MODULATION OF PROTEIN 4.1 BINDING TO INSIDE-OUT MEMBRANE-VESICLES BY PHOSPHORYLATION

The effect of phosphorylation on the binding of protein 4.1 to erythrocyte inside-out vesicles was investigated. Protein 4.1 was phosphorylated with casein kinase A, protein kinase C, and cAMP-dependent protein kinase. An analysis of the phosphopeptides generated by alpha-chymotryptic and tryptic digestion indicates these kinases phosphorylate similar as well as distinct domains within protein 4.1. All three enzymes catalyze the phosphorylation to varying degrees of the 46-, 16-, and 8-10-kDa fragments derived from limited chymotryptic cleavage. In addition, casein kinase A phosphorylates a 24-kDa domain, whereas protein kinase C phosphorylates a 30-kDa domain. Protein 4.1 phosphorylated by casein kinase A and protein kinase C, but not cAMP-dependent protein kinase, exhibits a reduced binding to KI-extracted inside-out vesicles. On the other band, phosphorylation of inside-out vesicles by casein kinase A does not affect their ability to bind protein 4.1. The inside-out vesicles, however, inhibit the phosphorylation of protein 4.1 by casein kinase A and protein kinase C, but not by cAMP-dependent protein kinase. These results suggest that casein kinase A and protein kinase C may modulate the binding of protein 4.1 to the membrane by phosphorylation of specific domains of the cytoskeletal protein. Since the 30-kDa domain has been suggested as a membrane-binding site, that phosphorylation by protein kinase C reduces the binding of protein 4.1 to inside-out vesicles is perhaps not surprising. On the other hand, the role of the casein kinase A substrate 24-kDa domain in membrane binding has not been established and needs to be examined. The results further suggest that the 16- and 8-10-kDa domains, which are phosphorylated by all three kinases, are probably not involved in the binding of protein 4.1 to membrane.