p34cdc2 kinase mRNA levels during the terminal differentiation of neurons (13), demonstration ofNF-H phosphorylation by p34Cdc2 kinase from nonneuronal sources, while intriguing, has uncertain physiologic relevance. The presence of consensus sequences of substrate for p34cdc2 kinase on vertebrate NF-H suggests the possibility that there may be cdc2-related kinases in the nervous system that phosphorylate these motifs.In this report, we present the purification, biochemical, and immunologic characterization of a p34cdc2-like kinase isolated from rat spinal cords. We demonstrate that dephosphorylated NF-H and NF-M, and synthetic peptides containing KSPXK sequences are highly effective substrates and suggest that NF protein may be one of the endogenous substrates for this cdc2-like kinase.MATERIALS AND METHODS Materials. Synthetic peptide analogs of KSP sequences were custom-synthesized by Peptide Technologies (Washington, DC). Peptide substrates of protein kinase A (kemptide) and S6 kinase (S6 substrate) were obtained from Peninsula Laboratories, protein kinase C, acetyl-myelin basic protein-(4-14), tyrosine kinase (RR-SRC), p34cdc2 kinase [21-mer peptide analog, simian virus 40 (SV40) large tumor (T) antigen], and microcystin LR were obtained from GIBCO/BRL. a-Casein and dephosphorylated casein were obtained from Sigma; histone (Hi), alkaline phosphatase (calf intestine), and phosphatase-labeled goat anti-rabbit IgG were obtained from Boehringer Mannheim. Antibodies directed against C-terminal (mouse) and PSTAIRE regions (human) of p34cdc2 kinase were purchased from Upstate Biotechnology (Lake Placid, NY).Extraction. Rat spinal cords (120 g, obtained from PelFreez Biologicals) were suspended in isotonic low-salt buffer [20 mM Tris-HCl/l mM EDTA/1 mM EGTA/1 mM dithiothreitol (DTT)/leupeptin (5 ,g/ml)/0.3 M sucrose at pH 7.5] maintained at 4°C. After removing the blood contaminants and meninges, the tissues were diced and homogenized, 1:5 (wt/vol), in high-salt extraction buffer (where 0.8 M KCl was substituted for sucrose) with a Polytron homogenizer (Brinkmann). During homogenization, 0.5 M phenylmethylsulfonyl fluoride in ethanol was added to a final concentration of 2.5 mM, and the preparation was stirred for 3 hr at 4°C. The homogenate was centrifuged (100,000 x g for 60 min), and the supernate was dialyzed against column-equilibrating buffer [CEB = 20 mM Tris HCl/l mM EDTA/1 mM EGTA/1 mM DTT/5% (vol/vol) glycerol, pH 7.5]. The dialysate was centrifuged 50,000 x g for 30 min, and the resulting supemate was used for further purification.Purification. The dialyzed supernate was applied to a P-il phosphocellulose column, equilibrated in CEB, and washed Abbreviations: SV40, simian virus 40; T, tumor; DTT, dithiothreitol; NF, neurofilament.

Cyclin‐dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and τ protein phosphorylation. This report describes the characterization of a 62‐kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full‐length cDNA was characterized coding for a 67.5‐kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose‐dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino‐terminal peptide‐specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.

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.