Neuronal cyclin-dependent kinase-5 phosphorylation sites in neurofilament protein (NF-H) are dephosphorylated by protein phosphatase 2A

J Neurochem. 1995 Jun;64(6):2681-90. doi: 10.1046/j.1471-4159.1995.64062681.x.

Abstract

Neurofilament (NF) protein [high molecular mass (NF-H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys-Ser-Pro) repeat motifs. There are two major types of KSP motifs in the NF-H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2-like kinase [cyclin-dependent kinase-5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF-H and lysine-rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF-H in a NF preparation, and a bacterially expressed C-terminal tail domain of NF-H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks--P1, P2, and P3--were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5-phosphorylated 32P-histone (H1), 32P-NF-H in the NF preparation, and 32P-NF-H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A-like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF-H.

MeSH terms

  • Animals
  • Cyclin-Dependent Kinase 5
  • Cyclin-Dependent Kinases*
  • Ethers, Cyclic / pharmacology
  • Histones / metabolism
  • Microcystins
  • Neurofilament Proteins / genetics
  • Neurofilament Proteins / metabolism*
  • Neurons / enzymology*
  • Okadaic Acid
  • Peptides, Cyclic / pharmacology
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphoric Monoester Hydrolases / metabolism
  • Phosphorylation
  • Protein Phosphatase 2
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rats
  • Repetitive Sequences, Nucleic Acid
  • Spinal Cord / enzymology

Substances

  • Ethers, Cyclic
  • Histones
  • Microcystins
  • Neurofilament Proteins
  • Peptides, Cyclic
  • neurofilament protein H
  • Okadaic Acid
  • microcystin
  • Cyclin-Dependent Kinase 5
  • Protein-Serine-Threonine Kinases
  • Cdk5 protein, rat
  • Cyclin-Dependent Kinases
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2
  • Phosphoric Monoester Hydrolases