| Abstrakt: |
Ligation of the α6β4integrin induces tyrosine phosphorylation of the β4cytoplasmic 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 β4that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr1440, or secondarily Tyr1422, interacts with the SH2 domain of Shc, whereas Tyr1526, or secondarily Tyr1642, 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 α6β4. In addition, mutation of β4Tyr1526, which binds to the PTB domain of Shc, but not of Tyr1422and Tyr1440, which interact with its SH2 domain, abolishes the activation of ERK by α6β4. Phenylalanine substitution of the β4tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of α6β4in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of β4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type β4. This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of β4containing phenylalanine substitutions at Tyr1422and Tyr1440, at Tyr1526and Tyr1642, or at all four tyrosine phosphorylation sites. These results suggest that β4Tyr1526interacts 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. |
