Negative Modulation of Membrane Localization of the Raf-1 Protein Kinase by Hyperphosphorylation
1997; Elsevier BV; Volume: 272; Issue: 7 Linguagem: Inglês
10.1074/jbc.272.7.3915
ISSN1083-351X
AutoresMarkus Wartmann, Paul Hofer, P Turowski, Alan R. Saltiel, Nancy E. Hynes,
Tópico(s)Cellular Mechanics and Interactions
ResumoThe serine/threonine-specific protein kinase Raf-1 plays a key role in mitogenic signal transduction by coupling Ras to the mitogen-activated protein (MAP) kinase cascade. Ras-mediated translocation to the plasma membrane represents a crucial step in the process of serum-stimulated Raf-1 kinase activation. The exact role of the multisite phosphorylation in Raf regulation, however, is not clear. We have previously reported that the mobility shift-associated hyperphosphorylation of Raf correlates with a reduction of serum-stimulated Raf kinase activity (Wartmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701).Here we show that incubation of serum-starved CHO cells with D609, a purported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-1 that is due to hyperphosphorylation on sites identical to those observed following mitogen stimulation. Subcellular fractionation analyses revealed that D609-induced mobility shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-1. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibitor of activation of the MAP kinase kinase MEK, prevented D609-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels. Taken together, these data suggest that mobility shift-associated hyperphosphorylation of Raf-1, by virtue of reducing the amount of plasma membrane-bound Raf-1, represents a negative feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade. The serine/threonine-specific protein kinase Raf-1 plays a key role in mitogenic signal transduction by coupling Ras to the mitogen-activated protein (MAP) kinase cascade. Ras-mediated translocation to the plasma membrane represents a crucial step in the process of serum-stimulated Raf-1 kinase activation. The exact role of the multisite phosphorylation in Raf regulation, however, is not clear. We have previously reported that the mobility shift-associated hyperphosphorylation of Raf correlates with a reduction of serum-stimulated Raf kinase activity (Wartmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701). Here we show that incubation of serum-starved CHO cells with D609, a purported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-1 that is due to hyperphosphorylation on sites identical to those observed following mitogen stimulation. Subcellular fractionation analyses revealed that D609-induced mobility shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-1. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibitor of activation of the MAP kinase kinase MEK, prevented D609-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels. Taken together, these data suggest that mobility shift-associated hyperphosphorylation of Raf-1, by virtue of reducing the amount of plasma membrane-bound Raf-1, represents a negative feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade. INTRODUCTIONRaf-1 is a ubiquitously expressed serine/threonine protein kinase that assumes a critical role in relaying proliferative and developmental signals initiated by cell-surface receptors to the nucleus (1Daum G. Eisenmann Tappe I. Fries H.W. Troppmair J. Rapp U.R. Trends. Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar, 2Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (897) Google Scholar, 3Rapp U.R. Oncogene. 1991; 6: 495-500PubMed Google Scholar, 4Li P. Wood K. Mamon H. Haser W. Roberts T. Cell. 1991; 64: 479-482Abstract Full Text PDF PubMed Scopus (112) Google Scholar). Genetic studies in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster as well as biochemical studies in vertebrate cells have elucidated that Raf-1 couples Ras to the MAP 1The abbreviations used are: MAPmitogen-activated proteinCHOChinese hamster ovaryERKextracellular signal-regulated protein kinaseFCSfetal calf serumGrb2growth factor receptor binding protein 2MEK(MAP kinase or ERK) kinaseMKKMAP kinase kinasePC-PLCphosphatidylcholine-specific phospholipase CPKAcyclic AMP-dependent protein kinasePP1protein phosphatase 1PP2Aprotein phosphatase 2AShcSrc homologue 2-containing proteinSOSson-of-sevenlessPAGEpolyacrylamide gel electrophoresis. kinase cascade consisting of Raf-1 itself, the dual specificity MAP kinase kinases MKK1 and MKK2 (also termed MEK-1 and MEK-2), and the extracellular signal-regulated protein kinases (ERKs) or MAP kinases (5Avruch J. Zhang X.F. Kyriakis J.M. Trends. Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (540) Google Scholar). The MAP kinases carry the signal to the nucleus, where they phosphorylate transcription factors capable of mediating changes in gene expression (6Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 7Blumer K.J. Johnson G.L. Trends. Biochem. Sci. 1994; 19: 236-240Abstract Full Text PDF PubMed Scopus (420) Google Scholar).While the regulation of MEK and MAP kinases by phosphorylation is well understood (8Alessi D.R. Saito Y. Campbell D.G. Cohen P. Sithanandam G. Rapp U. Ashworth A. Marshall C.J. Cowley S. EMBO J. 1994; 13: 1610-1619Crossref PubMed Scopus (464) Google Scholar, 9Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (836) Google Scholar, 10Zheng C.F. Guan K.L. EMBO J. 1994; 13: 1123-1131Crossref PubMed Scopus (297) Google Scholar), the molecular mechanism(s) involved in Raf-1 regulation remain more obscure. The best understood aspects of Raf-1 kinase regulation are the initial events that precede its mitogen stimulation. Thus, growth factor receptor-induced activation of the mammalian nucleotide exchange factor mSOS, mediated by adapter proteins such as Shc and Grb2, stimulates the conversion of Ras from the inactive, GDP-bound state to the active, GTP-bound state. Activated Ras in turn directly interacts with Raf-1, resulting in the translocation of Raf from the cytoplasm to the plasma membrane. These conclusions are based on the findings that Raf-1 physically interacts with Ras (11Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. Science. 1993; 260: 1658-1661Crossref PubMed Scopus (775) Google Scholar, 12Van Aelst L. Barr M. Marcus S. Polverino A. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6213-6217Crossref PubMed Scopus (503) Google Scholar, 13Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1656) Google Scholar, 14Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (581) Google Scholar) and that Raf-1 is associated with the plasma membrane in cells expressing oncogenic Ras (15Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (877) Google Scholar, 16Traverse S. Cohen P. Paterson H. Marshall C. Rapp U. Grand R.J. Oncogene. 1993; 8: 3175-3181PubMed Google Scholar, 17Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar). Importantly, Raf and Ras transiently interact in mammalian cells upon extracellular stimulation (18Hallberg B. Rayter S.I. Downward J. J. Biol. Chem. 1994; 269: 3913-3916Abstract Full Text PDF PubMed Google Scholar), providing a potential molecular basis for the transient membrane translocation of Raf observed in serum-stimulated cells (17Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar). The physical interaction of Raf-1 with activated Ras in vitro, however, is insufficient for stimulation of Raf-1 kinase activity (Ref. 19Zhang X.F. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Crossref PubMed Scopus (684) Google Scholar; data not shown). Artificial plasma membrane localization, on the other hand, is sufficient for Raf kinase activation in a Ras-independent manner (15Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (877) Google Scholar, 20Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (836) Google Scholar). Taken together, these observations suggest that membrane localization of Raf-1 is necessary for its activation and that the role of Ras is to recruit Raf-1 to the membrane for activation by an as yet elusive mechanism.Hyperphosphorylation of Raf-1 is a cellular response common to a wide range of physiological stimuli that activate the Raf-1/MEK/MAP kinase pathway and may be relevant to the process of Raf activity regulation (1Daum G. Eisenmann Tappe I. Fries H.W. Troppmair J. Rapp U.R. Trends. Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar, 3Rapp U.R. Oncogene. 1991; 6: 495-500PubMed Google Scholar, 4Li P. Wood K. Mamon H. Haser W. Roberts T. Cell. 1991; 64: 479-482Abstract Full Text PDF PubMed Scopus (112) Google Scholar). Phosphorylation of tyrosine residues 340 and 341 has been reported to be involved in the stimulation of Raf kinase activity in some cellular systems (21Morrison D.K. Kaplan D.R. Escobedo J.A. Rapp U.R. Roberts T.M. Williams L.T. Cell. 1989; 58: 649-657Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 22Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (300) Google Scholar, 23Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (520) Google Scholar). However, in many cellular systems, Raf kinase activation occurs in the apparent absence of Raf-1 tyrosine phosphorylation. In fact, even under circumstances when tyrosine phosphorylation is observed, the majority of phosphorylation events occur on serine residues (1Daum G. Eisenmann Tappe I. Fries H.W. Troppmair J. Rapp U.R. Trends. Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Thus, Raf-1 is phosphorylated in vivo at Ser-43, Ser-259, Ser-499, and Ser-621 (24Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1152) Google Scholar, 25McGrew B.R. Nichols D.W. Stanton Jr., V.P. Cai H. Whorf R.C. Patel V. Cooper G.M. Laudano A.P. Oncogene. 1992; 7: 33-42PubMed Google Scholar, 26Morrison D.K. Heidecker G. Rapp U.R. Copeland T.D. J. Biol. Chem. 1993; 268: 17309-17316Abstract Full Text PDF PubMed Google Scholar). While constitutive phosphorylation at Ser-621 might be necessary for Raf functionality, phosphorylation at Ser-259 and Ser-499 has been implicated in the protein kinase C-mediated activation of Raf (24Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1152) Google Scholar, 26Morrison D.K. Heidecker G. Rapp U.R. Copeland T.D. J. Biol. Chem. 1993; 268: 17309-17316Abstract Full Text PDF PubMed Google Scholar, 27Sözeri O. Vollmer K. Liyanage M. Frith D. Kour G. Mark III, G.E. Stabel S. Oncogene. 1992; 7: 2259-2262PubMed Google Scholar). Raf phosphorylated at Ser-43 by cAMP-dependent protein kinase displays a decreased affinity for Ras in vitro This could contribute to the negative regulation of the Raf-1/MEK-1/MAP kinase by cAMP-elevating agents observed in some cellular systems (28Burgering B.M. Bos J.L. Trends. Biochem. Sci. 1995; 20: 18-22Abstract Full Text PDF PubMed Scopus (290) Google Scholar). Furthermore, phosphorylation of Raf on Thr-269 mediated by a ceramide-activated protein kinase has been implicated in tumor necrosis factor-induced Raf kinase activation (29Yao B. Zhang Y. Delikat S. Mathias S. Basu S. Kolesnick R. Nature. 1995; 378: 307-310Crossref PubMed Scopus (303) Google Scholar).The exact molecular relationship between these phosphorylation events and those that are associated with the characteristic retardation of the electrophoretic mobility of Raf-1 following stimuli that activate the Ras/Raf-1/MEK/MAP kinase pathway is not clear. Initial experiments employing serine/threonine-specific phosphatases suggested a causal relationship between serine/threonine phosphorylation and Raf mobility shift as well as Raf kinase activation (30Kovacina K.S. Yonezawa K. Brautigan D.L. Tonks N.K. Rapp U.R. Roth R.A. J. Biol. Chem. 1990; 265: 12115-12118Abstract Full Text PDF PubMed Google Scholar). However, recent evidence argues against such a positive relationship between these two events (17Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar, 31Crespo P. Xu N. Daniotti J.L. Troppmair J. Rapp U.R. Gutkind J.S. J. Biol. Chem. 1994; 269: 21103-21109Abstract Full Text PDF PubMed Google Scholar, 32Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar, 33Ueki K. Matsuda S. Tobe K. Gotoh Y. Tamemoto H. Yachi M. Akanuma Y. Yazaki Y. Nishida E. Kadowaki T. J. Biol. Chem. 1994; 269: 15756-15761Abstract Full Text PDF PubMed Google Scholar, 34Williams N.G. Paradis H. Agarwal S. Charest D.L. Pelech S.L. Roberts T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5772-5776Crossref PubMed Scopus (81) Google Scholar). We have previously observed that the kinetics of mitogen-stimulated Raf kinase activation and Raf mobility shift are poorly correlated events. Thus, while mitogen-stimulated Raf-1 kinase activation occurs rapidly and is transient in nature, the decrease in Raf-1 protein mobility only becomes apparent at later times and coincides with a marked attenuation of mitogen-stimulated Raf-1 kinase activity (17Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar).Here we show that the mobility shift-associated hyperphosphorylation of Raf is associated with a decreased affinity of this form of Raf for the plasma membrane. Since plasma membrane localization is a positive determinant for Raf kinase activity, this post-translational modification might represent a molecular mechanism accounting, at least in part, for the attenuation of Raf kinase activity following mitogen stimulation. We further demonstrate that hyperphosphorylation of Raf can be blocked by a specific inhibitor of MEK activation and that this correlates with restoration of plasma membrane-bound Raf to control levels. Thus, the mobility shift-associated hyperphosphorylation of Raf is likely a consequence of activating the downstream components in the MAP kinase cascade and might represent a negative feedback mechanism contributing to the desensitization of this signaling pathway.DISCUSSIONThe molecular mechanism(s) involved in the regulation of Raf-1 protein kinase activity are complex and still incompletely understood. The initial events leading to activation of Raf-1 seem to involve Ras-mediated membrane translocation and phosphorylation on tyrosine residues (21Morrison D.K. Kaplan D.R. Escobedo J.A. Rapp U.R. Roberts T.M. Williams L.T. Cell. 1989; 58: 649-657Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 22Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (300) Google Scholar, 23Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (520) Google Scholar). Despite the obvious physiological importance, little is known about the mechanism(s) involved in turning Raf-1 kinase "off." Treatment of cells with a wide range of physiological stimuli results in hyperphosphorylation of Raf. While phosphorylation and activation of Raf by protein kinase C (24Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1152) Google Scholar, 27Sözeri O. Vollmer K. Liyanage M. Frith D. Kour G. Mark III, G.E. Stabel S. Oncogene. 1992; 7: 2259-2262PubMed Google Scholar) and/or tyrosine kinases such as Src and Lck (22Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (300) Google Scholar, 23Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (520) Google Scholar) might represent early receptor-stimulated events, these post-translational modifications are unlikely to account for the characteristic retardation of the mobility of Raf upon SDS-PAGE. The nature and significance of these mobility shift-associated hyperphosphorylation events have been controversial. Although experiments with serine/threonine-specific phosphatases initially suggested a stimulatory role for these post-translational modifications of Raf-1 (30Kovacina K.S. Yonezawa K. Brautigan D.L. Tonks N.K. Rapp U.R. Roth R.A. J. Biol. Chem. 1990; 265: 12115-12118Abstract Full Text PDF PubMed Google Scholar), recent evidence has challenged this hypothesis (17Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar, 31Crespo P. Xu N. Daniotti J.L. Troppmair J. Rapp U.R. Gutkind J.S. J. Biol. Chem. 1994; 269: 21103-21109Abstract Full Text PDF PubMed Google Scholar, 32Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar, 33Ueki K. Matsuda S. Tobe K. Gotoh Y. Tamemoto H. Yachi M. Akanuma Y. Yazaki Y. Nishida E. Kadowaki T. J. Biol. Chem. 1994; 269: 15756-15761Abstract Full Text PDF PubMed Google Scholar, 34Williams N.G. Paradis H. Agarwal S. Charest D.L. Pelech S.L. Roberts T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5772-5776Crossref PubMed Scopus (81) Google Scholar). First, the kinase activity negatively affected by phosphatase treatment in the experiments of Kovacina et al. (30Kovacina K.S. Yonezawa K. Brautigan D.L. Tonks N.K. Rapp U.R. Roth R.A. J. Biol. Chem. 1990; 265: 12115-12118Abstract Full Text PDF PubMed Google Scholar) is unlikely to reflect Raf-1, since the peptide used, Syntide-2, is not a Raf-1 substrate (49Force T. Bonventre J.V. Heidecker G. Rapp U. Avruch J. Kyriakis J.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1270-1274Crossref PubMed Scopus (68) Google Scholar). While the more recent demonstration of Raf-1 kinase inactivation following phosphatase treatment was performed using the physiological Raf-1 substrate MEK, it was not demonstrated whether this correlated with corresponding changes in the electrophoretic mobility of Raf-1 (50Dent P. Jelinek T. Morrison D.K. Weber M.J. Sturgill T.W. Science. 1995; 268: 1902-1906Crossref PubMed Scopus (172) Google Scholar). Furthermore, only highly purified Raf-1 devoid of the putative chaperone proteins hsp90 and 14-3-3, which form a complex with Raf-1 in vivo (17Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar, 51Stancato L.F. Chow Y.-H. Hutchison K.A. Perdew G.H. Jove R. Pratt W.B. J. Biol. Chem. 1993; 268: 21711-21716Abstract Full Text PDF PubMed Google Scholar, 52Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 53Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 54Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar), was susceptible to inactivation by phosphatase treatment. Second, a comparison of the time course of activation and mobility shift of Raf in response to various agonists has shown that these processes are poorly correlated (17Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar, 33Ueki K. Matsuda S. Tobe K. Gotoh Y. Tamemoto H. Yachi M. Akanuma Y. Yazaki Y. Nishida E. Kadowaki T. J. Biol. Chem. 1994; 269: 15756-15761Abstract Full Text PDF PubMed Google Scholar, 55Lisbona C. Alemany S. Calvo V. Fernandez-Renart M. Eur. J. Immunol. 1994; 24: 2746-2754Crossref PubMed Scopus (8) Google Scholar). Thus, we have previously demonstrated that the transient activation of Raf protein kinase in response to serum stimulation of cells correlated with a transient membrane translocation of Raf and occurred in the absence of a Raf mobility shift. On the contrary, in accordance with the the kinetics of serum-stimulated Raf hyperphosphorylation presented here, the appearance of the Raf mobility shift correlated with an attenuation of Raf kinase activity and was paralleled by a reduction in the amount of membrane-associated Raf (17Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar).Here we show that mobility shift-associated hyperphosphorylation of Raf induced by treatment of cells with D609, a PC-PLC inhibitor, correlates with a decrease in membrane-localized Raf below that observed in untreated cells. The results obtained in the cell-free reconstitution experiments demonstrated that this phenomenon is not due to effects of D609 on the plasma membrane. Importantly, hyperphosphorylated Raf from serum-treated cells displayed a similar decreased tendency to localize to the membrane fraction in this in vitro assay. Furthermore, comparison of the phosphopeptide maps revealed no significant differences in the phosphorylation state of Raf in D609- and mitogen-stimulated cells. Taken together, these results suggest that negative modulation of membrane localization mediated by mobility shift-associated hyperphosphorylation of Raf represents an important molecular mechanism that could account, at least in part, for the attenuation of Raf kinase activity that follows its initial mitogen activation.The results presented here imply that mobility shift-associated hyperphosphorylation does not directly regulate the catalytic activity of Raf, but rather exerts its effect indirectly, by modulating the ability of Raf to associate with the plasma membrane, the residence of the elusive Raf-"activating principle." Consistent with this hypothesis, dephosphorylation of mobility-shifted Raf, isolated from D609 or serum-treated cells, by incubation with either of the serine/threonine-specific protein phosphatases PP1 or PP2A, did not significantly alter the in vitro protein kinase activity of Raf (data not shown). Thus, total cellular Raf kinase activity might be the result of the stimulus-independent intrinsic catalytic activity of Raf and a component determined by the stimulus-dependent levels of a membrane-localized "activating principle," such as a putative lipid co-factor. Following serum starvation, the levels of this "activating principle" are likely low, but not necessarily absent. Fluctuations in the levels of the Raf activator in serum-starved cells might explain the varying degrees of reduction (0-89%) below basal Raf kinase activity observed in D609-treated cells. In serum-stimulated cells, on the other hand, the level of the Raf activator is likely significantly increased and might account for the intermediate, rather than basal or reduced, Raf kinase activity of mobility-shifted Raf observed at later times following mitogen stimulation.It has previously been demonstrated in NIH3T3 cells that inhibition of endogenous PC-PLC by D609 blocks activation of Raf-1 in response to mitogenic growth factors (56Cai H. Erhardt P. Troppmair J. Diaz Meco M.T. Sithanandam G. Rapp U.R. Moscat J. Cooper G.M. Mol. Cell Biol. 1993; 13: 7645-7651Crossref PubMed Scopus (119) Google Scholar). However, in contrast to our results obtained with CHO cells, Cai et al. (56Cai H. Erhardt P. Troppmair J. Diaz Meco M.T. Sithanandam G. Rapp U.R. Moscat J. Cooper G.M. Mol. Cell Biol. 1993; 13: 7645-7651Crossref PubMed Scopus (119) Google Scholar) failed to observe a Raf-1 mobility shift in response to treatment of serum-starved NIH3T3 cells with D609. This is probably due to the different cell system or the higher concentration of D609 (35 μg/ml) used in their experiments. While we observed a pronounced Raf protein mobility shift at low concentrations of D609, the shift was less pronounced at higher concentrations (>35 μg/ml; Fig. 1A). Thus, the inhibition of serum-stimulated Raf-1 kinase activation at concentrations of D609 higher than 35 μg/ml might not rely on the effect of hyperphosphorylation of Raf-1 proposed here but might be the consequence of inhibiting mitogen-stimulated PC-PLC activity. Furthermore, it remains to be established whether the D609-induced hyperphosphorylation of Raf-1 reported here is a consequence of inhibition of PC-PLC or whether D609 modulates targets distinct from PC-PLC.Several recent reports support the hypothesis that mobility shift-associated hyperphosphorylation of Raf is a feedback consequence of activation of the MAP kinase pathway. Hormone-induced activation of an estrogen-dependent form of oncogenic Raf-1, stably expressed in quiescent 3T3 cells, resulted in increased activity of MEK and, to a lesser extent, MAP kinase. Under these conditions, a mobility shift-associated hyperphosphorylation of the endogenous pool of Raf-1 was observed without a concomitant stimulation of Raf-1 kinase activity (32Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar). Ueki et al. (33Ueki K. Matsuda S. Tobe K. Gotoh Y. Tamemoto H. Yachi M. Akanuma Y. Yazaki Y. Nishida E. Kadowaki T. J. Biol. Chem. 1994; 269: 15756-15761Abstract Full Text PDF PubMed Google Scholar) reported that overexpression of MAP kinase in CHO cells overexpressing human insulin receptor (CHO/IR) led to hyperphosphorylation of Raf-1, which, significantly, was accompanied by a decrease in the MEK kinase activity of Raf-1. The clearest evidence arguing for an involvement of MEK or a downstream component in the hyperphosphorylation of Raf, however, has been provided by the demonstration that blocking activation of MEK by PD98059, or by overexpression of a dominant negative MEK mutant, abolishes the insulin-stimulated mobility shift of Raf in CHO/IR cells (57Waters S.B. Holt K.H. Ross S.E. Syu L.-J. Guan K.-L. Saltiel A.R. Koretzky G.A. Pessin J.E. J. Biol. Chem. 1995; 270: 20883-20886Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Furthermore, Alessi et al. (48Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar) demonstrated that PD98059 blocks the platelet-derived growth factor-stimulated hyperphosphorylation of Raf and, interestingly, prevents the attenuation of Raf kinase activity that occurred following its initial stimulation. Here, by the use of an agent that activates MAP kinase pathway components downstream of Raf in a PD968059-sensitive manner, we present further evidence in favor of the hypothesis that mobility shift-associated hyperphosphorylation of Raf in response to mitogens, and probably other stimuli, occurs in a negative feedback manner. First, time course analyses demonstrated that D609- and FCS-stimulated activation of ERK2 precedes, rather than ensues, the appearance of mobility-shifted Raf. The fact that D609-induced activation of ERK2 and hyperphosphorylation of Raf is PD98059-sensitive strongly suggests the involvement of a component of the MAP kinase pathway at a point distal to Raf in this phenomenon. While MEK-1 is unable to phosphorylate recombinant Raf-1 in vitro (data not shown) and thus is an unlikely Raf-1 kinase kinase, the classical MAP kinase isoforms are able to phosphorylate Raf-1 in vitro (Refs. 58Lee R. Cobb M.H. Blackshear P.J. J. Biol. Chem. 1992; 267: 1088-1092Abstract Full Text PDF PubMed Google Scholar and 59Anderson N.G. Li P. Marsden L.A. Williams N. Roberts T.M. Sturgill T.W. Biochem. J. 1991; 277: 573-576Crossref PubMed Scopus (83) Google Scholar; data not shown). While this is consistent with MAP kinase being a potential mediator of Raf-1 hyperphosphorylation in vivo, phosphorylation of Raf-1 by the classical MAP kinases in vitro fails to cause the characteristic mobility shift of Raf-1 (data not shown). Thus, it is likely that the putative Raf kinase kinase responsible for mobility shift-associated hyperphosphorylation of Raf lies downstream of ERK2. The molecular mechanism of how D609 leads to the activation of ERK2 (and MEK; data not shown) and whether D609 affects additional components in this pathway remains to be established. Differential modulation of component(s) downstream of ERK2 by D609 and FCS might explain the observation that, while D609 and FCS stimulated ERK2 activity with similar kinetics, the time course of appearance of mobility-shifted Raf elicited by D609 was somewhat slower than that induced by FCS.PKA phosphorylates Raf-1 on sites that either negatively affect the Raf-1/Ras interaction (i.e. Ser-43) or inhibit the catalytic activity of Raf (28Burgering B.M. Bos J.L. Trends. Biochem. Sci. 1995; 20: 18-22Abstract Full Text PDF PubMed Scopus (290) Google Scholar). PKA is thus a potential Raf kinase kinase whose activation might account for the phenomenon reported here. Indeed, comparison of the phosphopeptide maps presented here with those reported by Morrison and co-workers (26Morrison D.K. Heidecker G. Rapp U.R. Copeland T.D. J. Biol. Chem. 1993; 268: 17309-17316Abstract Full Text PDF PubMed Google Scholar) suggests that D609 as well as serum induce the phosphorylation of Raf on Ser-43 (Fig. 2, phosphopeptide 1 and/or 2). However the in vivo phosphorylation of this site is probably not mediated by PKA, since we di
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