CROSS-LINKING OF INTEGRINS INDUCES TYROSINE PHOSPHORYLATION OF THE PROTOONCOGENE PRODUCT VAV AND THE PROTEIN-TYROSINE KINASE SYK IN HUMAN FACTOR-DEPENDENT MYELOID CELLS

Attachment to extracellular matrix is important in the regulation of p roliferation and differentiation of hematopoietic stem and progenitor cells. Post-ligand occupancy events of integrin receptors in myeloid c ells are largely unknown. We examined early signaling events after sti mulation of integrin receptors (outside-in signal) using a cross-linki ng system in a growth factor-dependent myeloid cell line, M07e. alpha 4, alpha 5, and beta 1 integrin cross-linking induced a similar patter n of transient tyrosine phosphorylation of cellular proteins. The appr oximate 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-ph osphorylated 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-p hosphorylated by stimulation with Steel factor (SLF) and granulocyte m acrophage 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 al most equal to that by SLF stimulation, whereas that of Erk2 was much w eaker than with SLF stimulation. Upon integrin engagement, antibodies raised against Syk coprecipitated several tyrosine-phosphorylated prot eins. In vitro binding assays demonstrated that, among these Syk- asso ciated proteins, pp40, which differed from Erks, Crk, and Crk1, binds Syk through SH2 domains of Syk and is a prominent tyrosine-phosphoryla ted protein in integrin cross-linked cells. These results suggest that tyrosine phosphorylation of Vav and Erk2 in myeloid cells might be re gulated by both integrins and cytokines in the bone marrow microenviro nment, whereas Syk might be involved in a distinct pathway from that s hared between integrins and cytokines in myeloid cells.