Cross-linking of integrins induces tyrosine phosphorylation of the proto-oncogene product Vav and the protein tyrosine kinase Syk in human factor-dependent myeloid cells.

Attachment to extracellular matrix is important in the regulation of proliferation and differentiation of hematopoietic stem and progenitor cells. Post-ligand occupancy events of integrin receptors in myeloid cells are largely unknown. We examined early signaling events after stimulation of integrin receptors (outside-in signal) using a cross-linking system in a growth factor-dependent myeloid cell line, M07e, alpha 4, alpha 5, and beta 1 integrin cross-linking induced a similar pattern of transient tyrosine phosphorylation of cellular proteins. The approximate molecular weights of these phosphoproteins were M(r) 150,000, M(r) 120,000-125,000, M(r) 95,000, M(r) 70,000, M(r) 60,000, and M(r) 40,000-50,000. Vav, Syk, and Erk2 were identified as some of the tyrosine-phosphorylated proteins, and their weights were M(r) 95,000, M(r) 70,000, and M(r) 40,000-50,000, respectively. Erk2 and Vav were also tyrosine-phosphorylated by stimulation with Steel factor (SLF) and granulocyte macrophage colony-stimulating factor, whereas tyrosine phosphorylation of Syk was not induced by stimulation with these cytokines. The degree of tyrosine phosphorylation of Vav through integrin engagement was almost equal to that by SLF stimulation, whereas that of Erk2 was much weaker than with SLF stimulation. Upon integrin engagement, antibodies raised against Syk coprecipitated several tyrosine-phosphorylated proteins. In vitro binding assays demonstrated that, among these Syk-associated proteins, pp40, which differed from Erks, Crk, and Crkl, binds Syk through SH2 domains of Syk and is a prominent tyrosine-phosphorylated protein in integrin cross-linked cells. These results suggest that tyrosine phosphorylation of Vav and Erk2 in myeloid cells might be regulated by both integrins and cytokines in the bone marrow microenvironment, whereas Syk might be involved in a distinct pathway from the shared between integrins and cytokines in myeloid cells.