The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras.
The serine/threonine kinase Raf-1 is an essential component of the MAPK cascade. Activation of Raf-1 by extracellular signals is initiated by association with intracellular membranes. Recruitment of Raf-1 to membranes has been reported to be mediated by direct association with Ras and by the phospholipase D product phosphatidic acid (PA). Here we report that insulin stimulation of HIRcB fibroblasts leads to accumulation of Ras, Raf-1, phosphorylated MEK, phosphorylated MAPK, and PA on endosomal membranes. Mutations that disrupt Raf-PA interactions prevented recruitment of Raf-1 to membranes, whereas disruption of Ras-Raf interactions did not affect agonist-dependent translocation. Expression of a dominant-negative Ras mutant did not prevent insulin-dependent Raf-1 translocation, but inhibited phosphorylation of MAPK. Finally, the PA-binding region of Raf-1 was sufficient to target green fluorescent protein to membranes, and its overexpression blocked recruitment of Raf-1 to membranes and disrupted insulin-dependent MAPK phosphorylation. These results indicate that agonist-dependent Raf-1 translocation is primarily mediated by a direct interaction with PA and is independent of association with Ras.
Rizzo
Megan A
MA
Departments of Pharmacology and Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Shome
K
K
Watkins
S C
SC
Romero
G
G
D000818:Animals; D001692:Biological Transport; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D010712:Phosphatidic Acids / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000819:agonists / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D010712:Phosphatidic Acids; D011993:Recombinant Fusion Proteins; D011972:Receptor, Insulin; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins