MITOGEN-ACTIVATED PROTEIN-KINASES (ERK1,2) PHOSPHORYLATE LYS-SER-PRO (KSP) REPEATS IN NEUROFILAMENT PROTEINS NF-H AND NF-M

Mammalian neurofilament proteins, particularly midsized (NF-M) and hea vy (NF-H) molecular weight neurofilament proteins, are highly phosphor ylated in axons. Neurofilament function depends on the state of phosph orylation of the numerous serine/threonine residues in these proteins. Most phosphorylation occurs in the lys-ser-pro (KSP) repeats in the C -terminal tail domains of NF-H and NF-M. In our previous study, cyclin -dependent kinase 5 (cdk5) was shown to phosphorylate specifically the KSPXK repeats in rat NF-H. Because 80% of the repeats are of the KSPX XXK type, it was of interest to determine which kinase phosphorylates these motifs. Using a synthetic KSPXXXK peptide to screen for a specif ic kinase, we fractionated rat brain extracts by column chromatography and identified extracellular signal-regulated kinase (Erk2) activated by an upstream activator, the mitogen-activated protein kinase kinase MAPKK (MEK), by Western blot analysis, sequence identification, and i nhibition by a specific MEK inhibitor (PD 98059). The fraction contain ing Erk2, as well as bacterially expressed Erk1 and Erk2, phosphorylat ed all types of KSP moths in peptides (KSPXK, KSPXXK, KSPXXXK, and KSP XXXXK) derived from NF-M and NF-H. They also phosphorylated an express ed 24 KSPXXXK repeat NF-H polypeptide, an expressed NF-H as well as de phosphorylated native rat NF-H, and NF-M proteins with accompanying de creases in their respective electrophoretic mobilities. A comparative kinetic study of Erk2 and cdk5 phosphorylation of KSPXK and KSPXXXK pe ptides revealed that, in contrast to cdk5, which phosphorylated only t he KSPXK peptide, Erk2 could phosphorylate both. The preferred substra te for Erk2 was KSPXXXK peptide. The MEK inhibitor PD 98059 also inhib ited phosphorylation of NF-H, NF-M, and microtubule associated protein (MAP) in primary rat hippocampal cells and caused a decrease in neuri te outgrowth, suggesting that Erk1,2 may play an important role in neu rite growth and branching. These data suggest that neuronal Erk1 and E rk2 are capable of phosphorylating serine residues in diverse KSP repe at motifs in NF-M and NF-H.