Raf-1 phosphorylation sites. (A) Schematic diagram of Raf-1 showing known phosphorylation sites and potential kinases reported to phosphorylate these sites. The locations of the kinase domain, the Ras binding domain (RBD), the cysteine-rich domain (CRD), conserved regions 1–3 (CR 1–3), and the ATP binding site K375 are indicated. (B) Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 were metabolically labeled with ³²P for 2 h followed by stimulation with 100 ng/ml EGF or vehicle for 30 min. myc-Raf-1 proteins were immunoprecipitated and resolved using 7.5% SDS-PAGE. Ninety percent of the sample was analyzed by autoradiography (middle) and the remaining 10% were analyzed for myc-Raf-1 recovery (bottom). For quantifying ³²P incorporation in myc-Raf-1, the corresponding Raf-1 bands were excised from the gel and ³²P incorporation in Raf-1 was determined by Cherenkov counting (top). (C) myc-Raf-1 samples from B were subjected to phosphopeptide mapping as described in Materials and Methods. Representative autoradiograms of Raf-1 phosphopeptide maps (1 and 2) and a schematic representation of the phosphopeptide spots (3) are presented. The electrophoresis direction and the TLC chromatography orientation are indicated by arrows. “Ori” represents the spotting point. The location of phospho-S43, phospho-S621, and phospho-S259 peptides is indicated.

Identification of HSP-70 and HSC-70 as Raf-1 associated proteins. (A and B) Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 (lanes 2–4) or control vector (lane 1) were incubated with vehicle (lanes 1–3) or with 20 μM MEK inhibitor (U0126, lane 4) for 1 h before stimulation with vehicle (lane 2) or with 100 ng/ml EGF (lanes 1, 3, and 4) for 30 min. Cell lysates were precleared using mouse IgG pre-coupled to protein A beads for 90 min at 4°C and then immunoprecipitated using 9E10 myc antibody for 12 h at 4°C. After extensive washes, 90% of the sample was resolved using 7% SDS-PAGE and stained with Coomassie-Blue (A), whereas the remaining 10% were assayed for Raf-1 kinase activity using an MEK-ERK-coupled kinase assay (B). Bands corresponding to HSP-90, myc-Raf-1, and the 70-kDa doublet band below myc-Raf-1 were excised from the gel and analyzed using electrospray mass spectrometry either for protein identity, or in the case of myc-Raf-1, for phosphorylation site identification. The 90-kDa protein was confirmed to be HSP-90, and the 70-kDa doublet was identified as HSP-70 and HSC-70. For attaining enough Raf-1 protein samples for the phosphorylation site identification (4 to 5 μg), twenty 15-cm dishes and 50 μg of myc antibody were used per each point. The migration points of HSP-90, myc-Raf-1, and IgG are indicated. (C) Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 (lanes 2, 3, 5, and 6) or control vector (lanes 1 and 4) were stimulated with vehicle (lanes 2 and 5) or with 100 ng/ml EGF (lanes 1, 3, 4, and 6) for 30 min. Total cell extracts (lanes 1–3) and myc-immunoprecipitates (lanes 4–6) were resolved using 7.5% SDS-PAGE followed by immunoblot analysis using HSC-70 (top) HSP-70 (middle), or myc (bottom) antibodies. (D) Exponentially growing (lanes 1, 3, 4, 6, and 7) or heat-shock treated HEK-293 cells (30 min at 42°C, 6 h before cell extraction; lanes 2, 5, and 8) were lysed and cell extracts containing 10 mg of protein were used for immunoprecipitation s using HSC-70 (lanes 1 and 2), HSP-70 (lanes 4 and 5), or control IgG (lanes 3 and 6). The immunoprecipitates (lanes 1–6) and 50 μg of total cell extracts (lanes 7 and 8) were separated using 7.5% SDS-PAGE and immunoblotted for Raf-1.

Kinase activity comparison of Raf-1 phosphorylation site mutants. The indicated Raf-1 phosphorylation site mutants were expressed in COS-7 cells, and their kinase activity was assayed using the coupled Raf kinase assay. Cells were stimulated with 100 ng/ml EGF or vehicle for 15 min (left) or for 15 or 30 min as indicated (right). Presented are an autoradiogram showing ³²P incorporation in ERK (middle), a graphical representation of ³²P incorporation in ERK quantified using phosphor imaging (top), and a myc immunoblot showing myc-Raf-1 recovery (bottom).

Raf-1 S471 is critical for Raf-1 function in vivo. Serum-deprived COS-7 cells expressing myc-Raf-1 variants and FLAG-MEK (A) or HA-ERK (B and C), as indicated, were stimulated with 100 ng/ml EGF or vehicle for 15 min. Raf-1 activity in the cells was examined by determining phosphorylation at the activation sites of MEK (A) and ERK (B and C). MEK phosphorylation was determined both in FLAG-MEK immunoprecipitates (A, top two) and in total cell extracts (A, third from top). The endogenous MEK (endo), FLAG-MEK, and myc-Raf-1 variant expression in the cells was determined by anti-MEK (A, 4^th panel from top), anti-FLAG (A, fifth from top) or anti-myc (A, bottom) immunoblotting correspondingly. ERK phosphorylation was determined in HA-ERK immunoprecipitates (B and C, top). HA-ERK recovery in the immunoprecipitates (B and C, middle) and myc-Raf-1 variant expression in the cells (B and C, bottom) were determined by anti-HA and anti-myc immunoblotting, respectively.

B-Raf S578, corresponding to Raf-1 S471, is required for B-Raf kinase activation by EGF but not for the constitutive kinase activity induced by the V599E cancer associated mutation. Serum-deprived COS-7 cells expressing HA-ERK alone (lanes 1 and 2) or coexpressing wild-type FLAG-B-Raf (lanes 3 and 4), FLAG-B-Raf V599E (lanes 5 and 6), FLAG-B-Raf S578A (lanes 7 and 8), FLAG-B-Raf T588A (lanes 9 and 10), FLAG-B-Raf S578A/V599E (lanes 11 and 12) or FLAG-B-Raf T588A/V599E (lanes 13 and 14) were stimulated with 100 ng/ml EGF or vehicle for 15 min as indicated. B-Raf activity in the cells was assessed by HA immunoprecipitation and determining ERK phosphorylation at its activation sites (top). HA-ERK recovery and FLAG-B-Raf variant expression in the cells were determined by anti-ERK (middle) and anti-FLAG immunoblotting (bottom), respectively.

Dephosphorylation of Raf-1, primarily at EGF-induced sites, results in Raf-1 kinase inactivation. (A and B) Serum-deprived COS-7 cells expressing myc-Raf-1 S259A mutant were metabolically labeled with ³²P for 2 h, followed by stimulation with 100 ng/ml EGF (2–4) or vehicle (1) for 30 min. After myc immunoprecipitation, samples were incubated with vehicle (1, 2, and 4) or with 1 mM pS621 phosphopeptide corresponding to Raf-1 amino acids 613–627 to displace coassociated 14-3-3 (2) for 30 min at 4°C. After extensive washes, the samples were incubated with vehicle (1 and 4) or with protein phosphatase 1-γ (PP1-γ; 0.2 mU/ml, samples 2 and 3) for 30 min at 12°C. After extensive washes, 70% of the sample was analyzed by phosphopeptide mapping (A) and the remaining was assayed for Raf-1 kinase activity using an MEK-ERK-coupled kinase assay as described in Materials and Methods (B). Shown are representative autoradiograms of the phosphopeptide maps (A, 1–4) and a schematic representation of the phosphopeptide spots (A, 5; the location of the missing pS259 peptide spot is indicated with an arrow); and an autoradiogram of the Raf-1 kinase assay (B, middle), a myc-Raf-1 immunoblot (B, bottom), and a graphical representation of ³²P incorporation in ERK quantified using phosphorimaging (top, PI units are arbitrary phosphorimager units).

Identification of Raf-1 S29, S289, S296, S301, and S471/T481 as novel Raf-1 phosphorylation sites. (A) Schematic diagram of Raf-1 showing known phosphorylation sites (top) and the newly identified phosphorylation sites (bottom). (B) myc-Raf-1 proteins were purified as described in Figure 3A and subjected to electrospray mass spectrometry to identify phosphorylation sites. The result pertaining to identification of phosphorylation at a peptide corresponding to Raf-1 471-483 is presented. Samples were analyzed after either treatment with calf intestinal phosphatase (+CIP) or with vehicle (-CIP). The mass picks corresponding to Raf-1 471-483 mass, 1472.6, and its phosphorylated form, 1552.6, are indicated. The sequence of Raf-1 471-483 peptide is provided with its two potential phosphorylation sites indicated. (C) Alignment of Raf-1 455-495 region containing subdomain VIB of the Raf-1 kinase domain with other Raf family members. The locations of Raf-1 S471 and T481 as well as V492, corresponding to the B-Raf activating mutation site V599, are indicated with arrows. D-Raf-1 and CE-Raf (lin-45) are the Drosophila and C. elegans Raf proteins, correspondingly.

Raf-1 S471 is a critical site for Raf-1 kinase activity. Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 or the indicated myc-Raf-1 S471-substituted mutants (A and B), or the indicated myc-Raf-1 T481-substituted mutants (C and D) were stimulated with 100 ng/ml EGF or vehicle for 15 min. The myc-Raf-1 variants were immunoprecipitated and assayed for kinase activity in a coupled kinase assay, and ERK phosphorylation was determined either by ³²P incorporation (A and C) or by immunoblotting with phospho-ERK specific antibodies (B and D). Presented are an autoradiogram showing ³²P incorporation in ERK (A and C, middle), a graphical representation of ³²P incorporation in ERK quantified using phosphor-imaging (A and C, top), a myc immunoblot showing myc-Raf-1 recovery (bottom) and an immunoblot showing ERK phosphorylation (B and D, top).

Raf-1 S471 is required for the kinase activity of a constitutively active Raf form, Bxb-Raf, both in vivo and in vitro. (A and B) Serum-deprived COS-7 cells expressing HA-ERK (A) or FLAG-MEK (B) together with wild-type GST-Bxb-Raf or a mutant containing serine to alanine substitution at a site corresponding to Raf-1 S471 (S471A), as indicated, were stimulated with 100 ng/ml EGF or vehicle for 15 min. Raf activity in the cells was examined as in Figure 7 by determining phosphorylation of ERK in HA-ERK immunoprecipitates (A, top) and phosphorylation of MEK in total cell extracts (B, top). Shown are also HA-ERK recovery (A, middle) and FLAG-MEK (B, middle) and GST-Bxb-Raf (A and B, bottom) cellular expressions. (C) Serum-deprived COS-7 cells expressing wild-type GST-Bxb-Raf (lanes 1 and 2) or S471A GST-Bxb-Raf mutant (lanes 3 and 4) were stimulated with 100 ng/ml EGF or vehicle for 15 min. After GSH-pull-down, the kinase activity of the GST-Bxb-Raf forms was examined using the coupled Raf kinase assay. ERK and MEK phosphorylations were determined by phospho-ERK (bottom) and phospho-MEK (top) immunoblotting, respectively. GST-Bxb-Raf recovery was determined by anti-Raf-1 immunoblotting (middle).

Raf-1 S471 is required for Raf-1-MEK binding but not for Raf-1 dimerization or Raf-1 binding to Ras and 14-3-3. (A) COS-7 cells were transfected with FLAG-Ras G12V together with myc-Raf-1forms as indicated. myc-Raf-1 recovery in FLAG-Ras immunoprecipitates was determined using anti-myc immunoblotting (top). Also shown are myc-Raf expression in total cell lysates (middle) and FLAG-Ras recovery in the immunoprecipitates (bottom). (B) Serum-deprived COS-7 cells expressing a control GST vector or GST-Raf-1 fusion together with myc-Raf-1 variants as indicated were stimulated with 100 ng/ml EGF or vehicle for 15 min. myc-Raf-1 recovery in GST-pull-downs was determined using anti-myc immunoblotting (top). Also shown are myc-Raf-1 expression in total cell lysates (middle) and GST-fusion protein recovery in the GSH-pull-downs (bottom). (C) COS-7 cells were transfected with an empty vector (lane 1), GST control vector (lanes 2, 4, and 9), GST-MEK-1 (lanes 5–8 and 10), or GST-14-3-3 (lanes 11–14) together with the indicated myc-Raf-1 variants. myc-Raf-1 recovery in GST-pull-downs was determined using anti-myc immunoblotting (top). Also shown are GST-fusion protein recoveries in the GSH-pull-downs (middle) and myc-Raf-1 expression in total cell lysates (bottom).