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Phosphorylation of the High Molecular Weight Neurofilament Protein (NF-H) by Cdk5 and p35*

  • Dongming Sun
    Footnotes
    Affiliations
    Departments of Pathology, Anatomy & Cell Biology, and Columbia University College of Physicians and Surgeons, New York, New York 10032
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  • Conrad L. Leung
    Footnotes
    Affiliations
    Departments of Pathology, Anatomy & Cell Biology, and Columbia University College of Physicians and Surgeons, New York, New York 10032

    Departments of Biochemistry & Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032
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  • Ronald K.H. Liem
    Correspondence
    To whom correspondence should be addressed: Dept. of Pathology, Columbia University College of Physicians & Surgeons, 630 West 168th St., New York, NY 10032. Tel.: 212-305-4078; Fax: 212-305-5498;
    Affiliations
    Departments of Pathology, Anatomy & Cell Biology, and Columbia University College of Physicians and Surgeons, New York, New York 10032
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants NS15182 and AG13185 (to R. K. H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U50707.
    Supported as National Institutes of Health predoctoral trainees on Training Grants AG00189 and EY07105.
Open AccessPublished:June 14, 1996DOI:https://doi.org/10.1074/jbc.271.24.14245
      The high molecular weight neurofilament protein (NF-H) is highly phosphorylated in the axon. The phosphorylation sites have been identified as KSP (Lys-Ser-Pro) repeats in the tail domain of NF-H. These KSP sequences are present more than 50 times in the NF-H tail, and most of these sites are normally phosphorylated in vivo. These KSP sites can be further divided into two separate consensus sequences, KSPXK and KSPXY (where Y is not K). The extensive phosphorylation of NF-H has been proposed to play a critical role in the determination of axonal diameter. Recent studies have shown that Cdk5, a kinase related to the cell cycle-dependent kinase Cdc2, is expressed in the brain and associates with the cytoskeleton. In vitro phosphorylation studies have shown that Cdk5 in conjunction with its activator, p35, is able to phosphorylate histone H1, dephosphorylated NF-H, as well as a synthetic peptide with the repetitive KSP motif. We have cloned the cDNAs for rat Cdk5 and p35 by reverse transcription-polymerase chain reaction and cDNA library screening and studied the phosphorylation of NF-H both in vivo and in vitro. By transient transfection assays, we have shown that NF-H can only be extensively phosphorylated in the presence of both Cdk5 and p35. This phosphorylation can be inhibited by a Cdk5-dominant negative mutant, an observation which further supports that Cdk5 is a kinase that is able to phosphorylate NF-H. By immunoprecipitating Cdk5 and p35 from the transfected cells, we have been able to show that the KSPXK repeats are the preferred phosphorylation sites for Cdk5, while the KSPXY repeats are not directly phosphorylated by Cdk5 and p35.

      INTRODUCTION

      Neurofilaments are 8–10 nm fibrous structures present in all vertebrate axons. In mammals, neurofilaments consist of three protein subunits, known as high (NF-H),
      The abbreviations used are: NF-H, NF-M, and NF-L
      neurofilament high, middle, and low molecular weight proteins, respectively
      PAGE
      polyacrylamide gel electrophoresis
      Cdk
      cyclin dependent kinase
      PCR
      polymerase chain reaction
      RT
      reverse transcriptase
      kb
      kilobase(s)
      ECL
      enhanced chemiluminescence
      GSK3β
      glycogen synthase kinase 3β
      HA
      hemagglutinin.
      middle (NF-M), and low (NF-L) molecular weight neurofilament proteins (
      • Fliegner K.H.
      • Liem R.K.H.
      ,
      • Liem R.K.H.
      ,
      • Lee M.K.
      • Cleveland D.W.
      ,
      • Nixon R.A.
      • Sihag R.K.
      ). These three proteins belong to the intermediate filament protein family and are classified along with α-internexin as type IV intermediate filaments. Like all other intermediate filament proteins, neurofilament proteins contain an amino-terminal head domain, a central α-helical domain of ∼310 amino acids, and a carboxyl-terminal tail domain of variable length. The tail domains increase in length with increasing size of the neurofilament proteins and are extensively phosphorylated in NF-M and NF-H (
      • Carden M.J.
      • Schlaepfer W.W.
      • Lee V.M.-Y.
      ,
      • Julien J.P.
      • Mushynski W.E.
      ). The consensus sequence for phosphorylation has been identified as Lys-Ser-Pro (KSP) (
      • Lee V.M.-Y.
      • Otvos Jr., L.
      • Carden M.J.
      • Hollosi M.
      • Dietzschold B.
      • Lazzarini R.A.
      ,
      • Geisler N.
      • Vandekerckhove J.
      • Weber K.
      ,
      • Elhanany E.
      • Jaffe H.
      • Link W.T.
      • Sheeley D.M.
      • Gainer H.
      • Pant H.C.
      ), which can be further divided into two separate consensus sequences, KSPXK and KSPXY (where Y is not K). There are about 50 potential phosphorylation sites in the rat, mouse, and human NF-H carboxyl-terminal tail domains (
      • Chin S.S.M.
      • Liem R.K.H.
      ), although the relative numbers of KSPXK and KSPXY repeats differ markedly among different species. Phosphate determinations have shown that most of these sites are phosphorylated in vivo (
      • Geisler N.
      • Vandekerckhove J.
      • Weber K.
      ,
      • Julien J.P.
      • Mushynski W.E.
      ,
      • Wong J.
      • Hutchison S.B.
      • Liem R.K.H.
      ). Rat NF-M contains only 5 KSP sites (
      • Napolitano E.W.
      • Chin S.S.
      • Colman D.R.
      • Liem R.K.H.
      ), and human NF-M has 12 KSP sites (
      • Myers M.W.
      • Lazzarini R.A.
      • Lee V.M.
      • Schlaepfer W.W.
      • Nelson D.L.
      ). Antibodies have been raised which can distinguish phosphorylated and nonphosphorylated KSP epitopes (
      • Sternberger L.A.
      • Sternberger N.H.
      ). Several studies have shown that after extensive dephosphorylation by alkaline phosphatase, NF-H migrates more rapidly on SDS-PAGE (
      • Carden M.J.
      • Schlaepfer W.W.
      • Lee V.M.-Y.
      ,
      • Lee V.M.-Y.
      • Carden M.J.
      • Schlaepfer W.W.
      • Trojanowski J.Q.
      ). This characteristic mobility shift upon phosphorylation of NF-H and the antibodies specific for the phosphorylated and nonphosphorylated KSP epitopes are therefore useful tools for studying its phosphorylation.
      The functional role of neurofilament phosphorylation is still not completely clear. Early studies indicated that NF-H might function as a cross-linking protein whose phosphorylation was thought to be important for the formation of cross-linking bridges between neurofilaments (
      • Hirokawa N.
      • Glicksman M.A.
      • Willard M.B.
      ,
      • Liem R.K.
      • Pachter J.S.
      • Napolitano E.W.
      • Chin S.S.
      • Moraru E.
      • Heimann R.
      ). However, a later study showed that nonphosphorylated NF-H was also situated between intermediate filaments (
      • Hisanaga S.
      • Hirokawa N.
      ). More recently it has been suggested that the phosphate groups of NF-H may result in electrostatic repulsion, which in turn could increase axonal diameter (
      • Shaw G.
      ,
      • Carden M.J.
      • Trojanowski J.Q.
      • Schlaepfer W.W.
      • Lee V.M.-Y.
      ). Phosphorylation of NF-H has also been correlated with its ability to interact with microtubules (
      • Hisanaga S.
      • Kusubata M.
      • Okumura E.
      • Kishimoto T.
      ).
      The kinase(s) which phosphorylate the KSP sequences on NF-H are still not completely identified. A number of different protein kinases, including casein kinase (
      • Floyd C.C.
      • Grant P.
      • Gallant P.E.
      • Pant H.C.
      ), cyclic AMP-dependent protein kinase (
      • Dosemeci A.
      • Pant H.C.
      ), Ca2+-calmodulin-dependent protein kinase (
      • Vallano M.L.
      • Buckholz T.M.
      • DeLorenzo R.J.
      ), and protein kinase C (
      • Sihag R.K.
      • Nixon R.A.
      ) are able to phosphorylate the neurofilament proteins. However, NF-M is a better substrate than NF-H for all these protein kinases, and the characteristic mobility shift on SDS-PAGE due to phosphorylation of NF-H is not observed. Other studies have revealed protein kinases which are associated with neurofilaments, including a protein kinase activity from bovine spinal cord neurofilament-enriched preparations (
      • Dosemeci A.
      • Floyd C.C.
      • Pant H.C.
      ) and a 115-kDa neurofilament-associated kinase isolated by affinity chromatography using bacterially produced NF-H (
      • Xiao J.
      • Monteiro M.J.
      ). These protein kinases also only phosphorylate NF-H in a limited manner and there is no evidence that they phosphorylate NF-H at the KSP sites.
      Recent results have pointed to the family of cyclin-dependent protein kinases (Cdks) as candidate kinases for the KSP sites on NF-H. The cell cycle kinase Cdc2 was shown to phosphorylate neurofilaments (
      • Guan R.J.
      • Hall F.L.
      • Cohlberg J.A.
      ,
      • Hisanaga S.
      • Kusubata M.
      • Okumura E.
      • Kishimoto T.
      ) and cause the characteristic gel mobility shift of NF-H upon phosphorylation (
      • Hisanaga S.
      • Kusubata M.
      • Okumura E.
      • Kishimoto T.
      ). However, these results do not have much physiological relevance, since it is known that Cdc2 kinase is absent in terminally differentiated neurons. By using the polymerase chain reaction (PCR) with degenerate oligonucleotide primers corresponding to conserved regions of the Cdks, a family of novel Cdc2-related cDNA clones have been isolated (
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.H.
      ). Among them, PSSALRE is expressed in neuronal cell lines and brain, as well as other cell lines and organs (
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.H.
      ). This same kinase was also separately identified by another laboratory, shown to be able to bind cyclin D1 (
      • Xiong Y.
      • Zhang H.
      • Beach D.
      ) and renamed as Cdk5 (cyclin-dependent kinase 5). A number of recent studies have shown that Cdk5 is associated with the cytoskeleton and able to phosphorylate NF-H in vitro. (
      • Lew J.
      • Winkfein R.J.
      • Paudel H.K.
      • Wang J.H.
      ,
      • Shetty K.T.
      • Link W.T.
      • Jaffe H.
      • Wang J.
      • Pant H.C.
      ,
      • Hisanaga S.
      • Ishiguro K.
      • Uchida T.
      • Okumura E.
      • Okano T.
      • Kishimoto T.
      ). An additional protein with a molecular mass of 23–25 kDa was observed in most of the Cdk5 preparations. cDNA cloning showed that this 23–25-kDa protein is a degradation product of a larger protein, p35, which acts as a neural-specific regulatory subunit of Cdk5 (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ,
      • Lew J.
      • Huang Q.Q.
      • Zhong Q.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ). p35 associates physically with and activates Cdk5 kinase. Although it has no homology with cyclins, it serves a similar function in modulating the activity of Cdk5.
      In this study, we have cloned the cDNAs for both Cdk5 and p35 from rat brain and conducted NF-H phosphorylation studies by transient transfections and immunoprecipitation/kinase assays. We have demonstrated that NF-H can be phosphorylated only in the presence of both Cdk5 and p35. Furthermore, this phosphorylation is specific for the KSPXK sites.

      DISCUSSION

      Neurofilament phosphorylation has been postulated to play an important role in maintaining axonal caliber (
      • Fliegner K.H.
      • Liem R.K.H.
      ). The highly phosphorylated carboxyl-terminal tail domains of NF-H and NF-M are unique among intermediate filaments and are presumed to mediate neuron-specific functions, including maintaining the shape of the axon, which presumably requires a specialized cytoskeleton. The kinase(s) responsible for the phosphorylation of the NF-H tail domain have not been definitively identified. Several lines of evidence have pointed to Cdk5 as a candidate kinase for the phosphorylation of NF-H. Cdk5 is a kinase which was originally identified by homology to the cyclin-dependent kinase, Cdc2 (
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.H.
      ). Several other reports identified a protein kinase activity in the central nervous system, which was co-isolated with the cytoskeleton and upon further characterization turned out to be Cdk5 (
      • Hellmich M.R.
      • Pant H.C.
      • Wada E.
      • Battey J.F.
      ,
      • Lew J.
      • Beaudette K.
      • Litwin C.M.
      • Wang J.H.
      ,
      • Lew J.
      • Winkfein R.J.
      • Paudel H.K.
      • Wang J.H.
      ). In a number of these studies, NF-H or a synthetic peptide containing the KSP repeats was used as the substrate for these kinase assays. In addition, the microtubule-associated protein, τ, was shown to be a substrate for Cdk5 (
      • Arioka M.
      • Tsukamoto M.
      • Ishiguro K.
      • Kato R.
      • Sato K.
      • Imahori K.
      • Uchida T.
      ). It was noted from early studies that Cdk5 and its mRNA are expressed in a number of cell lines and tissues. However, immunoprecipitation experiments showed that Cdk5 kinase activity was only detected in brain (
      • Tsai L.H.
      • Takahashi T.
      • Caviness Jr., V.S.
      • Harlow E.
      ) suggesting the presence of a brain-specific activator. Two different activator proteins have been proposed for Cdk5. One of these, p35, is only expressed in brain (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ,
      • Lew J.
      • Huang Q.Q.
      • Zhong Q.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ), and phosphorylation of histone H1 by Cdk5 is observed when p35 and Cdk5 are co-transfected into non-neuronal cells. In vitro kinase assays using bacterially produced Cdk5 and p35 also showed that p35 is able to activate Cdk5 to phosphorylate histone H1 (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ). A second putative activator, p67, has been identified in purified kinase preparations containing Cdk5 and shown to activate bacterially expressed Cdk5 in in vitro kinase experiments (
      • Shetty K.T.
      • Kaech S.
      • Link W.T.
      • Jaffe H.
      • Flores C.M.
      • Wray S.
      • Pant H.C.
      • Beushausen S.
      ). p67 is identical to nsec-1 (rbsec1 or munc-18) and plays a role in exocytosis (
      • Pevshner J.
      • Scheller R.
      ), but has not been reported to be a kinase activator. In this report, we have focused our attention on the first of these activators, p35.
      In order to show that Cdk5 in the presence of p35 is able to phosphorylate NF-H in vivo, we performed transient transfections with various combinations of the Cdk5, p35, and NF-H expression constructs into the SW13 cell line. We have previously shown that NF-H is not significantly phosphorylated when transfected in fibroblasts. In the present study, we have also determined that in SW13 cells, NF-H remains predominantly in the nonphosphorylated state (Fig. 3B). Although Cdk5 is expressed in untransfected SW13 cells, the endogenous Cdk5 does not show any kinase activity as determined by the lack of phosphorylation of NF-H or histone H1, a commonly used substrate for cyclin-dependent kinases (Fig. 4A). By Northern blot analysis, we have determined that p35 mRNA is not expressed in SW13 cells, a result consistent with the observed lack of kinase activity from the endogenous Cdk5.
      Several lines of evidence presented in this study show that Cdk5, in conjunction with p35, is capable of phosphorylating NF-H in vivo. Co-transfections of Cdk5, p35, and NF-H resulted in highly phosphorylated NF-H as shown by the significant mobility shift of NF-H on SDS-PAGE (Fig. 3A). In addition to the mobility shift, NF-H, which has been co-transfected with Cdk5 and p35 is readily recognized by an antibody specific for the phosphorylated form of NF-H, but not by an antibody to the nonphosphorylated form of NF-H. Since it is possible that the phosphorylation of NF-H is not directly by Cdk5, but rather by a kinase, which is in turn activated by Cdk5, we performed the immunoprecipitation experiments. These experiments show that immunoprecipitated Cdk5 can phosphorylate NF-H provided p35 is present (Fig. 4). These experiments also show that of the two possible consensus sequences for NF-H phosphorylation, Cdk5 is able to phosphorylate only one sequence (KSPXK) and not the other (KSPXY). Another line of evidence for the identification of NF-H as a substrate for Cdk5 comes from the experiment with the dominant-negative Cdk5dn, which can compete with Cdk5 and reduce its kinase activity on NF-H.
      The data presented in this paper, as well as data from others using synthetic peptides, have demonstrated that the KSPXK repeats on NF-H are the preferred sites of phoshorylation by Cdk5. The lack of reactivity of SMI32 against exogenously expressed NF-H from cells co-transfected with Cdk5 and p35 is therefore somewhat surprising, since the KSPXK motif accounts for only ∼20% of the KSP sites of rat NF-H and might indicate that the SMI32 antibody is specific for this motif in the nonphosphorylated form. Alternatively, additional phosphorylation of NF-H on the KSPXY sites by Cdk5 (or by a different kinase) could occur only after prior phosphorylation of the KSPXK sites by Cdk5. It is therefore interesting to note that recent studies have shown that in vitro phosphorylation of recombinant τ by Cdk5 and by glycogen synthase kinase 3β (GSK3β) resulted in the phosphorylation of some of the same sites as τ from Alzheimer's disease-paired helical filaments (
      • Baumann K.
      • Mandelkow E.-M.
      • Biernat J.
      • Piwnica-Worsm H.
      • Mandelkow E.
      ,
      • Mandelkow E.-M.
      • Drewes G.
      • Biernat J.
      • Gustke N.
      • Van Lint J.
      • Vandenheede J.R.
      • Mandelkow E.
      ). This abnormal phosphorylation of τ has also been observed in cultured cells in which GSK3β and τ were overexpressed by transient transfections (
      • Loveston S.
      • Reynolds C.H.
      • Latimer D.
      • Davis D.R.
      • Anderton B.H.
      • Gallo J.-M.
      • Hanger D.
      • Mulot S.
      • Marquardt B.
      • Stabel S.
      • Woodgett J.R.
      • Miller C.C.
      ) In vitro experiments have shown that τ becomes a better substrate for GSK3β after it is first phosphorylated by Cdk5 (
      • Kobayashi S.
      • Ishiguro K.
      • Omori A.
      • Takamatsu M.
      • Arioka M.
      • Imahori K.
      • Uchida T.
      ). In our experiments, we could not detect any significant level of phosphorylation of NF-H by transient co-transfection with GSK3β (Fig. 3). However, it is possible that in the cells co-transfected with NF-H, Cdk5, and p35 constructs, further phosphorylation of NF-H by the endogenous GSK3β occurred after the KSPXK sequences were phosphorylated by Cdk5/p35. It is also interesting to note that although τ is a substrate of Cdk5 it contains a KSPXY sequence, but does not have a KSPXK sequence.
      Even though Cdk5 is related to the cyclin-dependent kinase family, it has several unique features, such as its expression in terminally differentiated neurons and the fact that its activator, p35, has nearly no homology to cyclins. We have observed no differences in the ability of rat p35 and bovine p23 (which corresponds to the carboxyl-terminal portion of p35) to activate Cdk5 in the co-transfection experiments. These results are consistent with the report showing kinase activity from the bacterially produced Cdk5 and truncated p35 (
      • Qi Z.
      • Huang Q.Q.
      • Lee K.Y.
      • Lew J.
      • Wang J.H.
      ) and support the notion that the carboxyl-terminal portion of p35 is sufficient to activate Cdk5. What role, if any, the amino-terminal part of p35 plays in regulating the Cdk5 activation remains an open question. Recently, a p35 isoform, which is able to activate Cdk5 in vitro, has been isolated from human brain (
      • Tang D.
      • Yeung J.
      • Lee K.-Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ). It will be interesting to see whether p35 belongs to a larger protein family and whether Cdk5 or other related kinases can be activated by different members of this family. The restricted expression of p35 to the central nervous system also raises the possibility that in the peripheral nervous system, other protein kinases and/or activators are present which phosphorylate NF-H.

      Acknowledgments

      We thank Dr. Gee Ching for editorial assistance and critical discussions on the manuscript and David Knowles for his assistance in preparation of the constructs and the purification of the bacterially expressed proteins.

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