Neuronal Cyclin‐Dependent Kinase‐5 Phosphorylation Sites in Neurofilament Protein (NF‐H) Are Dephosphorylated by Protein Phosphatase 2A

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.

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2

“…These data reveal a key role for Pin1 in regulating the dephosphorylation of cdc2-phosphorylated Dab2. Many substrates for cyclin-dependent kinases are dephosphorylated by protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) (Veeranna et al, 1995;Ajiro et al, 1996;Kwon et al, 1997;He et al, 2001a), which explains in part why inhibitors of PP1 and PP2A are potent tumor promoters (Haystead et al, 1989;Suganuma et al, 1990). We examined the abilities of the PP1/PP2A-selective inhibitor okadaic acid and the PP2B-selective inhibitor deltamethrin to block dephosphorylation of Dab2.…”

Downregulation of Neuronal cdk5/p35 in Opioid Addicts and Opiate-Treated Rats: Relation to Neurofilament Phosphorylation

“…Other studies also indicate that cdk5/p35 is a major kinase complex regulating the phosphorylation state of these cytoskeletal proteins (Sun et al, 1996;Qi et al, 1998). In turn, protein phosphatase 2Ac (PP2Ac) appears to be the most effective phosphatase in the nervous tissue in dephosphorylating the sites in NF-H known to be phosphorylated by cdk5 (Veeranna et al, 1995). It therefore appears that cdk5/p35 and PP2Ac may mutually regulate the phosphorylation state of NF in neurons (Grant et al, 2001).…”

Cdk5 Levels Oscillate during the Neuronal Cell Cycle

“…In addition to the brain, low levels of Cdk5 kinase activity are also present in the adult mouse prostate and embryonic limb buds (8). The function of Cdk5 has been widely investigated during development where it has a recognized role in the phosphorylation of a variety of cytoskeletal proteins (9,10). In recent years, additional roles for Cdk5 have been discovered in the post-mitotic neuron.…”