PMC

cAMP agonists inhibit the phosphorylation of Raf-1 on serine 338. COS-1 cells were transfected with pCMV5Raf-1 or pCMV5-RafS259A, serum starved overnight, and treated with 40 μM forskolin plus 50 μM IBMX for 20 min and 100 ng of TPA per ml for 15 min as indicated. (A) The Raf-1 proteins were immunoprecipitated with the crafVI antibody and immunoblotted with anti-phosphoserine 338 and anti-serine 259 antibodies. After being stripped, the blots were stained with crafVI antiserum.

Raf-1 is phosphorylated by PKA on serine 259. Shown are tryptic phosphopeptide maps of recombinant Raf-1 phosphorylated by PKA (Raf + PKA) in vitro (A), recombinant RafS259A phosphorylated by PKA (RafS259A + PKA) in vitro (B), a mixture of Raf + PKA and RafS259A + PKA (C), endogenous Raf-1 immunoprecipitated from serum-starved NIH 3T3 cells labeled with [³²P]orthophosphoric acid (D), endogenous Raf-1 from forskolin-treated cells (40 μM, 30 min) (E), and a mixture of panels A and E (F). Phosphorylation sites are indicated. Asterisks indicate spurious in vitro phosphorylation sites that do not occur in cells. In panels D and E the spots were quantified with a phosphorimager and the relative changes are indicated. Equal amounts of Raf-1 protein were loaded.

The role of Raf-1 serine 43. (A) cAMP agonists and TPA induce phosphorylation of S43. COS-1 cells were transfected with pCMV5-Raf-1, serum starved overnight, and treated with 40 μM forskolin plus 100 μM IBMX for 30 min or 100 ng of TPA per ml for 15 min. Raf-1 immunoprecipitates were sequentially immunoblotted with phospho-Raf-1 S43 and Raf-1 antibodies. (B) COS-1 cells transfected with pCMV5-Raf-1 or pCMV5-RafS43A were serum starved overnight and treated with 100 μM 8Cl-cAMP, 100 ng of TPA per ml, or 20 ng of EGF per ml for 30 min as indicated. For cotreatments 8Cl-cAMP was administered first (20 min). Cell lysates were incubated with Ras/GTP beads for 1 h, and the pulldowns were analyzed by immunoblotting with a Raf-1 antibody. In the lower panel cell lysates were examined for the expression of transfected Raf proteins. (B) An aliquot of the cell lysates was immunoprecipitated with crafVI antibody to assay kinase activity by using a linked assay with MEK and kinase-negative ERK (ERK⁻) as substrates. The kinase assays were blotted, autoradiographed (upper panel), and stained with crafVI (lower panel). vect., vector-transfected cells; S43A, pCMV5-RafS43A-transfected cells.

cAMP inhibits the ERK pathway at the level of Raf-1 through the induction of the phosphorylation of serine 259 in Raf-1. (A) RafS259-stimulated ERK activity is resistant to inhibition by cAMP. COS-1 cells were transfected with HA-ERK-2 plus pCMV5Raf-1 or pCMV5RafS259D. Cells were serum starved and treated with forskolin and IBMX as in Fig. . HA-ERK-2 was immunoprecipitated with an HA antibody, and activated ERK was detected by immunoblotting with a phosphospecific ERK antibody (upper panel). The blot was stripped and subsequently stained with an anti-ERK antiserum. (B) The cAMP-induced increase in Raf-1 serine 259 phosphorylation is followed by inhibition of ERK. COS-1 cells were transfected with a small amount (0.4 μg) of FLAG-tagged Raf-1 in order to facilitate the use of the phosphoserine 259-specific antibody. Growing cells were treated with 10 μM H-89 for 30 min followed by 40 μM forskolin plus 100 μM IBMX for the indicated time points (Fsk/IBMX). Cells were lysed, and FLAG-Raf-1 was immunoprecipitated with anti-FLAG antibodies and stained with a phosphoserine 259-specific antibody (upper panel). The blots were stripped and subsequently stained with a FLAG antibody (lower panel). ERK phosphorylation (upper panel) and expression (lower panel) were examined by blotting whole-cell lysates. (C) RafS259-induced reporter gene expression is refractory to inhibition by cAMP. NIH 3T3 cells were cotransfected with a GAL4-Elk-1 transcription factor, a CAT reporter gene under the control of GAL-4 DNA binding sites and Raf-1 or RafS259D. Cells were treated as indicated, and CAT expression was measured 8 h posttreatment.

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.