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. 2002 May;22(10):3237–3246. doi: 10.1128/MCB.22.10.3237-3246.2002

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Copyright © 2002, American Society for Microbiology

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FIG. 3. — Mutation of Raf-1 serine 259 impedes the downregulation of Raf kinase activity by PKA. (A) Sf-9 cells were coinfected with baculoviruses encoding Raf-1, the RafS259D, or the kinase-negative RafK375 M mutant in combination with Ras plus Lck and PKA as indicated. The kinase activity of Raf-1 immunoprecipitates was measured in a linked assay using MEK and kinase-negative ERK (ERK⁻) as substrates. con, no Raf added. (B) COS-1 cells were transiently transfected with Raf-1, RafS259A, or RafS259D cloned into pCMV5. On day 3 posttransfection serum-starved cells were preincubated with 25 μg of forskolin per ml for 30 min and then treated with TPA (100 ng/ml) plus EGF (20 ng/ml) for 15 min as indicated. Raf-1 was immunoprecipitated and subjected to a linked kinase assay using recombinant MEK and kinase-negative ERK as substrates. The data shown represent three independent experiments. The kinase reactions were blotted, autoradiographed, and subsequently stained with crafVI antiserum. (C) Kinase activity of a RafS43A/S259A double mutant. COS cells were transfected with the indicated expression plasmids. Cells were treated with 20 μM forskolin (Fsk) plus 100 μM IBMX for 30 min followed by 100 ng of TPA per ml for 15 min as indicated. Raf proteins were immunoprecipitated with crafVI and examined for kinase activity by the two-step kinase assay. An anti-Raf Western blot of the immunoprecipitates is shown in the lower panel. (D) Endogenous Raf-1 was immunoprecipitated by using the phosphoserine 259-specific antiserum from COS cells treated as indicated for panel C. The supernatants were subsequently immunoprecipitated with a non-phosphorylation-sensitive Raf-1 antiserum. The precipitates were immunoblotted with anti-phosphoserine 259 and the anti-Raf antisera. In the blots shown the loading of the anti-phosphoserine 259 immunoprecipitates was adjusted to equal levels of total Raf-1 protein, and the fold induction of serine 259 phosphorylation is given below. The graph shows a quantitation of the unadjusted immunoprecipitates. The fraction of Raf-1 immunodepleted by the phosphoserine 259 antiserum is plotted against the amount of total Raf-1 according to the following formula: (Raf immunodepleted by α-p259)/(Raf immunodepleted by α-p259 + Raf subsequently immunoprecipitated with α-Raf-1 antiserum). Quantification of blots was done with PDI scan.

FIG. 3. — Mutation of Raf-1 serine 259 impedes the downregulation of Raf kinase activity by PKA. (A) Sf-9 cells were coinfected with baculoviruses encoding Raf-1, the RafS259D, or the kinase-negative RafK375 M mutant in combination with Ras plus Lck and PKA as indicated. The kinase activity of Raf-1 immunoprecipitates was measured in a linked assay using MEK and kinase-negative ERK (ERK⁻) as substrates. con, no Raf added. (B) COS-1 cells were transiently transfected with Raf-1, RafS259A, or RafS259D cloned into pCMV5. On day 3 posttransfection serum-starved cells were preincubated with 25 μg of forskolin per ml for 30 min and then treated with TPA (100 ng/ml) plus EGF (20 ng/ml) for 15 min as indicated. Raf-1 was immunoprecipitated and subjected to a linked kinase assay using recombinant MEK and kinase-negative ERK as substrates. The data shown represent three independent experiments. The kinase reactions were blotted, autoradiographed, and subsequently stained with crafVI antiserum. (C) Kinase activity of a RafS43A/S259A double mutant. COS cells were transfected with the indicated expression plasmids. Cells were treated with 20 μM forskolin (Fsk) plus 100 μM IBMX for 30 min followed by 100 ng of TPA per ml for 15 min as indicated. Raf proteins were immunoprecipitated with crafVI and examined for kinase activity by the two-step kinase assay. An anti-Raf Western blot of the immunoprecipitates is shown in the lower panel. (D) Endogenous Raf-1 was immunoprecipitated by using the phosphoserine 259-specific antiserum from COS cells treated as indicated for panel C. The supernatants were subsequently immunoprecipitated with a non-phosphorylation-sensitive Raf-1 antiserum. The precipitates were immunoblotted with anti-phosphoserine 259 and the anti-Raf antisera. In the blots shown the loading of the anti-phosphoserine 259 immunoprecipitates was adjusted to equal levels of total Raf-1 protein, and the fold induction of serine 259 phosphorylation is given below. The graph shows a quantitation of the unadjusted immunoprecipitates. The fraction of Raf-1 immunodepleted by the phosphoserine 259 antiserum is plotted against the amount of total Raf-1 according to the following formula: (Raf immunodepleted by α-p259)/(Raf immunodepleted by α-p259 + Raf subsequently immunoprecipitated with α-Raf-1 antiserum). Quantification of blots was done with PDI scan.