Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition.
Exposure of cells to mitogens or growth factors stimulates Raf-1 activity through a complex mechanism that involves binding to active Ras, phosphorylation on multiple residues, and protein-protein interactions. Recently it was shown that the amino terminus of Raf-1 contains an autoregulatory domain that can inhibit its activity in Xenopus oocytes. In the present work we show that expression of the Raf-1 autoinhibitory domain blocks extracellular signal-regulated kinase 2 activation by the Raf-1 catalytic domain in mammalian cells. We also show that phosphorylation of Raf-1 on serine 338 by PAK1 and tyrosines 340 and 341 by Src relieves autoinhibition and that this occurs through a specific decrease in the binding of the Raf-1 regulatory domain to its catalytic domain. In addition, we demonstrate that phosphorylation of threonine 491 and serine 494, two phosphorylation sites in the catalytic domain that are required for Raf-1 activation, is unlikely to regulate autoinhibition. These results demonstrate that the autoinhibitory domain of Raf-1 is functional in mammalian cells and that its interaction with the Raf-1 catalytic domain is regulated by phosphorylation of serine 338 and tyrosines 340 and 341.
Tran
Nancy H
NH
Department of Integrative Biology and Pharmacology, University of Texas Houston Health Science Center, Houston, Texas 77030, USA.
Frost
Jeffrey A
JA
D000818:Animals; D020134:Catalytic Domain; D006801:Humans; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014162:Transfection; D014981:Xenopus; D054462:p21-Activated Kinases
D012694:Serine; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D054462:p21-Activated Kinases