REGULATION OF IN-VITRO PHOSPHORYLATION OF THE CASEIN KINASE-II SITES IN B-50 (GAP-43)

Casein kinase II (CKII) phosphorylates the rat neuronal growth-associa ted protein B-50 (GAP-43) at serines 191/192 and threonines 88, 89 and /or 95 both in vitro and in neuronal growth cones. Since little is kno wn concerning regulation of the phosphorylation of these sites, these studies were undertaken to characterize the factors which determine th e degree of B-50 phosphorylation by CKII in vitro, Phosphorylation of rat B-50 on serine and threonine residues by recombinant human CKII is stimulated by polylysine. Maximal stimulation occurs at 10 mu g/ml of polylysine, a concentration which has no effect on protein kinase C ( PKC)-mediated phosphorylation of B-50. Digestion with Staphylococcus a ureus V8 protease demonstrates CKII-mediated phosphorylation of B-50(1 -132) and the C-terminal fragment S3/S4. Phosphorylation of B-50 by ei ther CKII or PKC is inhibited by the N-terminal monoclonal antibody NM 2, while the C-terminal antibody NM6 has no effect on phosphorylation by either protein kinase. Protein phosphatase 2A dephosphorylates both the CKII and PKC sites, while protein phosphatases 2B and 1 are more selective for the PKC site. These results indicate that the phosphoryl ations of B-50 by CKII and PKC are determined by distinct regulatory s ignals in vivo. (C) 1998 Elsevier Science B.V.