A Phorbol Ester‐Sensitive Kinase Catalyzes the Phosphorylation of P₀ Glycoprotein in Myelin

The proposed structural protein of peripheral nerve myelin, P₀, has been shown to have several covalent modifications. In addition to being glycosylated, sulfated, and acylated, P₀ is phosphorylated, with the intracellular site of this latter addition being in question. By employing nerve injury models that exhibit different levels of P₀ biosynthesis in the absence and presence of myelin assembly, we have examined the cellular location of P₀ phosphorylation. It is demonstrated that there is comparable P₀ phosphorylation in both normal and crush‐injured adult rat sciatic nerves, although the level of biosynthesis of P₀ differs between these myelin maintaining and actively myelinating nerve models, respectively. The glycoprotein does not appear to be phosphorylated readily in the transected adult sciatic nerve, a preparation in which P₀ biosynthesis is observed but that lacks myelin membrane. These observations suggest that the modification is not associated with the biosynthesis or maturation of P₀ in the endoplasmic reticulum or Golgi, but that it instead occurs after myelin assembly. That P₀ phosphorylation occurs in the normal nerve even when translation is inhibited by cycloheximide treatment lends further support to this conclusion. P₀ is shown to be phosphorylated on one or more serine residues, with all or most of the phosphate group(s) being labile as evidenced by pulse‐chase analysis. Addition of a biologically active phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate or 4β‐phorbol 12, 13‐dibutyrate, substantially increases the extent of [³²P]orthophosphate incorporation into the glycoprotein of normal and crushed nerve but not transected nerve. Biologically inactive 4α‐phorbol 12, 13‐didecanoate has no effect on P₀ phosphorylation. Similarly, the addition of the cyclic AMP analog 8‐bromo‐cyclic AMP causes no appreciable changes in P₀ labeling. These findings indicate that the phorbol ester‐sensitive enzyme, protein kinase C, may be responsible for the phosphorylation of P₀ within the myelin membrane.