Tyrosine phosphorylation of the β 4 integrin cytoplasmic domain mediates Shc signaling extracellular signal-regulated kinase and antagonizes formation of hemidesmosomes

Abstract

Ligation of the α 6β 4 integrin induces tyrosine phosphorylation of the β 4 cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of β 4 that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr 1440, or secondarily Tyr 1422, interacts with the SH2 domain of Shc, whereas Tyr 1526, or secondarily Tyr 1642, interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by α 6β 4. In addition, mutation of β 4 Tyr 1526, which binds to the PTB domain of Shc, but not of Tyr 1422 and Tyr 1440, which interact with its SH2 domain, abolishes the activation of ERK by α 6β 4. Phenylalanine substitution of the β 4 tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of α 6β 4 in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of β 4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type β 4. This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of β 4 containing phenylalanine substitutions at Tyr 1422 and Tyr 1440, at Tyr 1526 and Tyr 1642, or at all four tyrosine phosphorylation sites. These results suggest that β 4 Tyr 1526 interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.