Single point mutations in the SH2 domain impair the transforming potential of vav and fail to activate proto-vav.
The importance of an intact Src Homology 2 (SH2) domain for transformation by members of the tyrosine kinase family, including v-src, c-src, c-abl, fps and fyn is well documented. To determine the role of the SH2 domain in transformation by a protein which is not a member of this family, we employed site directed mutagenesis to change four highly conserved residues in the SH2 domain of the vav oncogene and the vav proto-oncogene (proto-vav). Proto-vav encodes a protein that contains one SH2 domain and two Src Homology 3 (SH3) domains, in addition to a number of other motifs usually found in transcriptional factors and guanine nucleotide exchange factors. Substitution of arginine 629 to glycine (R629G) and arginine 647 to leucine (R647L) in vav did not impair its transforming potential in NIH3T3 fibroblasts. By contrast, substitutions of tryptophan 622 to arginine (W622R) and glycine 642 to valine (G642V) in the vavSH2, greatly reduced its transforming potential. Similar point mutations introduced in the SH2 domain of proto-vav did not activate the transforming potential of the normal gene. Interestingly, although all the vav SH2 mutant proteins were constitutively phosphorylated on tyrosine when expressed in NIH3T3 cells, they fail to bind to a phosphorylated epidermal growth factor receptor (EGFR), regardless of their transforming potential.
Katzav
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Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002471:Cell Transformation, Neoplastic; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009857:Oncogenes; D010766:Phosphorylation; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051567:Proto-Oncogene Proteins c-vav; D011519:Proto-Oncogenes; D013329:Structure-Activity Relationship
D018797:Cell Cycle Proteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; C496619:Vav1 protein, mouse; D004815:Epidermal Growth Factor; D066246:ErbB Receptors