac was of calcium-induced changes in spreading or Rac was by these we that elevation of intracellular calcium is to activate Rac in the of receptor stimulation. also suggest that intracellular calcium activation of Rac the pathway. we the of which is to intracellular calcium increase but calcium induced by of cells with the S. 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Rac activation be induced artificially either by calcium from intracellular or by the of extracellular calcium and was thus a of Rac activation induced by thrombin receptor was also be blocked by of intracellular calcium. Furthermore, thrombin Rac activation required phospholipase C whereas Rac These findings suggest that calcium induced by receptor activation of the are and necessary to activate Rac. Increased [Ca2+] i was to induce Rac activation in cell including cells and cells, but activate Rac in or It was C.M. K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that adhesion regulate Rac However, the that we between cell are to be of the formation of in cells, cells form In neutrophils, chemoattractant-induced Rac activation is independent of intracellular calcium (20Geijsen N. van Delft S. Raaijmakers J.A. Lammers J.W. Collard J.G. Koenderman L. Coffer P.J. 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