ERK-1 overexpression induces Raf-1 phosphorylation on several sites, including S289, 296, and 301. (A and B) COS-7 cells expressing wild-type myc-Raf-1 (lanes 1–4), myc-Raf-1 S289/296/301A (lanes 5–8), or myc-Raf-1 S259A (lanes 9–12) alone (lanes 1, 2, 5, 6, 9, and 10) or coexpressing HA-ERK-1 (lanes 3, 4, 7, 8, 11, and 12) were metabolically labeled with ³²P and were left untreated or were stimulated with 100 ng/ml EGF for 30 min as indicated. myc-Raf-1 proteins were immunoprecipitated as in Figure 1, and 10% of the sample (A) was separated using 7.5% SDS-PAGE. Shown is an autoradiogram representing ³²P incorporation in myc-Raf-1 (top) and a myc immunoblot representing myc-Raf-1 protein recovery (bottom). The remaining 90% of the samples were subjected to phosphopeptide mapping (B) as described in the legends of Figure 1 and in Materials and Methods. Representative autoradiograms of Raf-1 phosphopeptide maps (1–12) and a schematic representation of the phosphopeptide spots (13) are presented. The positions of phospho S43, S621, and S259 peptides are indicated (13). The positions of spots 4, 5, 10, 11, and 12 are indicated by arrows (13 and 8). Note that the maps of the Raf-1 S259A mutant lack the spot corresponding to the pS259 peptide (9–12, indicated by arrow).

ERK-1 can directly phosphorylate Raf-1 in vitro. (A and C) Approximately 500 ng of wild-type (lanes 3 and 4) or S289/296/301A myc-Raf-1 protein (lanes 5 and 6) or a mock sample (lanes 1 and 2) were purified by myc immunoprecipitation from serum-deprived COS-7 cells and incubated with recombinant bacterial ERK-1 (ba, lanes 2, 4, and 6) or with same ERK-1 activated in vitro by incubation with active MEK (ac, lanes 1, 3, and 5) as detailed in Materials and Methods. Ten percent of the sample was resolved using 7.5% SDS-PAGE (A, bottom) and assayed for Raf-1 recovery by myc-immunoblotting, and the remaining 90% (A, top) was used for Raf-1 phosphopeptide mapping (C, 1 and 2). Note that 1 contains a map of Raf-1 excised from lane 3, and 2 contains a map of Raf-1 excised from lane 5. Panel 3 is a schematic representation of the phosphopeptide spots in 1 and 2. (B) In a similar experiment as described in A, wild-type myc-Raf-1 (lanes 1 and 3–6) or myc-Raf-1 S289/296/301A (trp, lane 2) was incubated with recombinant ERK-1 activated in vitro by active MEK (lanes 1 and 2) or with the indicated combinations of recombinant active Raf-1 (produced from sf9 cells coinfected with vRas and vSrc) and recombinant bacterial MEK-1 (lanes 3–6). Lane 7 is a control lane containing only the recombinant MEK and active Raf-1. Presented are an autoradiogram showing ³²P incorporation in Raf-1 (left) and a myc immunoblot showing the recovery of myc-Raf-1 (right). Indicated are the migration positions of ³²P myc-Raf-1 and MEK (left) and of myc-Raf-1 protein (right).

ERK-1 phosphorylation stabilizes the active form of Raf-1 by attenuating its inactivation rate. (A) Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 (left, lanes 1–8) or S289/296/301A myc-Raf-1 (left, lanes 9–16) or coexpressing HA-ERK-1 (right) were treated with 100 ng/ml EGF for the indicated time points. After stimulation, Raf-1 kinase activity in the samples was assayed as described in Materials and Methods. Presented are a phospho-MEK immunoblot showing the level of MEK phosphorylation (middle), a myc-immunoblot showing Raf-1 recovery (bottom), and a bar graph showing a densitometric quantification of the phospho-MEK band (top). The migration positions of phospho-MEK and myc-Raf-1 are indicated. (B) Serum-deprived COS-7 cells expressing myc-Raf-1 were treated with vehicle (lanes 1–8) or 20 μM U0126 for 30 min before EGF treatment. The samples were analyzed for Raf-1 kinase activity as described in A. (C) Serum-deprived COS-7 cells expressing HA-ERK-1 (lanes 1 and 2) or coexpressing HA-ERK-1 and either wild-type myc-Raf-1 (lanes 3 and 4), myc-Raf-1 S259A (lanes 5 and 6), myc-Raf-1 S289/296/301A (lanes 7 and 8), or myc-Raf-1 S259/289/296/301A (lanes 9 and 10) were stimulated with EGF as indicated for 20 min, and total cell extracts were immunoblotted for phospho-ERK, pS296 Raf-1 and myc-Raf-1 as indicated. (D) Wild-type myc-Raf-1 (lanes 1–8), S471A myc-Raf-1 mutant (a kinase inactive Raf mutant, lanes 9–14), and S289/296/301A myc-Raf-1 (lanes 15–20) were immunopurified from serum-deprived COS-7 cells (lanes 1–3, 7, 9–11, 15, 16, and 19) or from cells stimulated with EGF for 15 min (lanes 4–6, 8, 12–14, 17, 18, and 20). Raf kinase activity in the samples was assayed directly (lanes 7, 8, 19, and 20, representing a 0-time point) or after incubation with vehicle (lanes 1, 4, 9, 12, 15, and 17), recombinant ERK-1 (ba, lanes 2, 6, 10, and 14) or recombinant ERK-1 activated in vitro with MEK-1 (ac, lanes 3, 5, 11, 13, 16, and 18) under conditions provided in the experimental procedures. Presented are a phospho-MEK immunoblot showing the level of MEK phosphorylation (top) and a myc-immunoblot showing myc-Raf-1 recovery (bottom). The migration positions of phospho-MEK and myc-Raf-1 are indicated. (E) Wild-type myc-Raf-1 purified from serum-deprived (lane 1) or EGF-stimulated cells (lanes 2–12) was incubated for the indicated time points in a complete kinase reaction buffer (lanes 4, 6, 8, 10, and 12) or in a kinase reaction buffer lacking ATP (lanes 3, 5, 7, 9, and 11) at 30°C and assayed for Raf-1 kinase activity as described in A. Bottom, a Coomassie blue staining presenting myc-Raf-1 recovery and GST-MEK amounts. Lane 13 is a control lane containing GST-MEK alone.

Identification of Raf-1 S289, 296, and 301 as novel in vivo phosphorylation sites. (A) A diagram depicting known and newly identified Raf-1 phosphorylation sites and potential kinases reported to phosphorylate these sites. Indicated are RBD, Ras binding domain; CRD, cysteine-rich domain, CR1–3, conserved region 1–3; and K375, the ATP binding site. (B) A diagram showing Raf-1 sequence corresponding to amino acids 283–309. Indicated are the three phosphorylation sites identified by mass spectrometry analysis of myc-Raf-1 purified from COS-7 cells. (C) Serum-deprived COS-7 cells expressing wild type (1, 2 and 6, lanes 1 and 2) or S289/296/301A (3, 4 and 6, lanes 3 and 4) myc-Raf-1 were metabolically labeled with ³²P and were left untreated (basal) or stimulated with 100 ng/ml EGF for 30 min. myc-Raf-1 proteins were immunoprecipitated with a myc-epitope tag antibody, and 90% of the sample was subjected to phosphopeptide map analysis as described in Materials and Methods. The recovery of phospho-myc-Raf-1 (6, autoradiogram, top) and myc-Raf-1 protein (6, anti-myc blot, bottom) was determined by separating the remaining 10% sample on a separate gel. Representative autoradiograms of Raf-1 phosphopeptide maps (1–4) and a schematic representation of the phosphopeptide spots (5) are presented. The electrophoresis direction and the TLC chromatography orientation are indicated by arrows. ori in 5 represents the origin/spotting point. The location of phospho S43, S621, and S259 peptides is indicated. The locations of spots 4 and 7 are indicated by arrows. (D) Spot 4 from 2 was excised and analyzed for phosphoamino acid composition by two-dimensional electrophoresis as described in Materials and Methods. Presented are a ninhydrin staining of the TLC plate (right) showing the migration points of standard phospho-serine (P-S), phospho-threonine (P-T), and phospho-tyrosine (P-Y) and an autoradiogramshowing the radiolabeled phosphoamino acids (indicated are the corresponding migration positions of P-S, P-T, P-Y, and the free phosphate).

MEK inhibition blocks Raf-1 phosphorylation on EGF- and ERK-1-induced sites. COS-7 cells expressing wild-type myc-Raf-1 alone (1, 2, 5, 6, and 10, lanes 1, 2, 5, and 6) or coexpressing HA-ERK-1 (3, 4, 7, 8, and 10, lanes 3, 4, 7, and 8) were treated with vehicle (1–4 and 10, lanes 1–4) or were incubated in the presence of 20 μM U0126 for 30 min before ³²P metabolic labeling. After the labeling, cells were left untreated (1, 3, 5, 7, and 10, lanes 1, 3, 5, and 7) or were stimulated with 100 ng/ml EGF for 30 min (2, 4, 6, 8, and 10, lanes 2, 4, 6, and 8). myc-Raf-1 phosphopeptide mapping was performed as in Figure 1C. Presented are representative autoradiograms of Raf-1 phosphopeptide maps (1–8), a schematic representation of the phosphopeptide spots (9), and a gel showing ³²P incorporation in myc-Raf-1 and myc-Raf-1 protein recovery (10). The positions of spots 4, 5, 7, 11, and 12 are indicated by arrows (8 and 9). Note the reduction in the intensity of the indicated spots, most notably in 6 and 8 compared with 2 and 4, respectively.

Phospho-S296 Raf-1 antibody demonstrates EGF-induced, ERK-dependent endogenous Raf-1 phosphorylation. (A) Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 (lanes 1 and 2), S289/296/301A myc-Raf-1 (lanes 3 and 4), or control vector (lanes 5 and 6) alone (lanes 1, 3, and 5) or coexpressing M2-FLAG-MEKDD (lanes 2, 4, and 6) were treated with vehicle (lanes 1, 3, and 5) or 100 ng/ml EGF for 30 min (lanes 2, 4, and 6). Then, 80 μg of total cell extract or an equivalent amount of myc-immunoprecipitates was separated on 7.5% SDS-PAGE and immunoblotted using anti-Raf-1pS296 antibody (top) or myc antibody (bottom). (B and C) Serum-deprived COS-7 cells (C) or myc-Raf-1 expressing COS-7 cells (B) pretreated with vehicle (lanes 1–7) or 10 μM U0126 for 18 h followed by a fresh dose 30 min before EGF stimulation (lanes 8–14) were treated with 100 ng/ml EGF for the indicated times. Total cell extracts were immunoblotted using the indicated antibodies. Indicated are the migration positions of myc-Raf-1 (B) and endogenous Raf-1 (C). (D) Serum-deprived COS-7 cells expressing myc-Raf-1 (lanes 1–8) or coexpressing myc-Raf-1 with HA-ERK-1 (lanes 9–24) pretreated with vehicle (lanes 1–16) or 10 μM U0126 for 18 h followed by a fresh dose 30 min before EGF stimulation (lanes 17–24) were treated with 100 ng/ml EGF for the indicated times. Total cell extracts were immunoblotted with anti-pS296 Raf-1 antibody. Indicated is the migration position of phospho-myc-Raf-1. The asterisks denote protein standard markers used for signal equalization between the two gels (lanes 1–8 and 9–24). (E) Serum-deprived COS-7 cells expressing myc-Raf-1 (lanes 1–7) or coexpressing myc-Raf-1 with ERK-1/2 RNAi (lanes 8–14) were treated with 100 ng/ml EGF for the indicated time points. Total cell extracts were immunoblotted with anti-pS296 Raf-1 antibody (top) or myc (bottom). Indicated are the migration positions of pS296-myc-Raf-1 (top) and myc-Raf-1 (bottom).

The ERK-phosphorylated Raf-1 population has increased specific kinase activity. (A) Serum-deprived COS-7 cells expressing a vector control (lanes 1, 2, 7, and 8), myc-Raf-1 (lanes 3–6, 9, and 10), or myc-Raf-1 S289/296/301A (lanes 11–14) were subjected to immunoprecipitation using myc antibodies (lanes 1–6, 11, and 12) or pS296 antibodies (lanes 7–10, 13, and 14). The immunoprecipitates were assayed for Raf-1 kinase activity as in Figure 6A. To help in evaluating Raf-1-specific kinase activity and standardizing for total Raf-1 amounts used in the kinase assay, 75% (lanes 3 and 4) or 25% (lanes 5 and 6) of the myc-Raf-1 immunoprecipitates were used in the kinase assays. Note that the total myc-Raf-1 used in the pS296 immunoprecipitates (lanes 9 and 10) is equal to the total myc-Raf-1 used in lanes 5 and 6, demonstrating up to fourfold increase in Raf-1 specific activity in the pS296 immunoprecipitated samples. The bottom panel represents the pS296-phosphorylated myc-Raf-1 recovery in the samples. (B) myc-Raf-1 from ³²P metabolically labeled COS-7 cells were immunoprecipitated using myc antibodies (samples 1 and 2) or pS296 antibodies (samples 3 and 4) and analyzed by 2D-phosphopeptide mapping (panels 1–4, the position of the ERK-induces sites 4 and 11 are indicated by arrows). The right part shows myc-Raf-1 recovery (bottom), myc-Raf-1 phosphorylation on S296 (middle), and total ³²P incorporation in myc-Raf-1 (top). Equal amounts of ³²P counts (1500 cpm) were used to generate the phosphopeptide maps.