cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization.

Abstract

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.

Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain

“…However, transfection experiments failed to demonstrate phosphorylation of NF-H by GSK3 (Sun et al, 1996), suggesting the existence of additional requirements for in vivo phosphorylation by this kinase. In contrast, evidence based on synthetic peptides and recombinant, truncated NP-H constructs suggests that cdk5 is more selective, phosphorylating only sites of the consensus S/TPX 1KX2, especially where X, is not acidic (Hisanaga et a!., 1991; Beaudette et a!., 1993;Shetty et a!., 1993;Sun et a!., 1996). Three of the S/TP sites in chicken NF-M conform to this motif (Ser 575, Ser552, and Ser715).…”

“…These include casein kinase I Hollander et al, 1996) and at least three different Pro-dependent kinases: protein kinase FA/glycogen synthase kinase-3 (GSK3; Guan et al, 1991), cyclin-dependent kinases (Hisanaga et al, 1991;Guan et al, 1992;Lew et a!., 1992;Shetty et al, 1993;Sun et a!., 1996), and mitogenactivated protein kinase (ERK kinase; Roder and Ingram, 1991;Chertoff et al, 1995). The phosphorylation sites are mostly in Ser residues in Lys-Ser-Pro (KSP) sequences (Geisler et a!., 1987;Lee et a!., 1988), which are repeated as many as 60 times in mammalian NF-H. Other, less frequently repeated, potential phosphorylation motifs have also been recognized, especially in NF-M, including KXSP and KXXSP (Shaw, 1991).…”

Cyclin-dependent Kinase-5 Is Involved in Neuregulin-dependent Activation of Phosphatidylinositol 3-Kinase and Akt Activity Mediating Neuronal Survival

“…Transfection studies using COS-7 cells show that these residues are also phosphorylated in vivo. Although it has been shown that Cdk5 specifically phosphorylates peptides containing X(S/T)PXK motifs (where X represents a neutral/basic residue) (55,56), the ability of Cdk5 to phosphorylate this novel (SPRXR) consensus sequence motif in ErbB3 raises the possibility that Cdk5 targets other proteins in the nervous system than X(S/T)PX(K/R). In addition, this study suggests that Cdk5 is involved in the regulation of receptor protein-tyrosine kinases affecting downstream signaling transduction pathways in neuronal development and survival (Fig.…”

Identification of Functional Domains in the Neuronal Cdk5 Activator Protein

“…The purified protein kinase contains 33-and 25-kDa subunits; the 33-kDa subunit was later identified as the Cdc2-related kinase Cdk5 (16). Cdk5 was similarly identified as a neuronal protein kinase capable of phosphorylating the KSPXK sequence motif in neurofilament proteins NF-H, NF-M (17)(18)(19), and the tau protein (20). Phosphorylation of tau by Cdk5 is particularly interesting because abnormally phosphorylated tau is the major component of the paired helical filaments, which accumulate in the brains of Alzheimer patients (21).…”