IDENTIFICATION OF ENDOGENOUSLY PHOSPHORYLATED KSP SITES IN THE HIGH-MOLECULAR-WEIGHT RAT NEUROFILAMENT PROTEIN

The high-molecular-weight neurofilament protein (NF-H) is highly phosp horylated in vivo, with estimates as high as 16-51 mol of P-i/mol of p rotein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxyterminal tail reg ion of the molecule. Because the extent and site-specific patterns of tail domain phosphorylation are believed to modulate neurofilament str ucture and function, it becomes essential to identify the endogenous s ites of phosphorylation. In this study, we have used selective proteol ytic cleavage procedures, P-i determinations, microsequencing, and mas s-spectral analysis to determine the endogenously phosphorylated sites in the NF-H tail isolated from rat spinal cord. Twenty Ser residues i n NF-H carboxy-terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation co uld be identified in any of the 11 ''non-KSP'' Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphory lated. In addition, a 27-kDa KSP-rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and fo und to contain between 30 and 35 mol of P-i/mol of protein. This domai n appeared to be highly resistant to endoproteinase Glu-C digestion, a lthough it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phos phorylation of the tail domain may contribute to neurofilament stabili ty in vivo. A neuronal-derived protein kinase that specifically phosph orylates only KSPXKX motifs in neurofilaments has been reported. The p resence of extensively phosphorylated KSPXXX repeats in NF-H in vivo s uggests the existence of yet another, unidentified kinase(s) with spec ificity for KSPXXX motifs.