THE ROLE OF PHOSPHORYLATION IN ACTIVATION OF THE ALPHA-6A-BETA-1 LAMININ RECEPTOR

The phorbol ester phorbol 12-myristate 13-acetate (PMA) induces phosph orylation of serine residues in the cytoplasmic domain of the alpha6A integrin subunit, as well as activation of the alpha6Abeta1 laminin re ceptor. We examined whether phosphorylation correlates with the induct ion of high affinity binding of laminin by the alpha6Abeta1 receptor. Two potential phosphorylation sites for protein kinase C, serine 1041 and serine 1048, are present in the cytoplasmic domain of the alpha6A subunit. We introduced point mutations into the alpha6A cDNA, replacin g either one or both of the serine residues with alanine. Wild-type an d mutant alpha6A cDNAs were transfected into K562 cells. All alpha6A s ubunit mutants were expressed at levels similar to those of wild-type alpha6A and formed heterodimers with endogenous beta1. Analysis of the phosphorylation state of wild-type and mutant alpha6A subunits in res ting K562 cells and after treatment with PMA showed that serine 1041, but not serine 1048, is the target residue of PMA-induced phosphorylat ion. Cells expressing alpha6A mutant subunits or wild-type alpha6A tra nsfectants all bound laminin in the presence, but not in the absence o f PMA; however, the extent of binding differed. Cells transfected with alpha6A containing the serine to alanine mutation showed a 2-3-fold h igher binding to laminin than cells transfected with alpha6A containin g serine 1041. The results indicate that phosphorylation of the alpha6 A cytoplasmic domain is not required for the induction of high affinit y of the alpha6Abeta1 receptor by PMA, and suggest that, in contrast, it may reduce the affinity of this integrin for ligand.