Phosphorylation of the high molecular weight neurofilament protein (NF-H) by Cdk5 and p35.
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.
Sun
D
D
Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Leung
C L
CL
Liem
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D016384:Consensus Sequence; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D017931:DNA Primers; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D011994:Recombinant Proteins; C089495:neuronal Cdk5 activator (p25-p35); C051774:neurofilament protein H; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases