The role of phosphorylation in activation of the alpha 6A beta 1 laminin receptor.
The phorbol ester phorbol 12-myristate 13-acetate (PMA) induces phosphorylation of serine residues in the cytoplasmic domain of the alpha 6A integrin subunit, as well as activation of the alpha 6A beta 1 laminin receptor. We examined whether phosphorylation correlates with the induction of high affinity binding of laminin by the alpha 6A beta 1 receptor. Two potential phosphorylation sites for protein kinase C, serine 1041 and serine 1048, are present in the cytoplasmic domain of the alpha 6A subunit. We introduced point mutations into the alpha 6A cDNA, replacing either one or both of the serine residues with alanine. Wild-type and mutant alpha 6A cDNAs were transfected into K562 cells. All alpha 6A subunit mutants were expressed at levels similar to those of wild-type alpha 6A and formed heterodimers with endogenous beta 1. Analysis of the phosphorylation state of wild-type and mutant alpha 6A subunits in resting K562 cells and after treatment with PMA showed that serine 1041, but not serine 1048, is the target residue of PMA-induced phosphorylation. Cells expressing alpha 6A mutant subunits or wild-type alpha 6A transfectants all bound laminin in the presence, but not in the absence of PMA; however, the extent of binding differed. Cells transfected with alpha 6A containing the serine to alanine mutation showed a 2-3-fold higher binding to laminin than cells transfected with alpha 6A containing serine 1041. The results indicate that phosphorylation of the alpha 6A cytoplasmic domain is not required for the induction of high affinity of the alpha 6A beta 1 receptor by PMA, and suggest that, in contrast, it may reduce the affinity of this integrin for ligand.
Hogervorst
F
F
The Netherlands Cancer Institute, Division of Cell Biology, Amsterdam.
Kuikman
I
I
Noteboom
E
E
Sonnenberg
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D006801:Humans; D007797:Laminin / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D017474:Receptors, Laminin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D007797:Laminin; D017474:Receptors, Laminin; D010768:Phosphoserine; D004247:DNA; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate