Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.

摘要: The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase, but the mechanisms of activation are incompletely understood. To dissect these mechanisms, wild-type and mutant proteins were studied in an vitro system with purified plasma membranes v-Ras- v-Src-transformed cells (transformed membranes). Wild-type (His)6- FLAG-Raf-1 activated a Ras- ATP-dependent manner by transformed membranes; however, that defective (K375M), lack vivo site(s) regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, do not bind (R89L), intact zinc finger (CC165/168SS) not. lacking putative sites for unidentified (S259A) C (S499A) apparently reduced efficiency. kinase(s) responsible Src may reside membrane, since GTP loading quiescent NIH 3T3 (parental membranes) induced de novo capacity Raf-1. Raf-1, possessing only basal activity, was parental absence loading. In contrast, Y340D, significant was, surprisingly, stimulated Ras-independent manner. results suggest phosphorylation be permissive further modulation another membrane factor, such as lipid. A factor(s) extracted methanol-chloroform Sf9 coexpressing SrcY527F significantly enhanced activity Y340D active inactive Our findings model wherein (i) associates Ras-GTP, (ii) is and/or (iii) increased cofactor.