NEURONAL CYCLIN-DEPENDENT KINASE-5 PHOSPHORYLATION SITES IN NEUROFILAMENT PROTEIN (NF-H) ARE DEPHOSPHORYLATED BY PROTEIN PHOSPHATASE 2A

Neurofilament (NF) protein [high molecular mass (NF-H)] is extensively phosphorylated in vivo, The phosphorylation occurs mainly in its char acteristic KSP (Lys-Ser-Pro) repeat motifs. There are two major types of KSP motifs in the NF-H tail domain: KSPXKX and KSPXXX. Recent studi es by two different laboratories have demonstrated the presence of a c dc2-like kinase [cyclin-dependent kinase-5 (cdk5)] in nervous tissue t hat selectively phosphorylates KSPXKX and XS/TXK motifs in NF-H and ly sine-rich histone (H1). This article describes the identification of p hosphatases dephosphorylating three different substrates: histone (H1) , NF-H in a NF preparation, and a bacterially expressed C-terminal tai l domain of NF-H, each containing KSPXKX repeats phosphorylated in vit ro 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 characteriz ed on the basis of biochemical, pharmacological, and immunochemical st udies. One of the three peaks was identified as PP2A, whereas the othe rs were mixtures of both PP2A and PP1. These three peaks could dephosp horylate cdk5-phosphorylated P-32-histone (H1), P-32-NF-H in the NF pr eparation, and P-32-NF-H tail fusion protein. These studies suggest th e involvement of PP2A or a PP2A-like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF-H.