TYROSINE PHOSPHORYLATION OF P95(VAV) IN MYELOID CELLS IS REGULATED BY GM-CSF, IL-3 AND STEEL FACTOR AND IS CONSTITUTIVELY INCREASED BY P210(BCR/ABL)

Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95(Vav) is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95(Vav) in myeloid cells. In the factor-dependent MO7e cell line, p95(Vav) was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95(Vav) Tyrosine phosphorylation of p95(Vav) was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate, Since many of the cytokines known to induce tyrosine phosphorylation of p95(Vav) are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95(Vav) with JAK kinases, p95(Vav) coprecipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95(Vav) in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95(Vav) (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95(Vav). GST-Vav-SH2, but not GST, also precipitated JAK1, JAK3 and Tyk2, suggesting that other JAK family kinases might interact with p95(Vav). These results suggest that tyrosine phosphorylation of p95(Vav) is potentially directly regulated by JAK kinases, and further suggest that Vav is broadly involved in signal transduction in myeloid cells initiated by many cytokines and the oncogene BCR/ABL.