Calcium/Calmodulin-dependent Protein Kinase II Binds to Raf-1 and Modulates Integrin-stimulated ERK Activation

Abstract

Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1. Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1. Integrins are widely expressed transmembrane receptors for extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; Mek, extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; PI3-K, phosphatidylinositol 3-kinase; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CaM, Ca2+/calmodulin; FCS, fetal calf serum; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; TFP, trifluoperazine W7, N-(6-aminohexyl)-5-chloro-1-nafthalene-sulfonamide; MBP, myelin basic protein.1The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; Mek, extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; PI3-K, phosphatidylinositol 3-kinase; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CaM, Ca2+/calmodulin; FCS, fetal calf serum; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; TFP, trifluoperazine W7, N-(6-aminohexyl)-5-chloro-1-nafthalene-sulfonamide; MBP, myelin basic protein. proteins that provide a physical linkage between ECM and cytoskeletal structures (1Hemler M.E. 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We demonstrated previously that the ECM protein fibronectin (FN) controls both cell proliferation and survival in thyroid cells in primary cultures and in the immortalized human thyroid cell line TAD-2 (8Vitale M. Di Matola T. Fenzi G. Illario M. Rossi G. J. Clin. Endocrinol. Metab. 1998; 83: 3673-3680PubMed Google Scholar, 9Vitale M. Illario M. Di Matola T. Casamassima A. Fenzi G. Rossi G. Endocrinology. 1997; 138: 1642-1648Crossref PubMed Scopus (34) Google Scholar). In TAD-2 cells, integrin activation by FN promotes the activation of p21Ras. Ras activates the serine-threonine kinase Raf-1, which in turn activates the extracellular signal-related kinase kinase (Mek) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk). This pathway represents a prototypical model of the pathway initiated by cell adhesion that leads to cell proliferation (10Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar, 11Schlaepfer D.D. Hunter T. J. Biol. Chem. 1997; 272: 13189-13195Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Integrin activation also induces phosphatidylinositol 3-kinase (PI3-K) activation and accumulation of PI3-K products (phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate) (12King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (382) Google Scholar, 13Illario M. Amideo V. Casamassima A. Andreucci M. Di Matola T. Miele C. Rossi G. Fenzi G. Vitale M. J. Clin. Endocrinol. Metab. 2003; 88: 260-269Crossref PubMed Scopus (29) Google Scholar). Activation of PI3-K by integrins regulates phosphorylation of Raf-1 Ser 338 through the serine/threonine kinase Pak-1, providing a co-stimulatory signal that enhances Raf-1 activation by Ras and increases stimulation of cell growth by integrins (14Chaudhary A. King W.G. Mattaliano M.D. Frost J.A. Diaz B. Morrison D.K. Cobb M.H. Marshall M.S. Brugge J.S. Curr. Biol. 2000; 10: 551-554Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). After integrin activation, increase of intracellular calcium concentration [Ca2+] i has been observed upon cell attachment to ECM or binding of anti-integrin antibodies to platelets, macrophages, neutrophils, osteoclasts, and embryonic stem cells (15Jaconi M.E. Theler J.M. Schlegel W. Appel R.D. Wright S.D. Lew P.D. J. Cell Biol. 1991; 112: 1249-1257Crossref PubMed Scopus (215) Google Scholar, 16Schwartz M.A. J. Cell Biol. 1993; 120: 1003-1010Crossref PubMed Scopus (217) Google Scholar, 17Coppolino M.G. Woodside M.J. Demaurex N. Grinstein S. St-Arnaud R. Dedhar S. Nature. 1997; 386: 843-847Crossref PubMed Scopus (346) Google Scholar). Elevation of [Ca2+] i upon activation of α7β1 integrin in skeletal muscle cells results from both inositol triphosphate-evoked Ca2+ release from sarcoplasmic/endoplasmic reticulum and extracellular Ca2+ influx through voltage-gated, l-type plasma membrane Ca2+ channels (18Kwon M.S. Park C.S. Choi K. Ahnn J. Kim J.I. Eom S.H. Kaufman S.J. Song W.K. Mol. Biol. Cell. 2000; 11: 1433-1443Crossref PubMed Scopus (102) Google Scholar). [Ca2+] i mediates important components of the integrin signaling pathway by controlling cell functions, such as migration in vascular smooth muscle cells, and phagocytosis and migration in macrophages (19Bilato C. Curto K.A. Monticone R.E. Pauly R.R. White A.J. Crow M.T. J. Clin. Investig. 1997; 100: 693-704Crossref PubMed Scopus (80) Google Scholar, 20Blystone S.D. Slater S.E. Williams M.P. Crow M.T. Brown E.J. J. Cell Biol. 1999; 145: 889-897Crossref PubMed Scopus (106) Google Scholar). These Ca2+ signals are mediated by Ca2+/calmodulin protein kinase II (CaMKII), a ubiquitous serine/threonine protein kinase that is activated by Ca2+ and calmodulin (CaM) to phosphorylate diverse substrates involved in metabolism, neurotransmitter release and cell cycle control. Emerging data on potential connections between Ca2+ signaling and the MAPK pathway in multiple cell systems lead us to explore possible cross-talk between these two signal pathways upon integrin activation. Indeed, it has recently been reported that pharmacological inhibition of CaM decreased epidermal growth factor-induced MAPK activity by interfering with Raf-1 (21Tebar F. Llado A. Enrich C. FEBS Lett. 2002; 517: 206-210Crossref PubMed Scopus (37) Google Scholar). In PC-12 cells, calcium and CaM were both necessary for the activation of Erk after epidermal growth factor receptor stimulation and TrkA stimulation by nerve growth factor. The same report suggested that CaM might regulate the full activation of Raf-1 after Ras activation (22Egea J. Espinet C. Soler R.M. Peiro S. Rocamora N. Comella J.X. Mol. Cell. Biol. 2000; 20: 1931-1946Crossref PubMed Scopus (46) Google Scholar). In hippocampal neurons, activation of N-methyl-d-aspartate-type glutamate receptors induces CaMKII phosphorylation that inhibits the Ras GTPase activating protein (p135 Syn-GAP) (23Chen H.J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Therefore, Syn-GAP represents another potential intersection point between the Ca2+ signaling and the MAPK pathway. Our data demonstrate that integrin stimulation by FN, besides activating the Ras/Raf/Mek/Erk pathway, also generates a Ca2+/CaMK signal that modulates the Ras/Erk pathway through the regulation of Raf-1 activity. Cell Culture—Thyroid TAD-2 cells and the hepatoma cell line Hep3B were cultured in a 5% CO2 atmosphere at 37 °C, in Dulbecco's modified Eagle's medium (4.5 g of glucose) and 10% fetal calf serum (FCS). When needed, the cells were serum-starved in 0.5% BSA-Dulbecco's modified Eagle's medium for 12–18 h before stimulation. To obtain FN or BSA coating, cell culture plates were filled with the appropriate FN (Collaborative Research, Bedford, MA) or BSA (Sigma) dilution in PBS, incubated overnight at 4 °C and washed with PBS. Immunoprecipitation and Western Blot Analysis and Antibodies— For immunoprecipitation, the cells were lysed in radioimmunoprecipitation assay buffer (50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 150 mm NaCl, 1% 0.5% mm 5 mm mm and mm to CaMKII Raf-1 and protein MA) were used to CaMKII or Raf-1 from of were also used as a control. For Western the cells were lysed in buffer mm pH 5% 1% and and proteins were by and to a membrane Bedford, were by 5% 1% 5% FCS, and washed and incubated for h at 4 °C with primary and washed and incubated for h with a protein were by an were antibodies to MAPK, MAPK, and were from was from p21Ras activity was by affinity a Ras activation assay 4 cells were lysed with buffer mm pH mm NaCl, 5% mm 5 mm EDTA, and 10% and incubated with 5 of a of Raf-1 Ras binding peptide for at 4 The were in and proteins were by to and with a Raf-1 Ras binding and were in a Ras activation assay were a and CaMKII and cells were lysed in of buffer J.M. Mol. Endocrinol. 2000; PubMed Scopus Google with mm and of the were in of of mm pH mm mm CaM, and mm peptide CaMK activity was by mm in the activity was in the of mm (Sigma) at a concentration of was used as a for CaMKII activation. The was for at °C, and of the were as previously J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). CaM and II were a from A. R. The CaMK and the CaM inhibitors trifluoperazine and were from The CaMKII specific is from the CaMKII protein S. S. A. 1998; PubMed Scopus Google and was by of an The peptide was also used as a control. of cells by were with by the cells with Dulbecco's modified Eagle's 0.5% BSA, and mm for at 37 The cells were washed for with mm in mm NaCl, mm mm and mm pH When cells were incubated with or was with a was at and was at and were by mm and or mm EDTA, and mm The concentration of Ca2+ was by the a for G. M. J. Biol. Chem. Full Text PDF PubMed Scopus (80) Google Scholar). Raf-1, and Mek-1 activity was by a Raf-1 assay Raf-1 was from of cell The were washed and incubated in the of and for at of the was incubated with of myelin basic protein in the of The was with and proteins were through a 10% on was with the of a activity was as was by specific antibodies were washed and incubated for at °C in mm mm mm and of in the of The proteins were through a 10% and was by To Mek-1 activity, Mek-1 was by specific antibodies and incubated with phosphorylation was by Western with antibodies to antibodies were from was as of with to was on multiple Integrin Activation by FN and CaMKII of CaMK binding of and a in intracellular Ca2+ concentration To integrin activation by FN such a Ca2+ [Ca2+] i was by in TAD-2 cells in in to soluble FN binding were incubated with soluble FN in the of the binding or in the of [Ca2+] i for and in After of FN binding or were to the In the cells with FN [Ca2+] i to increase after a and to for the of the a increase by The FN receptor peptide at inhibited the [Ca2+] i peptide not on [Ca2+] i not To the FN-induced [Ca2+] i increase CaMK activity, we the of CaMKII, a CaMK expressed in but in cell is by and used as a for of activation cells were FN or BSA, and CaMKII phosphorylation was by Western at only a between BSA and FN was with the of an CaMKII by phosphorylation was in the FN-stimulated cell Activation of by cell attachment to FN was by in kinase assay II as The cells were with FN or with a that induces a [Ca2+] i FN induced a increase of CaMK activity, a with that induced by These results demonstrate that FN an intracellular Ca2+ signal for activation of of CaMKII FN-induced Erk data that Ras/Erk and Ca2+/CaMK signalings are both by To the two pathways we first the of CaMK inhibitors on the phosphorylation of Erk, the of In this we used CaMK and two CaM inhibitors and CaM is as the Ca2+ for CaMK activation. TAD-2 cells were FN with different of W7, or TFP, and the of Erk-1/2 phosphorylation was by Western 10% and FN induced a increase of Erk-1/2 phosphorylation. was inhibitory at and FN-induced Erk-1/2 phosphorylation at 5 In and inhibitory To CaMK the II is for FN-stimulated Erk the were with the specific ant-CaNtide. This peptide is from the CaMKII and was cell by the inhibited FN-induced Erk-1/2 phosphorylation at a 5 The peptide inhibitory these data demonstrate that Erk phosphorylation is on CaMKII activation. and are for FN-stimulated cell proliferation and to of CaMKII for Erk phosphorylation by FN in Hep3B the by CaMK in the regulation of the and in the of cell growth is to the thyroid TAD-2 cells or is a we the of FN on and in an cell Erk-1/2 phosphorylation was by Western in Hep3B cells FN or BSA in of serum or in plates with 10% in TAD-2 cells, FN and Erk-1/2 a the of on CaMKII activation was in cells by FN and CaMKII phosphorylation at was by Western CaMKII was in both and the same of activated kinase was such stimulation the used in To CaMKII activity is necessary for Erk phosphorylation in the cells were FN with or W7, TFP, or and the of Erk-1/2 phosphorylation was by Western FN-stimulated Erk phosphorylation was inhibited by the CaMK the CaMKII or the CaM inhibitors and in as it was in TAD-2 These results demonstrate that in Hep3B cells, as in TAD-2 cells, CaMKII modulates the signaling pathway by integrin activation is not for CaMKII activation. also demonstrate that CaMKII activity is but not for Erk activation in both and in TAD-2 CaMK Akt Ras/Erk pathway activation, kinase is phosphorylated by PI3-K after integrin activation. To inhibition of CaMK modulates this pathway, TAD-2 cells were with FN in the of Akt phosphorylation in was not affected by the of the CaMK Modulation of the Erk Activation by CaMK of results that CaMK activity is for the FN-induced in two different cell we CaMK was for p21Ras activation by The activity of p21Ras was in from TAD-2 cells by FN with or and Cell were incubated with Raf-1 Ras binding to the binding to activated and was and by Western and integrin activated p21Ras. The CaMK and the CaM inhibitors and were on the of Ras activation by FN, that the CaMK in the pathway of CaMKII Raf-1 the CaMK and MAPK pathways of Erk and of this Raf-1 and as possible for CaMK We CaMKII was for FN-induced activation of Raf-1 in both TAD-2 and Hep3B cells Raf-1 activity was by a Raf-1 inositol kinase assay in from cells on BSA or FN or in the of or ant-CaNtide. FN stimulation induced a increase Raf-1 activity, an inhibited by the with or that Raf-1 activation CaMKII activity. To phosphorylation or a phosphorylation by CaMKII, was in the assay with and in In the of Mek, was not and the CaMKII not phosphorylation by FN-stimulated cell not CaMK on Raf-1, and were to a of CaMK inhibitors on Raf-1, and Raf-1, and Mek-1 were by specific antibodies from FN-stimulated and were in the assay with or with Raf-1 in the Raf-1 inositol kinase assay The of Mek-1 to phosphorylate in the of CaMK inhibitors was by Western that CaMK inhibitors on Raf-1, and FN Raf-1/CaMKII generates protein To CaMKII might associated with Raf-1 in a protein complex, CaMKII was in from cells on BSA or CaMKII and Raf-1 were by Western by specific antibodies In the of FN of CaMKII and Raf-1 was not in TAD-2 cells, it was in Hep3B In both cell lines, Raf-1 and CaMKII were after adhesion to The CaMKII inhibited both the and the FN-induced Raf-1/CaMKII These results that CaMKII, activated or activated by in the formation of a protein with Raf-1, or Raf-1 by CaMKII in CaMKII phosphorylate Raf-1, the was from TAD-2 cells and incubated in with CaMKII and or were in the and phosphorylated Raf-1 was by and by Raf-1 was phosphorylated by CaMKII and inhibited Raf-1 was the of CaMKII the the CaMKII binding for CaMKII it is on CaMKII. These data demonstrate that CaMKII phosphorylate Raf-1 in vitro. Our results demonstrate that activation of the integrin receptors by FN binding stimulates the Ras/Erk pathway and that this pathway is by Ca2+ signaling through the regulation of Raf-1 activity We demonstrated previously that FN binding to integrins induces and phosphorylation with the formation of the in the thyroid cell line This pathway mediates the cell growth as inhibition of Ras or FN-induced proliferation M. Amideo V. Casamassima A. Andreucci M. Di Matola T. Miele C. Rossi G. Fenzi G. Vitale M. J. Clin. Endocrinol. Metab. 2003; 88: 260-269Crossref PubMed Scopus (29) Google Scholar). suggested the existence of a between Ca2+ signaling and MAPK pathway at multiple and with possible these signals by integrin activation in cell Therefore, we to Ca2+ signaling and the Ras/Erk pathway and regulate cell To a Ca2+ signaling is by integrin activation in this cell we [Ca2+] i upon soluble FN [Ca2+] i increase is and upon in [Ca2+] i increase was and These results are in with the data in cells M.A. J. Cell Biol. 1993; 120: 1003-1010Crossref PubMed Scopus (217) Google Scholar). Integrins not kinase activity, and ECM binding leads to the The of integrins the by FN leads to the activation of and generates signals that a [Ca2+] i The of the [Ca2+] i upon the cell and cell in cells, of to in neutrophils, and in platelets, but on cell growth been reported M.A. J. Cell Biol. 1993; 120: 1003-1010Crossref PubMed Scopus (217) Google Scholar, M.T. S.D. J. Biol. 2002; Google Scholar, M.A. T. 1997; PubMed Google Scholar). in [Ca2+] i are by CaM, a ubiquitous Ca2+ binding protein that to and regulates such as In study, CaMKII was activated upon binding in TAD-2 cells, in Hep3B cells, CaMKII to phosphorylated in and FN to increase phosphorylation. The that the CaMKII phosphorylation in Hep3B cells were not in this we observed in primary cultures of and thyroid cells that activation of CaMKII integrin only in A. L. and M. of CaMKII not been and the of activating signals is a possible for the CaMKII phosphorylation in CaMKII is not to Erk-1/2 activation, as by the that Erk-1/2 results in phosphorylation only upon integrin activation in Hep3B In both TAD-2 and Hep3B cells, inhibition of CaMKII activity by the CaMK the CaM inhibitors and TFP, and the inhibitory peptide demonstrated that CaMKII activity is for Erk-1/2 phosphorylation induced by for a pathway in the activation of Erk-1/2 has been in a of and CaM are both necessary for Erk-1/2 stimulation by in for the activation of Erk after nerve growth factor stimulation in PC-12 it has been reported that CaM regulates at multiple the activation of Erk by stimulation of the epidermal growth factor that demonstrate CaMKII for Erk activity are (21Tebar F. Llado A. Enrich C. FEBS Lett. 2002; 517: 206-210Crossref PubMed Scopus (37) Google Scholar, J. Espinet C. Soler R.M. Peiro S. Rocamora N. Comella J.X. Mol. Cell. 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Cell Biol. 1999; 145: 889-897Crossref PubMed Scopus (106) Google Scholar, 1998; PubMed Scopus (46) Google Scholar). evidence that cell proliferation induced by integrins the activation of the Ca2+/CaMK pathway has not been In study, integrin activation induced CaMKII phosphorylation and with Raf-1 in thyroid In Hep3B cells, CaMKII phosphorylation not FN CaMKII and Raf-1 were associated in In both cell CaMK inhibitors and inhibited Raf-1 activity. a Raf-1 phosphorylation by CaMKII has not been the existence of for CaMKII Raf-1 phosphorylation by CaMKII a of S.D. Huang J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Indeed, in demonstrate that Raf-1 phosphorylated by CaMKII. this not demonstrate that Raf-1 is a CaMKII in it this Raf-1 is a of protein kinase protein kinase and with provide co-stimulatory signals that lead to Erk activation in to receptor signals D.K. R.E. Curr. Opin. Cell Biol. 1997; 9: PubMed Scopus Google Scholar). 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In data demonstrate CaMKII is activated after integrin CaMKII regulates Ras-stimulated Raf-1 activity to Erk-1/2 activation in the integrin and CaMKII is necessary for Erk activation. The of Raf-1 activity by CaMKII might not to integrin signaling but a and in the cross-talk between intracellular signaling of to the of Ca2+ and CaMKII in other signaling pathways that Raf-1 as a factor. We A. R. for of the and for providing us with CaM and R. for with and F. and M. for in the of the