Protein Kinase C-α Phosphorylation of Specific Serines in the Connecting Segment of the β4 Integrin Regulates the Dynamics of Type II Hemidesmosomes

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Abstract
Although the regulation of hemidesmosome dynamics during processes such as epithelial migration, wound healing, and carcinoma invasion is important, the mechanisms involved are poorly understood. The integrin α6β4 is an essential component of the hemidesmosome and a target of such regulation. Epidermal growth factor (EGF) can induce hemidesmosome disassembly by a mechanism that involves serine phosphorylation of the β4 integrin subunit. Using a combination of biochemical and mutational analyses, we demonstrate that EGF induces the phosphorylation of three specific serine residues (S1356, S1360, and S1364) located within the connecting segment of the β4 subunit and that phosphorylation on these residues accounts for the bulk of β4 phosphorylation stimulated by EGF. Importantly, phosphorylation of these serines is critical for the ability of EGF to disrupt hemidesmosomes. Using COS-7 cells, which assemble hemidesmosomes type II upon exogenous expression of the α6β4 integrin, we observed that expression of a β4 construct containing Ser→Ala mutations of S1356, S1360, and S1364 reduced the ability of EGF to disrupt hemidesmosomes and that this effect appears to involve cooperation among these phosphorylation sites. Moreover, expression of Ser→Asp mutants that mimic constitutive phosphorylation reduced hemidesmosome formation. Protein kinase C-α (PKC-α) is the kinase responsible for phosphorylating at least two of these serines, based on in vitro kinase assays, peptide mapping, and mutational analysis. Together, these results highlight the importance of serine phosphorylation in regulating type II hemidesmosome disassembly, implicate a cluster of serine residues within the connecting segment of β4, and argue for a key role for PKC-α in regulating these structures.