IN-VIVO PHOSPHORYLATION OF 2',3'-CYCLIC NUCLEOTIDE 3'-PHOSPHOHYDROLASE (CNP) - CNP IN BRAIN MYELIN IS PHOSPHORYLATED BY FORSKOLIN-SENSITIVEAND PHORBOL-ESTER-SENSITIVE PROTEIN-KINASES

2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated i n vivo, in brain slices and in a cell free system. Phosphoamino acid a nalysis 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 s lices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-(32)]ATP with cAMP (5 mu M) and cAMP (5 mu M) + ca talytic unit of cAMP dependent protein kinase dramatically increased C NP2 phosphorylation by 4- and 6-fold, respectively. It is feasible tha t CNP2 was predominantly phosphorylated on serine and/or threonine res idues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 in creased 2-fold by incubating brain slices with phorbol ester. Forskoli n and phorbol ester increased the phosphorylation of single, but disti nct, CNP peptides. We present the first biochemical evidence that CNP2 , on a protein mass basis, is far more heavily phosphorylated than CNP 1, suggesting there are more phosphorylation sites on CNP2 than CNP1 a nd that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site.