Selective synthesis of 2 ',3 '-cyclic nucleotide 3 '-phosphodiesterase isoform 2 and identification of specifically phosphorylated serine residues

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is a protein found abund antly in the cytoplasmic compartments of CNS myelin. Two isoforms of this p rotein, CNP1 and CNP2, are detectable, They differ by a 20-amino acid exten sion exclusive to CNP2. Additionally, CNP2 is essentially the only isoform to be phosphorylated in vivo. In this study, we examine the phosphorylation of CNP2 in transfected cells. CNP2 was selectively expressed ectopically i n 293T cells and labeled with (32)p. Immunoprecipitation of labeled CNP2 an d tryptic phosphopeptide mapping analyses identified serines 9 and 22 as th e major sites of phosphorylation. Only serine 22 was phosphorylated initial ly in oligodendrocyte-enriched cultures of neonatal rat brain glial cells. However, 4 beta-phorbol 12,13-dibutyrate (PDB) induced the phosphorylation of serine 9, thereby producing the same pattern seen in 293T cells. These r esults suggest that serine 9 is phosphorylated by a PDB-sensitive kinase, l ikely protein kinase C, and that serine 22 appears to be constitutively pho sphorylated.