ALTERED LOCALIZATION AND CYTOPLASMIC DOMAIN-BINDING PROPERTIES OF TYROSINE-PHOSPHORYLATED BETA(1) INTEGRIN

We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY) peptide) that represents a portion of the cytoplasmic domain of the beta(1) integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY beta(1) peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PY beta(1) peptide and the tyrosine-phosphorylated form of the intact beta 1( )subunit, but did not bind the nonphosphorylated beta(1) peptide, the nonphosphorylated beta(1) subunit or other unrelated tyrosine-phosphorylated proteins. The anti-PY beta(1) antibodies labeled the podosomes of Rous sarcoma virus-transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated beta(1) subunit appeared distinct from that of the beta(1) subunit. Adhesion plaques were stained by the anti-beta(1) subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3-kinase bound to the PY beta(1) peptide, but not to the nonphosphorylated beta(1) cytoplasmic peptide. Other SH2 domains did not bind to the PY beta(1) peptide. These results show that the phosphorylated form of the beta(1) integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the beta(1) subunit cytoplasmic domain may affect cellular signaling pathways.