THE CYSTEINE-RICH REGION OF RAF-1 KINASE CONTAINS ZINC, TRANSLOCATES TO LIPOSOMES, AND IS ADJACENT TO A SEGMENT THAT BINDS GTP-RAS

Different domains of the serine/threonine kinase, raf-1, were expresse d as fusion proteins with glutathione S-transferase (GST) in Escherich ia coli and purified to near homogeneity by affinity chromatography. A cysteine-rich domain of raf-1 was found to contain 2 mol of zinc (mol ar basis), similar to analogous cysteine-rich domains of protein kinas e C. GST-fusion proteins, containing the cysteine-rich domain of raf-1 , bound to liposomes in a phosphatidylserine-dependent manner. In cont rast to protein kinase C, the translocation of raf-1 was not dependent upon diacylglycerol, phorbol ester, or calcium, nor did raf-1 bind ph orbol esters. A GST-fusion protein encoding residues 1-147 of raf-1 bo und to normal GTP-ras with high affinity, but not to mutant GTP-Ala35 ras; no binding was detected to GDP-ras. The binding of a smaller fusi on protein (residues 1-130 of raf-1) was about 10-fold weaker, inferri ng that a 17-amino acid sequence represents a critical binding determi nant in intact raf-1. These residues are adjacent to the amino-termina l end of, and partially extend into, the cysteine-rich domain (amino a cids 139-184). A synthetic peptide corresponding to this 17-amino acid sequence blocked the interaction of raf-1 with ras. The function of t he cysteine-rich region of raf-1 homologous to protein kinase C is to promote translocation of raf-1 kinase to membranes and to form part of the high affinity binding site for GTP-ras.