In vivo phosphorylation of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases.
2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-32]ATP with cAMP (5 microM) and cAMP (5 microM)+catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site.
Agrawal
H C
HC
Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110.
Sprinkle
T J
TJ
Agrawal
D
D
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D009186:Myelin Sheath / Q000201:enzymology*; D010703:Phorbol Esters / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D010703:Phorbol Esters; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D005576:Colforsin; C008876:phorbol-12,13-didecanoate; D000242:Cyclic AMP; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D013755:Tetradecanoylphorbol Acetate