| Protein 4.1 plays an essential role in maintaining the erythrocyte membrane stability and cytoskeleton assembly. Protein 4.1 also serves as a substrate for multiple erythrocyte protein kinases. Therefore, it is of importance to examine the effects of phosphorylation on the function of protein 4.1.;We also showed that phosphorylation of protein 4.1 by casein kinase A and protein kinase C reduced the binding ability of protein 4.1 to the KI-extracted erythrocyte inside-out vesicles, whereas phosphorylation by cAMP-dependent protein kinase did not affect the binding. The different effects elicited by these kinases could be attributed to the different phosphorylation sites on protein 4.1. Using mild chymotrypsin digestion, we identified that protein kinase C is able to phosphorylate the membrane-binding 30-kDa domain. Casein kinase A predominantly phosphorylates the 24-KDa domain which may also be involved in binding to the membrane. Alternatively, cAMP-dependent protein kinase phosphorylate primarily the 16- and 10-kDa domains that appeared not to be involved in membrane-binding. Our results correlate the effect of phosphorylation to the structural function of protein 4.1, suggesting that phosphorylation of protein 4.1 by multiple kinases could provide an effective mechanism for the regulation of cytoskeletal protein interactions.;We demonstrated that the red cell metabolite, 2,3-diphosphoglycerate, selectively enhances the phosphorylation of protein 4.1 by protein kinase C, while inhibits the phosphorylation by cAMP-dependent protein kinase and casein kinase A. This stimulatory effect appeared to be specific to protein 4.1 because other protein kinase C substrates did not exhibit the same effect. |
