Serine 338 phosphorylation is dispensable for activation of c-Raf1.
Numerous extracellular agonists induce consecutive stimulation of Ras guanine nucleotide exchange factors, Ras and c-Raf1, as the starting point of the intracellular mitogen-activated protein kinase cascade. Recent data point to a more complex reaction pattern of this simple sequence. This study was aimed at elucidating the activation process of endogenous c-Raf1 in U937 cells. Treatment of permeabilized U937 cells with the nonhydrolyzable nucleotide guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) induced prolonged stimulation of Ras and c-Raf1 activity. Intriguingly, both signaling proteins expressed differential responses toward specific inhibitors of phosphoinositide 3-kinases and tyrosine kinases, which indicates diverse signaling reactions feeding into Ras and cRaf-1. Phosphorylation of c-Raf1 serine 338 by p21-activated kinase has been recently reported to contribute to phosphoinositide 3-kinase-dependent activation of c-Raf1. However, in U937 cells stimulation of c-Raf1 activity by GTPgammaS did not correlate with p21-activated kinase activity and Ser-338 phosphorylation. Thus Ser-338 phosphorylation appears dispensable for c-Raf1 activation under the conditions used. Together these data deny an essential role for serine 338 phosphorylation in c-Raf1 activation and disclose divergent signaling connections of Ras and c-Raf1 in U937 cells.
Oehrl
Wolf
W
Institute for Molecular Cell Biology, University of Jena, 07747 Jena, Germany.
Rubio
Ignacio
I
Wetzker
Reinhard
R
D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D019833:Genistein / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism; D020298:U937 Cells; D000077191:Wortmannin; D018631:ras Proteins / Q000378:metabolism*
D000730:Androstadienes; D004791:Enzyme Inhibitors; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014443:Tyrosine; D012694:Serine; D006160:Guanosine Triphosphate; D019833:Genistein; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D000077191:Wortmannin