Tyrosine phosphorylation: a critical component in the formation of hemidesmosomes

Our goal was to evaluate the role of tyrosine phosphorylation in the comple te formation of hemidesmosomes that occurs during development or during rem odeling after injury. A corneal organ culture system was used to study hemi desmosome formation as it would occur in an intact tissue. Phosphorylation of the integrin subunit beta 4 and bullous pemphigoid antigen-1 (BPAG-1) wa s examined, as these proteins are known to play a role in linking the elect ron-dense plaques along the basal surface with the intermediate filaments t o complete the formation of hemidesmosomes. Corneal epithelial sheets were placed on substrata that contained an intact basal lamina or basal laminae that had been either modified or removed. These constructs were incubated f or up to 18 h, and hemidesmosome formation was evaluated by using transmiss ion electron microscopy. When epithelial sheets were placed on intact basal laminae and incubated in the presence of the tyrosine kinase inhibitor gen istein (200 mu M), hemidesmosome formation was impaired. The formation of e lectron-dense regions was delayed, and no association of intermediate filam ents was detected. Results were confirmed by biochemical studies. When the epithelium and underlying proteins were extracted and immunoprecipitated wi th beta 4 or BPAG-1, tyrosine phosphorylation decreased in the presence of genistein. In addition, the phosphorylation of beta 4 decreased when epithe lial sheets were incubated on substrata from which the basal lamina had bee n removed or altered. Thus, a reduction in phosphorylation of tyrosine resi dues impairs the formation of mature hemidesmosomes, and substrata that fai l to support hemidesmosome formation also demonstrate decreased phosphoryla tion of tyrosine residues.