Mutagenic analysis of Vav reveals that an intact SH3 domain is required for transformation

Abstract

The vav proto-oncogene encodes a protein with multiple modulae domains that enable it to function as a mediator, linking tyrosine signaling to downstream events in hematopoietic cells. Circumstantial evidence suggests that protein-protein interactions exerted by two of these domains, the Src homology 2 (SH2) and the Src homology 3 (SH3), play an important role in the regulation of Vav activity. To study the relevance of the SH3 domain for the function of vav as a transforming gene, we have created several mutations in the SH3 domain located at its carboxy region. Substitution of the non-conserved aspartic acid 797 (to asparagine, D797N) retained the transforming potential of the vav oncogene, whereas substitutions of ®ve highly conserved amino-acids: alanine 789 (to asparagine, A789N), leucine 801 (to arginine, L801R), tryptophan 821 (to arginine, W821R), glycine 830 (to valine, G830V) and valine 837 (to glutamic acid, V837E) greatly reduced its transforming potential. The mutant proteins resemble Vav in many biochemical properties; however, while the transforming mutant protein (D797N) associates with several unidenti®ed proteins in a manner similar to that of Vav, the non-transforming mutant Vav proteins react very poorly with these proteins. Among the known Vav-interacting proteins, hnRNP-K associates with all mutant proteins except A789N and V837E whereas binding of Zyxin to any of the mutant proteins is not a ected. Taken together, our results clearly demonstrate that the SH3 domain has a positive e ect on vav activity and is needed for vav transformation. The vavSH3C associating protein(s) that are crucial for its activity as a transforming gene have probably not yet been identi®ed.

Critical Role of the Pleckstrin Homology and Cysteine-rich Domains in Vav Signaling and Transforming Activity

“…We found that the transforming activities of the ⌬N-186 Vav SH3(W636K) and ⌬N-186 Vav SH3(W636K/W820K) mutants were decreased 3-6-fold, respectively. In contrast to the decreased activity described in a previous study (28), we found that the ⌬N-186 Vav SH3(820K) mutant showed an enhanced focus-forming ability. However, when we deleted the entire SH3/SH2/SH3 domain (⌬N-186 Vav⌬C), we were surprised to see that it was enhanced significantly in transforming activity.…”

“…Previous studies reached conflicting conclusions regarding the importance of SH domains for Vav transforming activity. Whereas missense mutation of the SH domains caused a loss of activity (28,29), deletion of the SH domains indicated that these domains were dispensable for transforming activity. The basis for these different observations is not known.…”

“…This domain organization is conserved in all three Vav family proteins. Although introduction of missense mutations into the SH2 and SH3 domains caused a loss of Vav transforming activity (28,29), subsequent deletion analyses showed that all the SH2/SH3 domains were dispensable for the transforming activity of all three Vav proteins (10,13,23). The basis for these contrasting observations is not known.…”

Proteomic characterization of the cellular response to chemopreventive triterpenoid astragaloside IV in human hepatocellular carcinoma cell line HepG2

“…Knock-down of Vav3 causes decreased receptor transactivation in cancer cells (19). As far as the role of Vav3 in the cell malignant transformation is concerned, the C-terminal SH3 domains appear to be indispensable to its cell transforming activity, whereas the N-terminal domains of Vav3 play a rather negative role (69,70). Due to the lack of GEF domain, Vav3.1 protein does not catalyze the exchange of GDP/GTP.…”

BCR/ABL, mRNA translation and apoptosis

“…105,106 In addition, PKCd 107 and MAPK/ERK 108 phosphorylate hnRNP K on serine 302 and on serines 284/353, respectively. While hnRNP K-Vav interaction may be relevant for Vav-transforming activity, 109 the importance of hnRNP K phosphorylation by PKCd for hnRNP K function as regulator of mRNA metabolism is still unclear.…”