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 beta 1 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 subunits 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 non-phosphorylated 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.
Johansson
M W
MW
La Jolla Cancer Research Foundation, California 92037.
Larsson
E
E
Lüning
B
B
Pasquale
E B
EB
Ruoslahti
E
E
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses; D002448:Cell Adhesion; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000648:ultrastructure; D002472:Cell Transformation, Viral; D019012:Integrin beta1; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D019000:Phosphotyrosine; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D019012:Integrin beta1; D016023:Integrins; D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine