The protein products of the raf gene family are cytoplasmic serine/threonine protein kinases. They are active in the transmission of mitogenic signals from membrane-associated tyrosine kinase growth factor receptors to the nucleus. The Raf protein itself is phosphorylated, and its kinase is activated by several of the tyrosine kinases and other serine/threonine kinases. A series of point and deletion mutations have been used to identify the site of tyrosine phosphorylation and two of the major serine phosphorylation sites in the Raf kinase. They are Y230, S42, and S497/499. Several approaches to generate systems which produce activated Raf protein or the dominant negative raf mutant K375W conditionally have led to success. We have identified two point mutations responsible for the temperature-sensitive phenotype of the v-mil gene on an MH2 mutant stock. Both involve highly conserved residues found on all protein kinases. Incorporation of these mutations, either singly or in combination, into an activated raf background has conferred the temperature-sensitive phenotype. To study the effect of inactivating mutations, it is necessary to eliminate the endogenous Raf protein. To this end, we have generated vectors which express the antisense of the first 250 nucleotides of the raf mRNA. Constitutive expression of this antisense significantly lowers the number of colonies obtained after transfection compared to constructs expressing the sense sequence. Similar experiments have been done using other parts of the message with comparable results. Constructs which express these antisense RNAs, or the dominant negative raf mutant S375K under the control of either the heat-shock promoter or the glucocorticoid inducible MMTV-LTR, allow us to study their effects in a conditional system. Another approach to study the effect of wild-type and mutant Raf-1 on the signaling cascade is to analyze germinal vesicle breakdown in Xenopus oocytes following microinjection of the appropriate c-raf-1 mRNAs. To this end, we have obtained and sequenced a c-raf-l- homologous cDNA from Xenopus and have incorporated it, and several mutants, into an RNA expression vector.
