Protein kinase C-alpha phosphorylation of specific serines in the connecting segment of the beta 4 integrin regulates the dynamics of type II hemidesmosomes.
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 alpha 6 beta 4 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 beta 4 integrin subunit. Using a combination of biochemical and mutational analyses, we demonstrate that EGF induces the phosphorylation of three specific serine residues (S(1356), S(1360), and S(1364)) located within the connecting segment of the beta 4 subunit and that phosphorylation on these residues accounts for the bulk of beta 4 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 alpha 6 beta 4 integrin, we observed that expression of a beta 4 construct containing Ser-->Ala mutations of S(1356), S(1360), and S(1364) 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-alpha (PKC-alpha) 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 beta 4, and argue for a key role for PKC-alpha in regulating these structures.
Rabinovitz
Isaac
I
Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115, USA. irarabinov@caregroup.harvard.edu
Tsomo
Lobsang
L
Mercurio
Arthur M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology; D022002:Hemidesmosomes / Q000378:metabolism*; D006801:Humans; D039663:Integrin beta4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012694:Serine / Q000737:chemistry; D014162:Transfection
D039663:Integrin beta4; D011994:Recombinant Proteins; D012694:Serine; D004815:Epidermal Growth Factor; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha