Phosphorylation of the High Molecular Weight Neurofilament Protein (NF-H) by Cdk5 and p35

doi:10.1074/jbc.271.24.14245

Phosphorylation of the High Molecular Weight Neurofilament Protein (NF-H) by Cdk5 and p35*

From the Departments of Pathology, Anatomy & Cell Biology, and
^§ Biochemistry & Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032

^¶ To whom correspondence should be addressed:
Dept. of Pathology, Columbia University College of Physicians & Surgeons, 630 West 168th St., New York, NY 10032.
Tel.: 212-305-4078; Fax: 212-305-5498; E-mail: RKL2{at}columbia.edu

Abstract

The high molecular weight neurofilament protein (NF-H) is highly phosphorylated in the axon. The phosphorylation sites have been identified as KSP (Lys-Ser-Pro) repeats in the tail domain of NF-H. These KSP sequences are present more than 50 times in the NF-H tail, and most of these sites are normally phosphorylated in vivo. These KSP sites can be further divided into two separate consensus sequences, KSPXK and KSPXY (where Y is not K). The extensive phosphorylation of NF-H has been proposed to play a critical role in the determination of axonal diameter. Recent studies have shown that Cdk5, a kinase related to the cell cycle-dependent kinase Cdc2, is expressed in the brain and associates with the cytoskeleton. In vitro phosphorylation studies have shown that Cdk5 in conjunction with its activator, p35, is able to phosphorylate histone H1, dephosphorylated NF-H, as well as a synthetic peptide with the repetitive KSP motif. We have cloned the cDNAs for rat Cdk5 and p35 by reverse transcription-polymerase chain reaction and cDNA library screening and studied the phosphorylation of NF-H both in vivo and in vitro. By transient transfection assays, we have shown that NF-H can only be extensively phosphorylated in the presence of both Cdk5 and p35. This phosphorylation can be inhibited by a Cdk5-dominant negative mutant, an observation which further supports that Cdk5 is a kinase that is able to phosphorylate NF-H. By immunoprecipitating Cdk5 and p35 from the transfected cells, we have been able to show that the KSPXK repeats are the preferred phosphorylation sites for Cdk5, while the KSPXY repeats are not directly phosphorylated by Cdk5 and p35.

Footnotes

↵^‡ Supported as National Institutes of Health predoctoral trainees on Training Grants AG00189 and EY07105.
↵* This work was supported in part by National Institutes of Health Grants NS15182 and AG13185 (to R. K. H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U50707[GenBank].
↵¹ The abbreviations used are:

NF-H, NF-M, and NF-L

neurofilament high, middle, and low molecular weight proteins, respectively

PAGE

polyacrylamide gel electrophoresis

Cdk

cyclin dependent kinase

PCR

polymerase chain reaction

RT

reverse transcriptase

kb

kilobase(s)

ECL

enhanced chemiluminescence

GSK3β

glycogen synthase kinase 3β

HA

hemagglutinin.
- Received January 29, 1996.
- Revision received April 1, 1996.