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• W2131993341 abstract "We have shown previously (Villalonga, P., López- Alcalá, C., Bosch, M., Chiloeches, A., Rocamora, N., Gil, J., Marais, R., Marshall, C. J., Bachs, O., and Agell, N. (2001) Mol. Cell. Biol. 21, 7345–7354) that calmodulin negatively regulates Ras activation in fibroblasts. Hence, anti-calmodulin drugs (such as W13, trifluoroperazine, or W7) are able to induce Ras/ERK pathway activation under low levels of growth factors. We show here that cell treatment with protein kinase C (PKC) inhibitors abolishes W13-induced activation of Ras, Raf-1, and ERK. Consequently, PKC activity is essential for achieving the synergism between calmodulin inhibition and growth factors to activate Ras. Furthermore, whereas the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) does not induce Ras activation in 3T3 cells, activation is observed if calmodulin is simultaneously inhibited. This indicates that calmodulin is preventing Ras activation by PKC. Treatment of cells with epidermal growth factor receptor or platelet-derived growth factor receptor tyrosine kinase inhibitors does not abrogate the activation of Ras by calmodulin inhibition. This implies that epidermal growth factor receptor and platelet-derived growth factor receptor tyrosine kinase activities are dispensable for the activation of Ras by TPA plus W13, and, therefore, Ras activation is not a consequence of the transactivation of those receptors by the combination of the anti-calmodulin drug plus TPA. Furthermore, K-Ras, the isoform previously shown to bind to calmodulin, is the only one activated by TPA when calmodulin is inhibited. These data suggest that direct interaction between K-Ras and calmodulin may account for the inability of PKC to activate Ras in 3T3 fibroblasts.In vitro experiments showed that the phosphorylation of K-Ras by PKC was inhibited by calmodulin, suggesting that calmodulin-dependent modulation of K-Ras phosphorylation by PKC could be the mechanism underlying K-Ras activation in fibroblasts treated with TPA plus W13. We have shown previously (Villalonga, P., López- Alcalá, C., Bosch, M., Chiloeches, A., Rocamora, N., Gil, J., Marais, R., Marshall, C. J., Bachs, O., and Agell, N. (2001) Mol. Cell. Biol. 21, 7345–7354) that calmodulin negatively regulates Ras activation in fibroblasts. Hence, anti-calmodulin drugs (such as W13, trifluoroperazine, or W7) are able to induce Ras/ERK pathway activation under low levels of growth factors. We show here that cell treatment with protein kinase C (PKC) inhibitors abolishes W13-induced activation of Ras, Raf-1, and ERK. Consequently, PKC activity is essential for achieving the synergism between calmodulin inhibition and growth factors to activate Ras. Furthermore, whereas the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) does not induce Ras activation in 3T3 cells, activation is observed if calmodulin is simultaneously inhibited. This indicates that calmodulin is preventing Ras activation by PKC. Treatment of cells with epidermal growth factor receptor or platelet-derived growth factor receptor tyrosine kinase inhibitors does not abrogate the activation of Ras by calmodulin inhibition. This implies that epidermal growth factor receptor and platelet-derived growth factor receptor tyrosine kinase activities are dispensable for the activation of Ras by TPA plus W13, and, therefore, Ras activation is not a consequence of the transactivation of those receptors by the combination of the anti-calmodulin drug plus TPA. Furthermore, K-Ras, the isoform previously shown to bind to calmodulin, is the only one activated by TPA when calmodulin is inhibited. These data suggest that direct interaction between K-Ras and calmodulin may account for the inability of PKC to activate Ras in 3T3 fibroblasts.In vitro experiments showed that the phosphorylation of K-Ras by PKC was inhibited by calmodulin, suggesting that calmodulin-dependent modulation of K-Ras phosphorylation by PKC could be the mechanism underlying K-Ras activation in fibroblasts treated with TPA plus W13. extracellular signal-regulated kinase guanine nucleotide exchange factor GTPase-activating protein protein kinase C calmodulin mitogen-activated protein kinase/extracellular signal-regulated kinase epidermal growth factor EGF receptor CaM-binding protein fetal bovine serum platelet-derived growth factor PDGF receptor 12-O-tetradecanoylphorbol-13-acetate Tris-buffered saline Ras-binding domain of Raf-1 myelin basic protein fetal calf serum glutathione-S-transferase The three members of the Ras family of small GTPases, H-, N-, and K-Ras, are key regulators of signal transduction pathways that control cell proliferation, differentiation, survival, and apoptosis (1Downward J. Curr. Opin. Genet. Dev. 1998; 8: 49-54Crossref PubMed Scopus (506) Google Scholar, 2Khosravi-Far R. Campbell S. Rossman K.L. Der C.J. Adv. Cancer Res. 1998; 72: 57-107Crossref PubMed Google Scholar, 3Rebollo A. Martinez A. Blood. 1999; 94: 2971-2980Crossref PubMed Google Scholar). The molecular basis for such a great variety of cell responses controlled by Ras proteins relies on the fact that Ras is able to transduce signals from different extracellular stimuli, including growth factors, hormones, and cell-extracellular matrix contacts to many downstream effectors (4Katz M.E. McCormick F. Curr. Opin. Genet. Dev. 1997; 7: 75-79Crossref PubMed Scopus (274) Google Scholar, 5Olson M.F. Marais R. Semin. Immunol. 2000; 12: 63-73Crossref PubMed Scopus (87) Google Scholar). As a molecular switch, Ras cycles between a GTP-bound active and an inactive state when GTP is hydrolyzed to GDP. Active Ras interacts with and modulates the activity of effector proteins. The best characterized Ras effector is the serine/threonine kinase Raf, which leads to the activation of the extracellular signal-regulated kinase (ERK)1 pathway that plays a major role in cell proliferation and differentiation (6Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 7Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar, 8Marshall C. Curr. Opin. Cell Biol. 1999; 11: 732-736Crossref PubMed Scopus (141) Google Scholar). Other effectors for Ras include the lipid kinase phosphatidylinositol-3-kinase (PI3K), which is involved in cell survival, proliferation, and metabolism (9Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1716) Google Scholar, 10Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (966) Google Scholar, 11Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar), and the nucleotide exchange factors for Ral GTPase, RalGDS, Rlf, and Rlg (12Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (471) Google Scholar).Many molecules have been described as influencing the Ras GTP/GDP cycle, mainly through two distinct biochemical activities: 1) the guanine nucleotide exchange factors (GEFs) that regulate the replacement of the nucleotide bound to Ras, favoring the GTP-bound active state; or 2) the GTPase-activating proteins (GAPs), which increase Ras low intrinsic GTPase activity and thereby promote its inactivation. Following extracellular stimulation, GEFs are recruited to the plasma membrane through binding to a set of molecular adaptors, inducing transiently Ras-GTP complexes (13Downward J. Curr. Opin. Genet. Dev. 1992; 2: 13-18Crossref PubMed Scopus (48) Google Scholar, 14Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1751) Google Scholar). Distinct signals such as those transduced from tyrosine kinase receptors, G protein-coupled receptors, or integrin-induced cell attachment to the extracellular matrix all lead to Ras activation. In many cases this is achieved through the membrane recruitment of the Sos exchange factor, which in turn depends on the adaptor protein Grb-2. Autophosphorylation of tyrosine kinase receptors or activation of a variety of non-receptor tyrosine kinases such as Src, Pyk-2, or focal adhesion kinase in response to the activation of diverse G protein-coupled receptors or cell-extracellular matrix engagement all create phosphotyrosine residues that allow Grb-2/Sos binding and thus its recruitment to the vicinity of Ras at the plasma membrane, leading to its activation (15Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar,16Howe A. Aplin A.E. Alahari S.K. Juliano R.L. Curr. Opin. Cell Biol. 1998; 10: 220-231Crossref PubMed Scopus (582) Google Scholar).In addition, other mechanisms are able to link extracellular signals to Ras activation, such as phospholipase C activation and its consequent increase in diacylglycerol and intracellular Ca2+. Ca2+ induces the activation of RasGRF, another Ras guanine nucleotide exchange factor present mainly in neurones that lead to Ras activation (17Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (390) Google Scholar). In addition, together with diacylglycerol, Ca2+ is also able to activate RasGRPs, a RasGEF family present in lymphocytes and neurones (18Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (545) Google Scholar). In turn, protein kinase C (PKC) activation by diacylglycerol is also able to activate Ras in some cellular types. The activation of Ras through PKC-dependent GAP inhibition has been reported in T lymphocytes upon T cell receptor activation (19Downward J. Graves J.D. Warne P.H. Rayter S. Cantrell D.A. Nature. 1990; 346: 719-723Crossref PubMed Scopus (683) Google Scholar). However, this seems not to be the case for fibroblasts (20Burgering B.M. de Vries-Smits A.M. Medema R.H. van Weeren P.C. Tertoolen L.G. Bos J.L. Mol. Cell. Biol. 1993; 13: 7248-7256Crossref PubMed Scopus (151) Google Scholar), although PKC is able to induce Ras activation in B lymphocytes and COS cells (21Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (398) Google Scholar). GAP activity can also be regulated by binding to phosphorylated tyrosine kinase receptors (22Cleghon V. Gayko U. Copeland T.D. Perkins L.A. Perrimon N. Morrison D.K. Genes Dev. 1996; 10: 566-577Crossref PubMed Scopus (42) Google Scholar). In summary, a highly interconnected network of multiple pathways regulates Ras activation in response to extracellular signals.The Ca2+-binding protein calmodulin (CaM), which acts as a second messenger in cellular signal transduction pathways and regulates cell proliferation (23Klee C. Vanaman T. Adv. Protein Chem. 1982; 35: 213-321Crossref PubMed Scopus (733) Google Scholar, 24Schulman H. Curr. Opin. Cell Biol. 1993; 5: 247-253Crossref PubMed Scopus (173) Google Scholar, 25Lu K.P. Means A.R. Endocr. Rev. 1993; 14: 40-58Crossref PubMed Scopus (269) Google Scholar, 26Herget T. Broad S. Rozengurt E. Eur. J. Biochem. 1994; 225: 549-556Crossref PubMed Scopus (23) Google Scholar), has been shown to be one of the molecules involved in the modulation of Ras activity. We have shown that CaM down-regulates the Ras/Raf/MEK/ERK pathway in fibroblasts (27Bosch M. Gil J. Bachs O. Agell N. J. Biol. Chem. 1998; 273: 22145-22150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 28Villalonga P. López-Alcalá C. Bosch M. Chiloeches A. Rocamora N. Gil J. Marais R. Marshall C.J. Bachs O. Agell N. Mol. Cell. Biol. 2001; 21: 7345-7354Crossref PubMed Scopus (162) Google Scholar), and other groups have shown that CaM is able to inhibit in vitroEGFR (29San Jose E. Benguria A. Geller P. Villalobo A. J. Biol. Chem. 1992; 267: 15237-15245Abstract Full Text PDF PubMed Google Scholar) activity and the shedding of EGF-like growth factors (30Diaz-Rodriguez E. Esparis-Ogando A. Montero J.C. Yuste L. Pandiella A. Biochem. J. 2000; 346: 359-367Crossref PubMed Scopus (56) Google Scholar). Although low doses of growth factors are not able to induce activation of the Ras/Raf/MEK/ERK pathway in fibroblasts, activation is observed if CaM is simultaneously inhibited, indicating that CaM is preventing the activation of this pathway under basal conditions. Furthermore, CaM inhibition in fibroblasts leads to a more sustained level of ERK activation following serum stimulation. CaM functions are mediated by its association with specific target proteins named CaM-binding proteins (CaMBPs), which are regulated upon CaM binding (31Bachs O. Agell N. Carafoli E. Cell Calcium. 1994; 16: 289-296Crossref PubMed Scopus (75) Google Scholar, 32Agell N. Aligue R. Alemany V. Castro A. Jaime M. Pujol M.J. Rius E. Serratosa J. Taules M. Bachs O. Cell Calcium. 1998; 23: 115-121Crossref PubMed Scopus (49) Google Scholar). We have recently demonstrated that K-RasB is a CaMBP and that this Ras isoform is specifically activated when serum-starved NIH3T3 cells are treated with a CaM inhibitor, suggesting a direct relation between CaM binding to Ras and Ras inhibition by CaM (28Villalonga P. López-Alcalá C. Bosch M. Chiloeches A. Rocamora N. Gil J. Marais R. Marshall C.J. Bachs O. Agell N. Mol. Cell. Biol. 2001; 21: 7345-7354Crossref PubMed Scopus (162) Google Scholar). However, the activation of Ras by CaM inhibition is low in the absence of growth factors, increasing dramatically in the presence of low levels of mitogens. We have analyzed here the intracellular elements that are cooperating with CaM inhibition to induce Ras activation. Our results show that PKC activity is responsible for this synergism and that CaM is preventing the activation of Ras by PKC in 3T3 fibroblasts.DISCUSSIONAn emerging theme in signal transduction is the fact that the activation of a pathway per se is not enough to explain its biological effects. To understand these effects, it is crucial to achieve a comprehensive knowledge of the spatiotemporal pattern of pathway activation. A paradigm for this is the activation of the Ras/ERK pathway, which leads to cell proliferation, cell cycle arrest, or differentiation depending on the timing and intensity of its activation (40Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar, 41Roovers K. Assoian R.K. Bioessays. 2000; 22: 818-826Crossref PubMed Scopus (423) Google Scholar). Although the molecular mechanisms involved in Ras activation have been intensively studied over the past decade, it is not yet completely understood which mechanisms govern Ras inactivation and, hence, its global signaling output in response to given stimuli. We have previously shown that CaM is essential for the impairment of Ras activation at low concentrations of growth factors in fibroblasts. Furthermore, CaM is important to ensure an appropriate signaling level of the ERK cascade in these cells, as CaM inhibition prior to mitogenic stimulation leads to a more sustained ERK activation and to accumulation of the CDK inhibitor p21cip1 (27Bosch M. Gil J. Bachs O. Agell N. J. Biol. Chem. 1998; 273: 22145-22150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). CaM is able to specifically impair K-Ras activation but not the activation of the other Ras isoforms, and this is most probably through its direct binding to GTP-bound K-RasB (28Villalonga P. López-Alcalá C. Bosch M. Chiloeches A. Rocamora N. Gil J. Marais R. Marshall C.J. Bachs O. Agell N. Mol. Cell. Biol. 2001; 21: 7345-7354Crossref PubMed Scopus (162) Google Scholar). To gain insight into the mechanism of how CaM is able to down-regulate the Ras/ERK pathway, we have analyzed the nature of the signals that are essential to induce the activation of this pathway in cooperation with CaM inhibition.As we had shown previously, low doses of serum, EGF, PDGF, and bombesin were all able to induce ERK activation in the presence of CaM inhibitors (28Villalonga P. López-Alcalá C. Bosch M. Chiloeches A. Rocamora N. Gil J. Marais R. Marshall C.J. Bachs O. Agell N. Mol. Cell. Biol. 2001; 21: 7345-7354Crossref PubMed Scopus (162) Google Scholar). These results suggested that an intracellular regulator of the Ras/ERK pathway, activated by all the above stimuli, was able to activate the Ras/ERK pathway when CaM was inhibited. Protein kinase C has been shown to be a regulator of the Ras/ERK signaling pathway that is able to regulate different levels of this pathway. In many cell types, conventional PKC activation by phorbol ester treatment induces a potent activation of ERK, but there are multiple upstream inputs of PKC in this pathway regarding the cell type. For instance, examples of the activation of Ras, Raf, or MEK by PKC have all been described using different cell systems (20Burgering B.M. de Vries-Smits A.M. Medema R.H. van Weeren P.C. Tertoolen L.G. Bos J.L. Mol. Cell. Biol. 1993; 13: 7248-7256Crossref PubMed Scopus (151) Google Scholar, 21Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (398) Google Scholar, 42Schonwasser D.C. Marais R.M. Marshall C.J. Parker P.J. Mol. Cell. Biol. 1998; 18: 790-798Crossref PubMed Scopus (674) Google Scholar,43Nakafuku M. Satoh T. Kaziro Y. J. Biol. Chem. 1992; 267: 19448-19454Abstract Full Text PDF PubMed Google Scholar). Therefore, we tested whether PKC was involved in the activation of ERK that CaM inhibitors were able to induce in the presence of low concentrations of growth factors in both NIH3T3 and Swiss 3T3 cells. Incubation with the broad spectrum PKC inhibitor GF109203X blocked ERK activation induced by W13 in Swiss 3T3 cells. Similarly, in NIH3T3 cells treated with either GF109203X, Ro-0432, or, chronically, TPA, the activation of ERK by W13 was also blocked. These results suggested that PKC was necessary for the observed activation of ERK under our experimental conditions.CaM most probably regulates the Ras/ERK pathway at the level of Ras, because we have shown that CaM binds specifically to GTP-bound K-RasB and that W13 treatment ultimately leads to K-Ras activation. However, the requirement for PKC activity to observe ERK activation in response to CaM inhibition could merely reflect the ability of PKC to activate different levels of this pathway downstream of Ras. To address this issue, we analyzed the effects of PKC inhibition on the activation of Raf-1 and Ras by W13 treatment in NIH3T3. As we had shown previously (27Bosch M. Gil J. Bachs O. Agell N. J. Biol. Chem. 1998; 273: 22145-22150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), treatment with W13 induces Raf-1 kinase activity in serum-starved NIH3T3 cells. Interestingly, PKC inhibition completely prevented W13-induced activation of Raf-1. These results indicated that the PKC-dependent step in W13-induced ERK activation was at least at the level of Raf-1. We finally tested whether Ras activation by W13 was also PKC-dependent. PKC inhibition in serum-starved NIH3T3 completely prevented the activation of Ras by W13 under both 0 and 0.5% FCS. This indicated that PKC was necessary for CaM inhibition to induce Ras activation, thereby placing PKC-dependence at the level of Ras.PKC activity was therefore necessary for the observed activation of the Ras/ERK pathway in response to CaM inhibition under low concentrations of growth factors. Altogether, these results suggested that CaM was preventing Ras activation by PKC in these cells. Thus, under CaM-inhibited conditions, different sources of PKC activity such as the growth factors tested to date were able to induce Ras activation. We therefore analyzed whether PKC activation by TPA treatment alone instead of growth factor treatment was able to cooperate with CaM inhibitors to induce Ras activation in NIH3T3 cells. Interestingly, in these cells PKC activation by TPA is not able to promote Ras activation as measured by the “classic” nucleotide labeling method, although it induces potently ERK activation (20Burgering B.M. de Vries-Smits A.M. Medema R.H. van Weeren P.C. Tertoolen L.G. Bos J.L. Mol. Cell. Biol. 1993; 13: 7248-7256Crossref PubMed Scopus (151) Google Scholar). Using the RBD pull-down method, we confirmed that TPA was unable to induce Ras activation in NIH3T3 cells. The inability of PKC activity alone to promote Ras activation ruled out the possibility that the effect of W13 was to induce PKC activation and, consequently, activation of the Ras/ERK pathway. In sharp contrast, TPA treatment in a CaM-inhibited background induced a robust activation of Ras at levels comparable with those achieved by PDGF treatment. These results suggested that CaM was essential in preventing the activation of Ras by PKC in these cells. Regarding the physiological role of this effect of CaM on Ras activation by PKC, this would be relevant in circumstances wherein cells need to keep PKC active but Ras should be down-regulated. For instance, after proliferative activation of the cells, PKC activity is more sustained than that of Ras. We hypothesize that CaM is essential for turning off Ras in an environment wherein PKC activity is high, such as in mitogenically stimulated fibroblasts. If PKC activity was not uncoupled to Ras activation in these cells, an excessively sustained ERK activity could lead to cell cycle arrest through p21cip1 up-regulation.It has been reported that CaM inhibitors can induce cleavage of membrane-bound proteins (30Diaz-Rodriguez E. Esparis-Ogando A. Montero J.C. Yuste L. Pandiella A. Biochem. J. 2000; 346: 359-367Crossref PubMed Scopus (56) Google Scholar), a process that is also influenced by PKC (37Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (474) Google Scholar, 38Schlondorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar, 44Pandiella A. Bosenberg M.W. Huang E.J. Besmer P. Massague J. Biol. Chem. 1992; 267: 24028-24033Abstract Full Text PDF PubMed Google Scholar), leading to the possibility that TPA, together with W13, was inducing Ras/ERK activation via the shedding of membrane-bound, pro-growth factors. The inhibition of both the EGF and PDGF receptor tyrosine kinases demonstrated that Ras activation by TPA together with W13 was independent of the kinase activity of these tyrosine kinase receptors and thus independent of their extracellular stimulation or intracellular transactivation by a PKC-dependent mechanism.As we had shown previously, CaM interacts specifically with K-Ras and inhibits its activation (28Villalonga P. López-Alcalá C. Bosch M. Chiloeches A. Rocamora N. Gil J. Marais R. Marshall C.J. Bachs O. Agell N. Mol. Cell. Biol. 2001; 21: 7345-7354Crossref PubMed Scopus (162) Google Scholar). We therefore investigated whether PKC activation, together with CaM inhibition, was able to differentially activate Ras isoforms. As expected, TPA plus W13 induced K-Ras activation nicely but did not induce H- or N-Ras activation. This indicated that the interplay between CaM and PKC in regulating Ras at the molecular level was highly specific, as it was restricted only to K-Ras (consistent with our previous observations). Two models for the possible cooperation between CaM inhibition and PKC activity in K-Ras activation that are in concordance with the results presented up to now are shown in Fig. 7.The mechanistic links lying between PKC and Ras still remain to be characterized in most of the cells types in which Ras is activated by PKC, as this has only been elucidated in T lymphocytes. In these cells, TPA stimulation leads to a very potent activation of Ras, and this has been shown to rely on GAP inhibition (19Downward J. Graves J.D. Warne P.H. Rayter S. Cantrell D.A. Nature. 1990; 346: 719-723Crossref PubMed Scopus (683) Google Scholar). It should be noted, however, that lymphocytes express RasGRPs, a novel class of nucleotide exchange factors that are regulated by diacylglycerol, and will probably cooperate in the activation of Ras by phorbol esters in lymphocytes (45Genot E. Cantrell D.A. Curr. Opin. Immunol. 2000; 12: 289-294Crossref PubMed Scopus (145) Google Scholar). Although there are a few studies of other cell types that suggest that PKC induces GAP inhibition (46Schubert C. Carel K. DePaolo D. Leitner W. Draznin B. J. Biol. Chem. 1996; 271: 15311-15314Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 47Choudhury S. Krishna M. Bhattacharya R.K. Cancer Lett. 1996; 109: 149-154Crossref PubMed Scopus (10) Google Scholar, 48Nori M. L'Allemain G. Weber M.J. Mol. Cell. Biol. 1992; 12: 936-945Crossref PubMed Scopus (66) Google Scholar), there is still no compelling evidence to propose a general role for GAP inhibition in Ras activation by PKC other than in T lymphocytes. Moreover, there is very little information regarding isoform specificity in Ras regulation by PKC, as this has only been investigated in COS cells. In these cells, TPA induces H-, N-, and K-Ras activation (21Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (398) Google Scholar), highlighting the mechanistic differences that must exist between COS and NIH3T3 cells. In NIH3T3 cells, it has been proposed that TPA induces Ras/ERK pathway activation upstream of Shc phosphorylation and its association with Grb-2 (49El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, these conclusions are made on the basis of the use of dominant negative Ras and Sos proteins, but no measurements of Ras activation are shown. We have clearly demonstrated that TPA does not induce Ras activation in NIH3T3 cells, and the use of RasN17 has proven not to be a reliable tool when studying Ras involvement in a signaling pathway (21Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (398) Google Scholar, 50Stewart S. Guan K.L. J. Biol. Chem. 2000; 275: 8854-8862Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).A strikingly direct link between PKC and K-RasB but not the other Ras isoforms was shown several years ago when PKC induced the direct phosphorylation of K-RasB (51Ballester R. Furth M.E. Rosen O.M. J. Biol. Chem. 1987; 262: 2688-2695Abstract Full Text PDF PubMed Google Scholar). Our results showed that this phosphorylation could be inhibited in vitro by CaM. Consequently, we favor the hypothesis that PKC phosphorylation of K-Ras is somehow activating its downstream signaling and that this effect would be prevented by CaM binding to K-Ras (Fig.8 b). However, the physiological consequences of this phosphorylation are not yet clear, and further work is needed to prove this hypothesis. The three members of the Ras family of small GTPases, H-, N-, and K-Ras, are key regulators of signal transduction pathways that control cell proliferation, differentiation, survival, and apoptosis (1Downward J. Curr. Opin. Genet. Dev. 1998; 8: 49-54Crossref PubMed Scopus (506) Google Scholar, 2Khosravi-Far R. Campbell S. Rossman K.L. Der C.J. Adv. Cancer Res. 1998; 72: 57-107Crossref PubMed Google Scholar, 3Rebollo A. Martinez A. Blood. 1999; 94: 2971-2980Crossref PubMed Google Scholar). The molecular basis for such a great variety of cell responses controlled by Ras proteins relies on the fact that Ras is able to transduce signals from different extracellular stimuli, including growth factors, hormones, and cell-extracellular matrix contacts to many downstream effectors (4Katz M.E. McCormick F. Curr. Opin. Genet. Dev. 1997; 7: 75-79Crossref PubMed Scopus (274) Google Scholar, 5Olson M.F. Marais R. Semin. Immunol. 2000; 12: 63-73Crossref PubMed Scopus (87) Google Scholar). As a molecular switch, Ras cycles between a GTP-bound active and an inactive state when GTP is hydrolyzed to GDP. Active Ras interacts with and modulates the activity of effector proteins. The best characterized Ras effector is the serine/threonine kinase Raf, which leads to the activation of the extracellular signal-regulated kinase (ERK)1 pathway that plays a major role in cell proliferation and differentiation (6Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 7Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar, 8Marshall C. Curr. Opin. Cell Biol. 1999; 11: 732-736Crossref PubMed Scopus (141) Google Scholar). Other effectors for Ras include the lipid kinase phosphatidylinositol-3-kinase (PI3K), which is involved in cell survival, proliferation, and metabolism (9Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1716) Google Scholar, 10Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (966) Google Scholar, 11Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar), and the nucleotide exchange factors for Ral GTPase, RalGDS, Rlf, and Rlg (12Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (471) Google Scholar). Many molecules have been described as influencing the Ras GTP/GDP cycle, mainly through two distinct biochemical activities: 1) the guanine nucleotide exchange factors (GEFs) that regulate the replacement of the nucleotide bound to Ras, favoring the GTP-bound active state; or 2) the GTPase-activating proteins (GAPs), which increase Ras low intrinsic GTPase activity and thereby promote its inactivation. Following extracellular stimulation, GEFs are recruited to the plasma membrane through binding t" @default.
• W2131993341 created "2016-06-24" @default.
• W2131993341 creator A5008409533 @default.
• W2131993341 creator A5010711016 @default.
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• W2131993341 date "2002-10-01" @default.
• W2131993341 modified "2023-09-29" @default.
• W2131993341 title "Calmodulin Prevents Activation of Ras by PKC in 3T3 Fibroblasts" @default.
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