The pleckstrin homology (PH) domain is a conserved protein module present in diverse signal transducing proteins. To investigate the function of the PH domain of the Ras exchanger Sos, we have generated a recombinant (His)6‐tagged PH domain from human Sos1 (PH‐Sos). Here we show that PH‐Sos binds with high affinity(1.5 μM) to lipid vesicles containing the negatively charged phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2). When microinjected into serum‐deprived rat embryo fibroblasts or COS cells, PH‐Sos displays a homogenous subcellular distribution. However, PH‐Sos rapidly accumulates in the plasma membrane following serum stimulation and, under these conditions, is localized preferentially to the leading edge of motile cells. Surprisingly, the membrane localization of PH‐Sos is not dependent on its ability to bind PIP2. Overexpression of the PH domain of Sos has a pronounced dominant‐negative effect on serum‐induced activation of the Ras signaling pathway. These results suggest that the PH domain of Sos participates in regulating the inducible association of Sos with the membrane, and indicate the presence of specific ligands that interact with this domain to bring about the activation of Ras.

The nuclear oncoprotein v-Myb is a transcriptional activator in both animal cells and the budding yeast Saccharomyces cerevisiae. Previous studies have suggested that an acidic domain of approximately 50 amino acids (amino acids 204-254 of v-Myb) is necessary and sufficient for transcriptional activation by v-Myb, c-Myb and GAL4-Myb fusion proteins. However, we find that first, none of the acidic residues within this region is essential for transcriptional activation in either animal cells or yeast. Second, transcriptional activation requires cooperation among multiple domains of v-Myb. In animal cells, transcriptional activation by v-Myb requires a central domain (amino acids 234-295), a C-terminal domain (amino acids 295-356), plus either of two more N-terminal domains (amino acids 163-197 or 198-232); in yeast, it requires the central domain plus either the C-terminal domain or a more N-terminal domain (amino acids 163-233). Third, although various subsets of these domains are sufficient for transcriptional activation by v-Myb, all of the domains must be present for transformation of primary hematopoietic cells. These results demonstrate that transcriptional activation by v-Myb is not sufficient for oncogenic transformation.

The v-myb oncogene and its cellular homolog c-myb encode sequence-specific DNA-binding proteins which regulate transcription from promoters containing Myb-binding sites in animal cells. We have developed a Saccharomyces cerevisiae system to assay transcriptional activation by v-Myb and c-Myb. In yeast strains containing integrated reporter genes, activation was strictly dependent upon both the Myb DNA-binding domain and the Myb recognition element. BAS1, an endogenous Myb-related yeast protein, was not required for transactivation by animal Myb proteins and by itself had no detectable effect on a Myb reporter gene. Deletion analyses demonstrated that a domain of v-Myb C terminal to the previously mapped Myb transcriptional activation domain was required for transactivation in animal cells but not in S. cerevisiae. The same domain is also required for the efficient transformation of myeloid cells by v-Myb. In contrast to results in animal cells, in S. cerevisiae the full-length c-Myb was a much stronger transactivator than a protein bearing the oncogenic N- and C-terminal truncations of v-Myb. These results imply that negative regulation of c-Myb by its own termini requires an additional animal cell protein or small molecule that is not present in S. cerevisiae.