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 hand, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Chao
T S
TS
Department of Biochemistry, College of Medicine, University of Illinois, Chicago 60612.
Tao
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D001345:Autoradiography; D002918:Chymotrypsin / Q000737:chemistry; D003598:Cytoskeletal Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism*; D009479:Neuropeptides; D010449:Peptide Mapping; D010766:Phosphorylation; D014357:Trypsin / Q000737:chemistry
D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D002918:Chymotrypsin; D014357:Trypsin