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-associated 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 known concerning regulation of the phosphorylation of these sites, these studies were undertaken to characterize the factors which determine the 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 microg/ml of polylysine, a concentration which has no effect on protein kinase C (PKC)-mediated phosphorylation of B-50. Digestion with Staphylococcus aureus V8 protease demonstrates CKII-mediated phosphorylation of B-501-132 and the C-terminal fragment S3/S4. Phosphorylation of B-50 by either CKII or PKC is inhibited by the N-terminal monoclonal antibody NM2, 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 phosphorylations of B-50 by CKII and PKC are determined by distinct regulatory signals in vivo.
Dokas
L A
LA
Department of Medicine, Division of Neurology, Room 1458, Ruppert Health Center, 3120 Glendale Avenue, Medical College of Ohio, Toledo, OH 43614-5809, USA. ldokas@gemini.mco.edu
Ting
S
S
Edgar
M A
MA
Oestreicher
A B
AB
Gispen
W H
WH
DeGraan
P N
PN
D000818:Animals; D000911:Antibodies, Monoclonal; D047390:Casein Kinase II; D019922:GAP-43 Protein / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D019606:Phosphoamino Acids / Q000032:analysis; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D019922:GAP-43 Protein; D019606:Phosphoamino Acids; D011994:Recombinant Proteins; D011107:Polylysine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases