Phosphorylation of P₀ Glycoprotein in Peripheral Nerve Myelin

Abstract: The P₀ protein in mammalian PNS myelin is known to undergo several posttranslational modifications, such as glycosylation, acylation, sulfation, and phosphorylation. Phosphorylation of purified P₀ protein in vitro was studied comparatively using three enzymes, i.e., calcium/phospholipid‐dependent protein kinase (protein kinase C), calcium/calmodulin‐dependent protein kinase II (CaM kinase II), and the catalytic subunit of cyclic AMP‐dependent protein kinase (A kinase). The phosphorylation of P₀ protein by CaM kinase II was the greatest, followed by that by protein kinase C; phosphorylation by A kinase, however, was much lower. In order to identify phosphorylation sites, P₀ protein was phosphorylated with [³²P]ATP and each kinase and then digested with lysylendopeptidase. The resulting phosphopeptides were isolated by HPLC. Subsequent amino acid sequence analysis and comparison with the known sequence of P₀ protein revealed that Ser¹⁸¹ and Ser²⁰⁴ were strongly phosphorylated by both protein kinase C and CaM kinase II. In addition, Ser²¹⁴ was also phosphorylated by protein kinase C, but not by CaM kinase II. Because all of these sites are located in the cytoplasmic domain of P₀ protein, phosphorylation may be important for maintenance of the major dense line of PNS myelin.