Mitogen-Activated Protein Kinases (Erk1,2) Phosphorylate Lys-Ser-Pro (KSP) Repeats in Neurofilament Proteins NF-H and NF-M

Abstract

Mammalian neurofilament proteins, particularly midsized (NF-M) and heavy (NF-H) molecular weight neurofilament proteins, are highly phosphorylated in axons. Neurofilament function depends on the state of phosphorylation 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 KSPXXXK type, it was of interest to determine which kinase phosphorylates these motifs. Using a synthetic KSPXXXK peptide to screen for a specific 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 inhibition by a specific MEK inhibitor (PD 98059). The fraction containing Erk2, as well as bacterially expressed Erk1 and Erk2, phosphorylated all types of KSP motifs in peptides (KSPXK, KSPXXK, KSPXXXK, and KSPXXXXK) derived from NF-M and NF-H. They also phosphorylated an expressed 24 KSPXXXK repeat NF-H polypeptide, an expressed NF-H as well as dephosphorylated native rat NF-H, and NF-M proteins with accompanying decreases in their respective electrophoretic mobilities. A comparative kinetic study of Erk2 and cdk5 phosphorylation of KSPXK and KSPXXXK peptides revealed that, in contrast to cdk5, which phosphorylated only the KSPXK peptide, Erk2 could phosphorylate both. The preferred substrate for Erk2 was KSPXXXK peptide. The MEK inhibitor PD 98059 also inhibited phosphorylation of NF-H, NF-M, and microtubule-associated protein (MAP) in primary rat hippocampal cells and caused a decrease in neurite outgrowth, suggesting that Erk1,2 may play an important role in neurite growth and branching. These data suggest that neuronal Erk1 and Erk2 are capable of phosphorylating serine residues in diverse KSP repeat motifs in NF-M and NF-H.

Chronic CXCL10 alters the level of activated ERK1/2 and transcriptional factors CREB and NF-κB in hippocampal neuronal cell culture

“…However, sustained ERK1/2 activation has been associated with neuronal death (Alessandrini et al, 1999;Murray et al, 1998;Runden et al, 1998). Abnormal activation of the ERK1/2 pathway has also been implicated in the pathogenesis of Alzheimer's disease (Alessandrini et al, 1999;Drewes et al, 1992;Knowles et al, 1999;Perry et al, 1999;Trojanowski et al, 1993;Veeranna et al, 1998). In our studies, the level of activated and total ERK1/2 was increased but there was no evidence supporting a negative effect of the elevated levels on the hippocampal cultures.…”

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

“…Immunoprecipitation and Kinase Assays-Kinase assays were performed as described previously (30). Briefly, 7 days in culture (DIC) primary rat cortical neurons were infected with p25, p35, Cdk5 wild type, dominant negative Cdk5, and p5 using the lentiviral gene delivery as described earlier.…”

Signal transduction cascades and hepatitis B and C related hepatocellular carcinoma

“…When activated, extracellular signal-regulated kinase (ERK), a MAPK, contributes to growth factor-stimulated mitogenesis and gene expression. 22,24 The binding of PY-IRS-1 to p85 stimulates glucose uptake, 25 inhibits apoptosis by activating Akt/protein kinase B, [26][27][28] and inhibits glycogen synthase kinase-3␤ (GSK-3␤). [29][30][31] The net re- Fig.…”