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• W2044481116 abstract "The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase. The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase. mitogen-activated protein kinase/extracellular signal-regulated kinase kinase protein phosphatase 2A colony-stimulating factor-1 hemagglutinin cytomegalovirus epidermal growth factor glutathione S-transferase The Raf-1 kinase is an important intermediate in the transduction of proliferative signals, and its activation may be a key event in the development of a wide range of tumors (1.Monia B.P. Johnston J.F. Geiger T. Muller M. Fabbro D. Nat. Med. 1996; 2: 668-675Crossref PubMed Scopus (446) Google Scholar). Activated Raf-1 can regulate the mitogen-activated protein kinase network by phosphorylating and activating MEK1; within the mitogen-activated protein kinase cascade, Raf interacts physically with MEK-1 via its kinase domain and with GTP-loaded Ras via its N terminus (2.Daum G. Eisenmann-Tappe I. Fries H.W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (488) Google Scholar). Activated Ras is the best studied activator of Raf-1. It binds to Raf-1 with high affinity and mediates its translocation from the cytosol to the plasma membrane, where activation takes place (3.Marshall M. Mol. Reprod. Dev. 1995; 42: 493-499Crossref PubMed Scopus (86) Google Scholar, 4.McCormick F. Mol. Reprod. Dev. 1995; 42: 500-506Crossref PubMed Scopus (98) Google Scholar). Artificial tethering of Raf-1 to the cell membrane results in partial activation, which can be further enhanced by mitogenic stimulation, suggesting that at the cell membrane Raf-1 is exposed to both constitutive and mitogen-regulated activators (5.Diaz B. Barnard D. Filson A. MacDonald S. King A. Marshall M. Mol. Cell. Biol. 1997; 17: 4509-4516Crossref PubMed Scopus (165) Google Scholar, 6.King A.J. Sun H. Diaz B. Barnard D. Miao W. Bagrodia S. Marshall M.S. Nature. 1998; 396: 180-183Crossref PubMed Scopus (386) Google Scholar, 7.Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (528) Google Scholar, 8.Mason C.S. Springer C.J. Cooper R.G. Superti-Furga G. Marshall C.J. Marais R. EMBO J. 1999; 18: 2137-2148Crossref PubMed Scopus (369) Google Scholar). Mitogenic stimulation of cells typically induces hyperphosphorylation of Raf-1 and a retardation of its migration on SDS gels. This hyperphosphorylation correlates with the down-regulation of Raf-1 kinase activity (9.Wartmann M. Davis R.J. J. Biol. Chem. 1994; 269: 6695-6701Abstract Full Text PDF PubMed Google Scholar, 10.Wartmann M. Hofer P. Turowski P. Saltiel A.R. Hynes N.E. J. Biol. Chem. 1997; 272: 3915-3923Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and may be implemented by a negative feedback mechanism depending on MEK activity (10.Wartmann M. Hofer P. Turowski P. Saltiel A.R. Hynes N.E. J. Biol. Chem. 1997; 272: 3915-3923Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 11.Alessi 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 (3259) Google Scholar). Serines 43, 621, and 259 are phosphorylated in resting fibroblasts, albeit to different degrees (12.Morrison D.K. Heidecker G. Rapp U.R. Copeland T.D. J. Biol. Chem. 1993; 268: 17309-17316Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of all three residues has been implicated in the negative regulation of Raf-1. Phosphorylation of serine 43 interferes with Ras binding and consequently with Ras-mediated activation (3.Marshall M. Mol. Reprod. Dev. 1995; 42: 493-499Crossref PubMed Scopus (86) Google Scholar). Phosphorylated serine 259 and serine 621 represent binding sites for 14-3-3 adaptor proteins (13.Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1195) Google Scholar, 14.Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (349) Google Scholar), whose function in Raf-1 activation is controversial. While bivalent binding to Ser259 and Ser621 has been suggested to maintain Raf-1 in an inactive conformation (15.Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (40) Google Scholar, 16.Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (390) Google Scholar), reversible association with 14-3-3 facilitates Ras-dependent activation in vivo and in vitro (17.Roy S. McPherson R.A. Apolloni A. Yan J. Lane A. Clyde-Smith J. Hancock J.F. Mol. Cell. Biol. 1998; 18: 3947-3955Crossref PubMed Scopus (116) Google Scholar). In particular, binding to the Ser(P)621site appears to be necessary for kinase activity (16.Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (390) Google Scholar, 18.Thorson J.A., Yu, L.W. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar), a finding that contrasts with the studies indicating that phosphorylation of this site by PKA in vitro is inhibitory (19.Mischak H. Seitz T. Janosch P. Eulitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar). Therefore, the significance of Ser621 phosphorylation is still in question. Its investigation is hampered by the fact that Ser621 is essential for the catalytic function of Raf-1 and cannot be replaced by other amino acids without loss of kinase activity (19.Mischak H. Seitz T. Janosch P. Eulitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar, 20.Baek K.H. Fabian J.R. Sprenger F. Morrison D.K. Ambrosio L. Dev. Biol. 1996; 175: 191-204Crossref PubMed Scopus (22) Google Scholar). Serine 259 can be phosphorylated by protein kinase B, another Ras effector activated in parallel with Raf, and this phosphorylation correlates with the down-regulation of kinase activity (21.Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (912) Google Scholar). Consistent with an inhibitory role of Ser(P)259, mutation of this residue moderately activates the Raf-1 kinase in cultured cells (16.Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (390) Google Scholar,22.Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (190) Google Scholar); the corresponding point mutants display a gain of function phenotype in Drosophila (15.Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (40) Google Scholar, 20.Baek K.H. Fabian J.R. Sprenger F. Morrison D.K. Ambrosio L. Dev. Biol. 1996; 175: 191-204Crossref PubMed Scopus (22) Google Scholar). Taken together, these data raise the possibility that dephosphorylation of negative regulatory residues plays a role in Raf-1 activation. Protein phosphatase 2A (PP2A) is a major form of serine/threonine phosphatase involved in the regulation of signal transduction, growth, and development (23.Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (600) Google Scholar). This class of enzymes consists of a heterotrimer that exists in multiple forms. The core components of all trimeric forms are the 36-kDa catalytic subunit (PP2AC) and the 65-kDa regulatory subunit (A subunit, PR65). This core heterodimer is ubiquitous, and it forms complexes with “variable” subunits of cellular origin (some of which are expressed in a tissue- and/or development-restricted manner) as well as with transforming viral antigens (24.Pallas D.C. Shahrik L.K. Martin B.L. Jaspers S. Miller T.B. Brautigan D.L. Roberts T.M. Cell. 1990; 60: 167-176Abstract Full Text PDF PubMed Scopus (459) Google Scholar, 25.Yang S.I. Lickteig R.L. Estes R. Rundell K. Walter G. Mumby M.C. Mol. Cell. Biol. 1991; 11: 1988-1995Crossref PubMed Scopus (206) Google Scholar, 26.Sontag E. Fedorov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (461) Google Scholar). Association with variable subunits of cellular and viral origin occurs via the N-terminal leucine-rich repeats of PR65 (27.Ruediger R. Roeckel D. Fait J. Bergqvist A. Magnusson G. Walter G. Mol. Cell. Biol. 1992; 12: 4872-4882Crossref PubMed Scopus (122) Google Scholar) and confers distinct properties to the enzyme (28.Kamibayashi C. Mumby M.C. Adv. Protein Phosphatases. 1995; 9: 195-210Google Scholar). Recently, PP2A has been shown to form a complex with Ca2+/calmodulin-dependent protein kinase IV (29.Westphal R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (224) Google Scholar) as well as with PAK1, PAK3, and p70 S6 kinase (30.Westphal R.S. Coffee Jr., R.L. Marotta A. Pelech S.L. Wadzinski B.E. J. Biol. Chem. 1999; 274: 687-692Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The isolated catalytic subunit can associate with casein kinase 2α (31.Heriche J.K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-955Crossref PubMed Scopus (250) Google Scholar). Where investigated (29.Westphal R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (224) Google Scholar), PP2A has been shown to contribute to the inactivation of the associated kinase. Here we show that the PP2A inhibitor okadaic acid inhibits full fledged Raf-1 activation. This effect is mediated by a change in the phosphorylation of Ser259 of Raf-1. In addition, Raf-1 forms stable complexes with PP2A heterodimers. Our results are the first indication that PP2A may support the activation of an associated kinase and highlight the intimate relationship between kinases and phosphatases, which we are just beginning to understand. BAC-1.2F5 cells (32.Morgan C. Pollard J.W. Stanley E.R. J. Cell. Physiol. 1987; 130: 420-427Crossref PubMed Scopus (151) Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 0.63 nm purified mouse recombinant mitogen colony-stimulating factor-1 (CSF-1) or 15% L-cell conditioned medium (33.Stanley E.R. Methods Enzymol. 1985; 116: 564-587Crossref PubMed Scopus (167) Google Scholar) as a source of murine CSF-1. COS-1 and COS-7 cells were grown in RPMI 1640 or Dulbecco's modified Eagle's medium supplemented with glutamine and 10% fetal calf serum. COS cells were transfected by electroporation (0.5–1 × 107 cells/cuvette, 240 V, 960 microfarads, 10 μg of plasmid DNA). The plasmids used were the pCMV5 vector, pCMV.HAcatα (amino acid 9 to the stop of human PP2ACα cloned downstream of a HA tag in pCMV), PRC/CMV.HA65a (amino acid 3 to the stop of human PR65a cloned downstream of a HA tag in pRC/CMV), pCMV-HA-ERK (courtesy of Michael Karin, UCSD), pCMV5Raf-1, and a S259A Raf-1 mutant (pCMV5-S259A). Cells were harvested 2 days after transfection. Under these conditions, protein expression increased linearly between 1 and 10 μg of transfected plasmid DNA. Confluent cultures were starved for 18 h prior to stimulation with recombinant CSF-1 (BAC-1.2F5 cells, 6.3 nm mouse recombinant CSF-1 or 63 nm human recombinant CSF-1 (Chiron Co.) or EGF (COS-1, 33 nm, 10 min). In selected experiments, cells were incubated with okadaic acid (100 nm, 45 min) prior to growth factor stimulation. N-terminal His6-tagged human PP2AC was expressed in the bacterial strain M15[pREP4], and the fusion polypeptide was purified by Ni2+-nitrilotriacetic acid chromatography (Qiagen). Balb/c × CBA F1 mice (∼3 months old) were immunized by subcutaneous injection with 100 μg of bacterially expressed PP2AC fusion protein emulsified with an equal volume of Titremax Gold (CytRx Corp.). The immunization was repeated four times at three monthly intervals, and then the mice rested for 6 months. A further 100 μg of fusion protein in phosphate-buffered saline plus 0.1% SDS was injected intraperitoneally at 6 and 3 days prior to sacrifice. Hybridoma lines were then established by fusing splenocytes from the immunized animal with the myeloma line Sp2/0-Ag14 by polyethylene glycol treatment and conventional procedures. Cell lines were screened for antibody production by a modified dot blot procedure using the bacterially expressed fusion protein (34.Dilworth S.M. Horner V.P. J. Virol. 1993; 67: 2235-2244Crossref PubMed Google Scholar) and cloned three times before tissue culture fluid containing the monoclonal antibody was harvested. Clone F2.8F5 was typed as an IgG2K species using Isotype strips (Roche Molecular Biochemicals) and confirmed as specific for PP2AC by Western blot analysis using total cell lysates. Using deletion mutants of PP2Ac (35.Evans D.R. Myles T. Hofsteenge J. Hemmings B.A. J. Biol. Chem. 1999; 274: 24038-24046Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), the epitope has been mapped close to the C-terminal end of PP2AC. Cells were lysed in solubilization buffer (10 mm Tris-Cl, 50 mm NaCl, 1% Triton X-100, 30 mm sodium pyrophosphate, 100 μm Na3VO4, 1 mm phenylmethylsulfonyl fluoride). Insoluble material was removed by centrifugation (15,000 rpm, 30 min, 4 °C). Immunoprecipitation was performed exactly as described previously (36.Baccarini M. Li W. Sbarba P.D. Stanley E.R. Receptor. 1991; 1: 243-259PubMed Google Scholar). A rabbit polyclonal antiserum raised against a carboxyl-terminal peptide of v-raf (SP63; CTLTTSPRLPVF) was used to immunoprecipitate Raf-1 molecules. A mouse monoclonal antibody against PP2AC (F2 8F5) was used to immunoprecipitate PP2A. HA-tagged PR65α and PP2AC were immunoprecipitated with the HA-epitope-specific monoclonal antibody 12CA5. Immunocomplexes were collected following incubation (1–3 h at 4 °C) with protein A-Sepharose beads (Sigma). For Western blotting, cell lysates (25 μg/lane) or immunoprecipitates were separated by 7.5% SDS-PAGE prior to electrophoretic transfer onto Hybond C super (Amersham Pharmacia Biotech). The blots were probed with rabbit polyclonal antisera directed against Raf-1, PP2AC (antibody C1–20), PR65α (37.Turowski P. Fernandez A. Favre B. Lamb N.J. Hemmings B.A. J. Cell Biol. 1995; 129: 397-410Crossref PubMed Scopus (136) Google Scholar), p55α and -β (38.Hendrix P. Turowski P. Mayer-Jaekel R.E. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 7330-7337Abstract Full Text PDF PubMed Google Scholar), or p72 plus p130 (39.Hendrix P. Mayer-Jackel R.E. Cron P. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 15267-15276Abstract Full Text PDF PubMed Google Scholar) or with monoclonal antibodies against Raf-1 (PBB.1; Ref. 40.Kolch W. Weissinger E. Mischak H. Troppmair J. Showalter S.D. Lloyd P. Heidecker G. Rapp U.R. Oncogene. 1990; 5: 713-720PubMed Google Scholar), PP2AC (F2 8F5), or PR 65 (C5 9E10) prior to incubation with horseradish peroxidase-conjugated secondary antibodies and exposure to the ECL substrate. All blotting reagents were from Amersham Pharmacia Biotech. The blots were stripped according to the manufacturer's instruction. Raf-1 kinase activity was measured as the ability of immunoisolated Raf-1 to phosphorylate recombinant, catalytically inactive MEK-1 (MEK−) or to activate recombinant MEK-1 in coupled assays using MBP (41.Alessi D.R. Cohen P. Ashworth A. Cowley S. Leevers S.J. Marshall C.J. Methods Enzymol. 1995; 255: 279-290Crossref PubMed Scopus (155) Google Scholar) as the end point of the assay. The GST-Raf-1 fusions used in this study have been previously described (42.Li S. Janosch P. Tanji M. Rosenfeld G.C. Waymire J.C. Mischak H. Kolch W. Sedivy J.M. EMBO J. 1995; 14: 685-696Crossref PubMed Scopus (155) Google Scholar). The proteins were expressed by standard techniques in the baculovirus Sf9 cell system and purified as described previously (43.Macdonald S.G. Crews C.M. Wu L. Driller J. Clark R. Erikson R.L. McCormick F. Mol. Cell. Biol. 1993; 13: 6615-6620Crossref PubMed Scopus (203) Google Scholar) with the exception that 1% Triton X-100 was added before binding to GST-agarose. For pull-down assays, 100–200 ng of GST-Raf-1 fusion proteins were incubated with either 1 mg of whole cell lysates or 200 ng of purified heterodimer, PR65α, or PP2AC for 3–6 h at 4 °C. The complexes were recovered by centrifugation and washed in solubilization buffer containing 0.03% SDS, 10 mmdithiothreitol, 0.5 m NaCl prior to Western blot analysis. PP2A heterodimer used in this study was provided by Prof. J. Goris (Leuven, Belgium). Catalytic subunit of PP2A was kindly provided by Dr. Lisa Ballou (I.M.P., Vienna). Both forms of the enzyme were purified from rabbit skeletal muscle (44.Stone S.R. Hofsteenge J. Hemmings B.A. Biochemistry. 1987; 26: 7215-7220Crossref PubMed Scopus (121) Google Scholar). Recombinant PR65α was kindly provided by Dr. P. Turowski. 32P-Labeling of cells was performed as described previously (36.Baccarini M. Li W. Sbarba P.D. Stanley E.R. Receptor. 1991; 1: 243-259PubMed Google Scholar). Cell lysis and immunoprecipitation of Raf-1 proteins were performed as described above. 32P-Labeled proteins were resolved by 7.5% SDS-PAGE, extracted from the gels, and subjected to digestion with sequencing grade trypsin (Promega) according to the manufacturer's instructions prior to phosphopeptide mapping. Tryptic peptides were separated in the first dimension by electrophoresis using pH 8.9 buffer and in the second dimension (chromatography) using a buffer containingn-butanol/pyridine/acetic acid/water (12:10:3:15). Chromatography was allowed to proceed for 20 h. The amount of Raf-1 contained in the immunoprecipitates used for the mapping of the phosphotryptic peptides was determined by immunoblotting an aliquot of the immunoprecipitates, and it was equal in all samples. Phosphotryptic peptide mapping was repeated twice with comparable results. We have studied Raf-1 phosphorylation and activation in BAC-1.2F5 macrophages stimulated by CSF-1 (45.Buscher D. Hipskind R.A. Krautwald S. Reimann T. Baccarini M. Mol. Cell. Biol. 1995; 15: 466-475Crossref PubMed Scopus (165) Google Scholar, 46.Krautwald S. Buscher D. Kummer V. Buder S. Baccarini M. Mol. Cell. Biol. 1996; 16: 5955-5963Crossref PubMed Scopus (43) Google Scholar). The addition of CSF-1 to quiescent BAC-1.2F5 cells induced robust and transient activation of Raf-1 (Fig. 1 A; Ref. 45.Buscher D. Hipskind R.A. Krautwald S. Reimann T. Baccarini M. Mol. Cell. Biol. 1995; 15: 466-475Crossref PubMed Scopus (165) Google Scholar). Peak (16-fold) Raf-1 activation correlated with the transient dephosphorylation of serine 621, the main residue phosphorylated in quiescent macrophages (Fig. 1 B, b). Conversely, the decay of Raf-1 activity to a 4-fold stimulation 15 min post-CSF-1 treatment was accompanied by the hyperphosphorylation of serines 43 and 621. A number of minor yet unidentified residues (spots 2–4) as well as Ser259 (Fig. 1 B, c) were also phosphorylated at this time. Pretreatment with okadaic acid (100 nm; at this concentration a specific inhibitor of PP2A but not PP1α (47.Favre B. Turowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13856-13863Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar)), caused a slight increase in basal kinase activity (2-fold) and in Raf-1 phosphorylation in general. Notably, both the peptide pattern and the amount of activity associated with the Raf-1 immunoprecipitates are identical in cells treated with okadaic acid alone and in cells treated with CSF-1 for 15 min (Fig. 1, Aand B, compare c and d). This suggests that both positive and negative regulatory phosphorylation sites are targets of an okadaic acid-sensitive phosphatase and that the balance between phosphorylation of the positive and negative regulatory sites determines the extent of Raf-1 activation. Remarkably, in the presence of okadaic acid, maximal Raf-1 stimulation by CSF-1 was prevented, and only a moderate (5–6-fold) activation could be obtained. Inhibition of early serine 621 dephosphorylation and hyperphosphorylation of several other sites (most prominently serine 259) could be observed concomitantly. Phosphorylation increased further after 15 min of CSF-1 stimulation; by this time, Raf-1 activation had decayed despite the continuous presence of the phosphatase inhibitor (Fig. 1, Aand B). Although these changes are complex, it can be appreciated that Ser259 and Ser621 are less phosphorylated (hypophosphorylated) in cells treated with CSF-1 alone than in cells treated with mitogen plus okadaic acid and that this “hypophosphorylation” correlates with maximal kinase activation. Thus, an okadaic acid-sensitive phosphatase is involved in Raf-1 dephosphorylation and activation in macrophages. To determine whether okadaic acid influenced Raf-1 stimulation by receptor tyrosine kinases other than the CSF-1 receptor, we analyzed the effect of the inhibitor on COS-1 cells stimulated with EGF. In agreement with the data shown in Fig. 1 A, okadaic acid significantly decreased EGF-mediated activation of wild type Raf-1 (Fig. 2 A). To assess whether this effect depended on Ser259, COS-1 cells transfected with a Ser259 → Ala Raf-1 mutant (RafS259A) were treated with EGF in the presence or absence of okadaic acid. As described previously (12.Morrison D.K. Heidecker G. Rapp U.R. Copeland T.D. J. Biol. Chem. 1993; 268: 17309-17316Abstract Full Text PDF PubMed Google Scholar, 15.Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (40) Google Scholar, 16.Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (390) Google Scholar, 20.Baek K.H. Fabian J.R. Sprenger F. Morrison D.K. Ambrosio L. Dev. Biol. 1996; 175: 191-204Crossref PubMed Scopus (22) Google Scholar, 22.Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (190) Google Scholar), the basal activity of RafS259A was modestly increased with respect to wild type Raf-1. EGF efficiently stimulated RafS259A, but in contrast with the wild type, activation of RafS259A was not decreased by pretreatment with okadaic acid (Fig.2 B). These data confirm the importance of the okadaic acid-sensitive phosphatase in Raf-1 activation by receptor tyrosine kinases and identify Ser259 as the Raf-1 site relevant for activation. The effect of okadaic acid on the activation of wild type Raf-1 in EGF-treated COS-1 cells was less dramatic than the one observed in BAC-1.2F5 macrophages stimulated by CSF-1. It is possible that the importance of dephosphorylation in Raf-1 activation may vary depending on the cell type; alternatively, Raf-1 overexpression might adversely affect the outcome of the experiment, as is often the case when multienzyme complexes are involved (see below). Our data are consistent with the hypothesis that mitogen treatment induces a kinase that inactivates Raf-1 by phosphorylating Ser259 and is counteracted by an okadaic acid-sensitive phosphatase. The strength of Raf-1 activation (or the proportion of Raf-1 molecules that can be activated) would depend on the balance between the activity of these two enzymes. Such a regulation would be particularly opportune in the case of Raf-1, whose moderate activation elicits proliferation, while a strong, prolonged Raf-1 signal leads to cell cycle arrest (48.Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (419) Google Scholar, 49.Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (577) Google Scholar). In line with this hypothesis, while this manuscript was in preparation, activated protein kinase B was shown to phosphorylate Ser259 of Raf-1, to suppress the activation of the mitogen-activated protein kinase cascade, and to shift a cell line from cell cycle arrest to proliferation (21.Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (912) Google Scholar). Okadaic acid specifically blocked Raf-1 activation at a concentration that selectively inhibits PP2A (47.Favre B. Turowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13856-13863Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). We therefore examined whether PP2A associated physically with Raf-1. Both the catalytic (PP2AC) and the regulatory (PR65) subunit of the PP2A core enzyme were detected in Raf-1 immunoprecipitates prepared from quiescent as well as CSF-1-treated cells. Neither PP2AC nor PR65 could be detected in precipitates prepared with nonimmune sera (lanes 1 and 2) or with protein A (Fig.3 A). In addition, significant amounts of Raf-1 were present in immunoprecipitates prepared with a monoclonal antibody against PP2AC but not with nonimmune mouse IgG (Fig. 3 B), demonstrating the specificity of the interaction. The ubiquitously expressed PP2A core heterodimer binds to different variable subunits of cellular or viral origins, which are involved in regulating its substrate specificity and/or localization. None of the cellular variable subunits tested (p55α and -β, and p72 plus p130) was present in Raf-1 immunoprecipitates (not shown). Consistent findings were obtained in fibroblasts stimulated with EGF (not shown). We next verified the interaction between Raf-1 and the PP2A heterodimer in COS-7 cells transfected with vectors directing the expression of HA-tagged PP2A subunits (Fig.4 A). Anti-HA immunoprecipitates from cells transfected with the HA-tagged PP2AC contained low amounts of endogenous PR65 and Raf-1 (Fig. 4 A,lane 1). Anti-HA immunoprecipitates from cells transfected with the HA-PR65, on the other hand, contained significant amounts of endogenous PP2AC subunit and more Raf-1 (Fig. 4 A, compare lanes 1 and 2). Therefore, the amount of Raf-1 detected correlated with the amount of heterodimer present in the anti-HA immunoprecipitates. To confirm the specificity of the interaction between the HA-tagged PP2A subunits and Raf-1, we monitored the presence of Raf-1 in anti-HA immunoprecipitates from cells overexpressing HA-ERK (Fig. 4 B). Neither endogenous (lane 3) nor overexpressed Raf-1 (lane 4) could be detected in these immunoprecipitates. The Raf-1-PP2A complex formation was further analyzed by in vitro reconstitution experiments with purified proteins (Fig.4 C). GST-tagged Raf was expressed in Sf-9 cells alone or in combination with v-Ras plus Lck in order to activate it (Raf*). Raf proteins immobilized on glutathione-Sepharose beads were incubated with PR65, PP2AC, or the heterodimer PR65-PP2AC. Consistent with the lack of effect of mitogens on in vivo complex formation, we did not observe significant differences between PP2A binding to Raf or Raf*. PP2AC displayed only weak binding to Raf-1. In contrast, both PR65 and the core heterodimer associated strongly. Thus, as it is the case for cellular and viral subunits (28.Kamibayashi C. Mumby M.C. Adv. Protein Phosphatases. 1995; 9: 195-210Google Scholar), PR65 probably plays the key role in the association between Raf-1 and the PP2A heterodimer. This association, however, is not likely to be direct. Raf-1 does not interact with PR65, PP2AC, or p55 in the yeast two-hybrid system, 2J. Rüth, V. Janssens, J. Goris, and M. Baccarini, unpublished observation. and size fractionation experiments indicate that in vivo Raf-1 and PP2A are part of a large protein complex (data not shown). Additional proteins, and possibly a variable subunit not detected in our experiments, might also be present in small amounts in the purified enzyme preparations used in the GST pull-down experiments and might be facilitating or even mediating the interaction observed in vitro. In this context, a variable subunit of PP2A has been recently shown to positively regulate Ras signaling upstream ofraf during vulval development in Caenorhabditis elegans (50.Sieburth D.S. Sundaram M. Howard R.M. Han M. Genes Dev. 1999; 13: 2562-2569Crossref PubMed Scopus (69) Google Scholar). Concentrations of okadaic acid that specifically affect PP2A reduced Raf-1 activation, and PP2A was found in Raf-1 immunoprecipitates from quiescent and mitogen-treated cells. Therefore, while an effect of the drug on other phosphatases cannot be formally excluded, PP2A presumably represents the okadaic acid-sensitive phosphatase involved in Raf-1 regulation. Our current working model is that Raf-1-associated PP2A facilitates kinase activation by maintaining Ser259 in a dephosphorylated state and thereby preventing the formation of inactive 14-3-3-Raf-1 complexes (14.Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 15.Rommel C. Radziwill G. Moelling K. Hafen E. Mech. Dev. 1997; 64: 95-104Crossref PubMed Scopus (40) Google Scholar, 16.Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (390) Google Scholar, 17.Roy S. McPherson R.A. Apolloni A. Yan J. Lane A. Clyde-Smith J. Hancock J.F. Mol. Cell. Biol. 1998; 18: 3947-3955Crossref PubMed Scopus (116) Google Scholar, 18.Thorson J.A., Yu, L.W. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar). This may permit the activation of a larger number of Raf-1 molecules and prolong it by counteracting the mitogen-induced kinase (probably protein kinase B) that phosphorylates Ser259. Ultimately, the Ser259 kinase must outpace PP2A to terminate Raf-1 activation. Inhibition of PP2A by okadaic acid has been reported to selectively impair Raf-dependent transformation (51.Sakai R. Ikeda I. Kitani H. Fujiki H. Takaku F. Rapp U. Sugimura T. Nagao M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9946-9950Crossref PubMed Scopus (54) Google Scholar). Furthermore, in genetically dissectable organisms, hypomorphic alleles of PP2A suppress the effects of activated Raf (52.Wassarman D.A. Solomon N.M. Chang H.C. Karim F.D. Therrien M. Rubin G.M. Genes Dev. 1996; 10: 272-278Crossref PubMed Scopus (107) Google Scholar) or enhance the loss of function phenotype of Raf mutations (50.Sieburth D.S. Sundaram M. Howard R.M. Han M. Genes Dev. 1999; 13: 2562-2569Crossref PubMed Scopus (69) Google Scholar). On the basis of these results, PP2A might have been considered either a Raf-1 effector or a positive regulator of Raf-1 activation. Our findings provide a mechanistic explanation for these observations. By identifying PP2A as a positive regulator of Raf-1, our data define a new function for this phosphatase and add a new facet to the complexity of Raf-1 regulation." @default.
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• W2044481116 title "Raf-1-associated Protein Phosphatase 2A as a Positive Regulator of Kinase Activation" @default.
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