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Identification of a Neuronal Cdk5 Activator-binding Protein as Cdk5 Inhibitor*

  • Yick-Pang Ching
    Footnotes
    Affiliations
    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
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  • Andy S.H. Pang
    Affiliations
    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
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  • Wing-Ho Lam
    Affiliations
    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
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  • Robert Z. Qi
    Footnotes
    Affiliations
    Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 1N4, Canada
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  • Jerry H. Wang
    Correspondence
    To whom correspondence should be addressed. Tel.: 852-2358-8701; Fax: 852-2358-1552
    Affiliations
    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
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  • Author Footnotes
    * This work was supported by a grant from the Research Grants Council of Hong Kong and Hong Kong AoE of Molecular Neuroscience.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.
    § Current address: Inst. of Molecular Biology, Hong Kong University, Pokfulam, Hong Kong, China.
    ‖ Current address: Inst. of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore.
Open AccessPublished:March 06, 2002DOI:https://doi.org/10.1074/jbc.C200032200
      Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35nck5a or p25nck5a), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35nck5a-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35nck5a could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85·Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35nck5akinase.
      Cdk
      cyclin-dependent protein kinase
      Nck5a
      neuronal cyclin-dependent protein kinase 5 activator
      Nclk
      neuronal Cdc2-like kinase
      GST
      glutathione S-transferase
      FL
      full length
      BF
      binding fragment
      PBS
      phosphate-buffered saline
      MOPS
      4-morpholinepropanesulfonic acid
      CKI
      cyclin kinase inhibitor
      Cyclin-dependent protein kinases (Cdks)1 play critical roles in the regulation of cell division (
      • Morgan D.O.
      ). As the name implies, functional Cdks require binding of a cyclin for kinase activity. In addition to depending on cyclin for activity, Cdk activities are regulated by complex mechanisms including protein phosphorylation and association with specific Cdk inhibitors. There are two Cdk inhibitor families: INK4 and Kip/Cip. Crystallography analysis has shown that the INK4 family member p16INK4a causes the kinase inhibition by direct association to Cdk4 (
      • Jeffrey P.D.
      • Tong L.
      • Pavletich N.P.
      ). On the other hand, p27Kip/Cip binds to both Cdk2 and cyclin A in the Cdk2·cyclin A complex to inhibit the kinase activity (
      • Pavletich N.P.
      ).
      Cyclin-dependent kinase 5 is unique among Cdks in many respects. Unlike most other Cdks, Cdk5 has no known function in the cell division cycle but is involved in the regulation of neuronal differentiation and neurocytoskeleton dynamics (

      Deleted in proof

      ,

      Deleted in proof

      ,
      • Dhavan R.
      • Tsai L.H.
      ,
      • Lew J.
      • Wang J.H.
      ,

      Deleted in proof

      ,
      • Grant P.
      • Sharma P.
      • Pant H.C.
      ,

      Deleted in proof

      ). There are two mammalian Cdk5 activators: neuronal Cdk5 activator, p35nck5a, and neuronal Cdk5 activator isoform, p39nck5ai; both Nck5a and Nck5ai are expressed predominantly in neurons of central nervous systems (
      • Lew J.
      • Qi Q.Q.
      • Huang Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ,
      • Tang D.
      • Yeung J.
      • Lee K.Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ). While Nck5a and Nck5ai are homologous proteins, they show little or no sequence similarity to cyclins. Members of both Cdk inhibitor families have been tested and shown to have no activity toward Cdk5. Biochemical analysis of Cdk5 in bovine brain extract suggests the existence of an inhibitory factor that exists together with Cdk5 and p35nck5a in a protein complex (
      • Lee K.Y.
      • Rosales J.L.
      • Tang D.
      • Wang J.H.
      ). However, the molecular identity of the factor has not been established. InSaccharomyces cerevisiae, there is a cyclin-dependent protein kinase, Pho85, and its cyclin partner Pho80, which have been shown to be the functional homologues of mammalian Cdk5 and Nck5a, respectively (
      • Huang D.
      • Patrick G.
      • Moffat J.
      • Tsai L.H.
      • Andrews B.
      ). In addition, there is a specific Pho80/Pho85 inhibitor protein, Pho81, whose mammalian functional homologue is not known.
      Recently we have used the yeast two-hybrid system to screen for the p35nck5a-associated protein (
      • Qi Z.
      • Tang D.
      • Zhu X.
      • Fujita Donald J.
      • Wang J.H.
      ). Among the positive clones are three clones, called C42, C48, and C53, whose cDNA sequences are novel (
      • Qi Y.P.
      • Ching Z.
      • Wang J.H.
      ). In the present study, we show that one of the novel proteins, C42, displays an inhibitory effect on Cdk5 kinase activity. Interestingly, while both the full-length C42 and the C42 fragment obtained from the yeast two-hybrid screen can bind Nck5a, only the full-length protein has Cdk5 inhibitory activity. Using deletion mutants, we have mapped the inhibitory domain of C42 to a 135-amino acid region, which shows a significant homology to the inhibitory region of Pho81.

      DISCUSSION

      Previously we have used the yeast two-hybrid screen to isolate three novel p35-binding proteins, named C42, C48, and C53, and showed that they bind to p35nck5a·Cdk5 complex (
      • Qi Y.P.
      • Ching Z.
      • Wang J.H.
      ). In this study, we demonstrate that the full-length C42, but not the C42 fragment from the yeast two-hybrid screen, possesses potent inhibitory activity toward Cdk5. We have carefully ruled out a number of artifacts that may give rise to an apparent kinase inhibition, such as the contamination of protease, ATPase, or phosphatase activity in the C42 sample. Affinity precipitation using bacterially expressed proteins indicates that either full-length C42 or C42 fragment can form a ternary complex with Cdk5 and Nck5a. Immunoprecipitation analysis shows that the ternary complexes can also exist in HeLa cell lysates. These results strongly suggest that C42 is a Cdk5 inhibitor. In fact, we have carried out transfection experiments showing that ectopic expression of the full-length C42 in NG108 neuroblastoma cells markedly suppressed the differentiation of the cells in differentiating medium, whereas the expression of C42-BF had significantly less effect.
      Y.-P. Ching, W.-H. Lam, and J. H. Wang, unpublished observation.
      The amino acid sequence of C42 shows no sequence similarity to proteins of either CKI family. The mechanism of Cdk5 kinase inhibition of C42 also appears to distinguish the protein from the known CKIs. While CKIs of the Kip/Cip family interact through the kinase catalytic subunit, members of the INK4 family undergo physical interactions with both the kinase and the cyclin subunits. In contrast, C42 appears to inhibit Cdk5 activity by interacting exclusively with the regulatory subunit Nck5a (or Nck5ai). Affinity binding experiments have shown that C42 undergoes high affinity binding to both Nck5a (or Nck5ai) and the Nck5a·Cdk5 complex but not to monomeric Cdk5. The inhibitory activity of C42 shows a strict specificity toward Nck5a or Nck5ai rather than the kinase subunit. This may be demonstrated by using Cdk2 instead of Cdk5 as the inhibition target. Only the p35nck5a-activated Cdk2 but not the cyclin A-activated Cdk2 can be inhibited by C42.
      While there is a good correlation between C42 inhibition activity and the ability of the protein to undergo high affinity binding to Cdk5 activator proteins, the inhibition activity is not solely due to the protein binding. Both full-length C42 and the C42 fragment bind to Nck5a with high affinity, but only the full-length C42 displays kinase inhibitory activity. Despite the fact that the other two novel Nck5a-binding proteins, C48 and C53, share the same Nck5a binding region of C42, they did not exhibit Nck5a inhibitory activity. These observations have led us to speculate that the full-length C42 contains a kinase inhibitory domain in addition to the high affinity Nck5a-binding domain. Deletion analysis has suggested the existence of a kinase inhibitory domain distinct from the Nck5a-binding domain of C42, a suggestion subsequently confirmed from sequence comparison between C42 and the recently identified Cdk inhibition domain in Pho81 (see below). A simple model to account for the existence of both Nck5a-binding and kinase inhibition domains is that while kinase inhibition depends solely on the kinase inhibitory domain, C42 uses the high affinity Nck5a-binding domain to increase the affinity of the inhibitory domain-enzyme interaction. The fact that the inhibitory domain can only inhibit about 60% of the kinase activity may suggest that the Nck5a-binding domain is required for achieving high inhibitory activity. This is also in agreement with the observation that the concentration of the inhibitory domain required for kinase inhibition is about an order of magnitude higher than that of full-length C42.
      A number of observations suggest that C42 is related to Pho81, a Cdk inhibitor that is specific for Pho85/Pho80, the yeast homologue of Cdk5/Nck5a. The kinase inhibitor domain of Pho81 has recently been mapped (

      Deleted in proof

      ). Sequence alignment of C42 and Pho81 has identified a region in C42 showing significant similarity to the kinase inhibitory domain of Pho81. The C42 deletion mutant containing only this region of the protein displayed Cdk5 inhibitory activity. In addition to structural similarity, C42 and Pho81 are similar in their Cdk inhibition mechanisms. Results of complementary assays in yeast suggested that Cdk5 could associate with Pho80 to form an active kinase and that the active kinase responded to Pho81 in yeast growing in low phosphate medium. Like C42, Pho81 exerts its kinase inhibitory activity through interaction with the regulatory subunit Pho80, rather than with Pho85, the catalytic (Cdk) subunit. The kinase inhibition by C42 is strongly dependent on the order of addition of Cdk5 to Nck5a and Cdk5 (Fig. 1B). Intriguingly a ternary complex of Cdk5, Nck5a, and C42 can be formed under either the inhibiting or the noninhibiting conditions. Based on their functional analysis of Pho81 and Pho85/Pho80 interaction in yeast cells, Huang et al. (

      Deleted in proof

      ) suggested that Pho81 might form either an active or an inactive ternary complex with Pho85/Pho80 kinase in high and low phosphate medium. Thus, from these various lines of evidence, it is suggested that C42 and Pho81 may be grouped into a new family of CKIs.

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