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Pctaire1 Interacts with p35 and Is a Novel Substrate for Cdk5/p35*

  • Kai Cheng
    Footnotes
    Affiliations
    From the Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
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  • Zhen Li
    Footnotes
    Affiliations
    From the Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
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  • Wing-Yu Fu
    Affiliations
    From the Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
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  • Jerry H. Wang
    Affiliations
    From the Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
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  • Amy K.Y. Fu
    Affiliations
    From the Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
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  • Nancy Y. Ip
    Correspondence
    Recipient of the Croucher Foundation Senior Research Fellowship. To whom correspondence should be addressed: Dept. of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China. Tel.: 852-2358-7304; Fax: 852-2358-2765;
    Affiliations
    From the Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
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  • Author Footnotes
    * This work was supported in part by the Research Grants Council of Hong Kong (HKUST 6103/00M and HKUST 6091/01M) (to N. Y. I.) and the Hong Kong Jockey Club.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.
    ‡ These authors contributed equally to this work.
Open AccessPublished:June 25, 2002DOI:https://doi.org/10.1074/jbc.M201161200
      Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that plays important roles during central nervous system development. Cdk5 kinase activity depends on its regulatory partners, p35 or p39, which are prominently expressed in the central nervous system. We have previously demonstrated the involvement of Cdk5 in the regulation of acetylcholine receptor expression at the neuromuscular junction, suggesting a novel functional role of Cdk5 at the synapse. Here we report the identification of Pctaire1, a member of the Cdk-related kinase family, as a p35-interacting protein in muscle. Binding of Pctaire1 to p35 can be demonstrated by in vitrobinding assay and co-immunoprecipitation experiments. Pctaire1 is associated with p35 in cultured myotubes and skeletal muscle, and is concentrated at the neuromuscular junction. Furthermore, Pctaire1 can be phosphorylated by the Cdk5/p25 complex, and serine 95 is the major phosphorylation site. In brain and muscle of Cdk5 null mice, Pctaire1 activity is significantly reduced. Moreover, Pctaire1 activity is increased following preincubation with brain extracts and phosphorylation by the Cdk5/p25 complex. Taken together, our findings demonstrate that Pctaire1 interacts with p35, both in vitroand in vivo, and that phosphorylation of Pctaire1 by Cdk5 enhances its kinase activity.
      Cdk
      cyclin-dependent kinase
      GST
      glutathioneS-transferase
      NMJ
      neuromuscular junction
      PBS
      phosphate-buffered saline
      MBP
      myelin basic protein
      MOPS
      4-morpholinepropanesulfonic acid
      Cyclin-dependent kinase 5 (Cdk5),1 a member of the Cdk family, is involved in cellular functions that are not related to the regulation of cell cycle progression (
      • Lew J.
      • Beaudette K.
      • Litwin C.M.E.
      • Wang J.H.
      ). Whereas other members of the family associate with cyclins, the serine/threonine kinase activity of Cdk5 requires the association with one of its regulatory units, p35 or p39. A C-terminal proteolytic product of p35, p25, has also been shown to activate Cdk5 (
      • Dhavan R.
      • Tsai L.H.
      ). Although Cdk5 is ubiquitous in mammalian tissues, the expression of its activators is largely restricted to neurons. The temporal and spatial specificity of Cdk5 kinase activity is therefore because of the neuronal expression of p35 and p39 (
      • Ishiguro K.
      • Kobayashi S.
      • Omore A.
      • Takamatsu M.
      • Yonekura S.
      • Anzai K.
      • Imahori K.
      • Uchida T.
      ,
      • Lew J.
      • Huang Q.Q., Qi, Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S.
      • Chae T.
      • Harlow E.
      ,
      • Zheng M.
      • Leung C.L.
      • Liem R.K.
      ). Recent studies demonstrate that p35 transcript and p35-associated Cdk5 kinase activity can also be detected in muscle (
      • Fu A.K.Y., Fu, W.Y.
      • Cheung J.
      • Tsim K.W.K., Ip, F.C.F.
      • Wang J.H.
      • Ip N.Y.
      ,
      • Fu W.Y., Fu, A.K.Y.
      • Lok K.-C., Ip, F.C.F.
      • Ip N.Y.
      ). Whereas the best characterized role for Cdk5 is in regulating neuronal migration and axonal guidance as well as cytoskeletal dynamics, there is also evidence that links Cdk5 activity to the regulation of membrane transport and dopamine signaling (
      • Dhavan R.
      • Tsai L.H.
      ).
      In addition to its known functions in neuronal development, recent evidence suggests a novel role for Cdk5 in the modulation of postsynaptic functions. For example, Cdk5 regulates the expression of acetylcholine receptors in muscle during the formation of the neuromuscular junction (NMJ) (
      • Fu A.K.Y., Fu, W.Y.
      • Cheung J.
      • Tsim K.W.K., Ip, F.C.F.
      • Wang J.H.
      • Ip N.Y.
      ). Cdk5 and its activators are localized to the NMJ in adult muscle and mediate the actions of neuregulin by associating with the ErbB receptors (
      • Fu A.K.Y., Fu, W.Y.
      • Cheung J.
      • Tsim K.W.K., Ip, F.C.F.
      • Wang J.H.
      • Ip N.Y.
      ). In addition to playing a key role at the NMJ, Cdk5 has recently been demonstrated to regulateN-methyl-d-aspartic acid receptors in cultured hippocampal neurons, suggesting that Cdk5 is also involved in modulating postsynaptic functions in the central nervous system (
      • Li B.S.
      • Sun M.K.
      • Zhang L.
      • Takahashi S., Ma, W.
      • Vinade L.
      • Kulkarni A.B.
      • Brady R.O.
      • Pant H.C.
      ).
      To further elucidate the functional roles of Cdk5 at the NMJ, it is important to examine the proteins interacting with the active Cdk5/p35 complex in postsynaptic sites. In this study, we have used p35 as bait to perform a yeast two-hybrid screen using a muscle cDNA library. Pctaire1, a member of the Cdk-related protein kinase family, is identified as a p35-interacting protein. Pctaire1 and its related proteins, Pctaire2 and -3, are 50–60-kDa proteins consisting of a core kinase domain flanked by unique N-terminal and C-terminal domains. The catalytic domain of Pctaire proteins contains all the motifs and amino acid residues conserved in the Cdk family (
      • Meyerson M.
      • Enders G.H., Wu, C.L., Su, L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.H.
      ). It has been suggested that the N-terminal domains of Pctaire define the specific function of the particular Pctaire family members (
      • Hirose T.
      • Kawabuchi M.
      • Tamaru T.
      • Okumura N.
      • Nagai K.
      • Okada M.
      ). In this study, we demonstrate that Pctaire1 can be phosphorylated by the Cdk5/p35 complex. Two of the potential Cdk5 phosphorylation sites for Pctaire1 reside on the N-terminal region that shows no amino acid homology with other Cdks. Our results show that one of them, serine 95, is the major phosphorylation site. Taken together, our findings demonstrate that p35 and Pctaire1 interact with each other, both in vitro and in vivo, and that Pctaire1 is a novel substrate of Cdk5. Whereas Cdk5/p25 alone cannot directly activate Pctaire1, we provide evidence that phosphorylation of Pctaire1 by the Cdk5 complex enhances its kinase activity.

      DISCUSSION

      In the present study, Pctaire1 was identified as an interacting protein with p35 in a yeast two-hybrid screen. Our findings demonstrate that p35 and Pctaire1 interact with each other, both in vitro and in vivo. The full interaction between p35 and Pctaire1 requires the presence of the N-terminal, core kinase, and C-terminal regions, suggesting that the three-dimensional structure of Pctaire1 is important for its association with p35. This finding is consistent with previous reports on the interaction between Pctaire1 and p11 or 14-3-3 proteins (
      • Sladeczek F.
      • Camonis J.H.
      • Burnol A.F.
      • Le Bouffant F.
      ,
      • Le Bouffant F.
      • Capdevielle J.
      • Guilemot J.C.
      • Sladeczek F.
      ), i.e. both the N- and C-terminal domains are required. We also found that Pctaire1 can be phosphorylated by Cdk5/p25 on serine 95, and is therefore a novel substrate of active Cdk5. Although the precise significance of this interaction remains to be elucidated, our findings suggest that Cdk5 regulates the kinase activity of Pctaire1.
      Based on the yeast two-hybrid screen, previous studies have identified several proteins that interact with Cdk5/p35, including neurofilament, tau, cables, and nudel (
      • Qi Z.
      • Tang D.M.
      • Zhu X.D.
      • Fujita D.J.
      • Wang J.H.
      ,
      • Zukerberg L.R.
      • Patrick G.N.
      • Nikolic M.
      • Humbert S., Wu, C.L.
      • Lanier L.M.
      • Gertler F.B.
      • Vidal M.
      • Van Etten R.A.
      • Tsai L.H.
      ,
      • Niethammer N.
      • Smith D.S.
      • Ayala R.
      • Peng J., Ko, J.
      • Lee M.
      • Morabito M.
      • Tsai L.H.
      ). Our identification of Pctaire1 as a novel substrate for Cdk5 provides the first demonstration of a Cdk-related kinase, other than Cdk5, that interacts with p35. Analysis of the deduced amino acid sequence of Pctaire1, a Cdc2-like kinase, identifies the presence of domains conserved among Cdks, including kinase subdomains with ∼52% similarity to Cdc2 kinases (
      • Le Bouffant F.
      • Capdevielle J.
      • Guilemot J.C.
      • Sladeczek F.
      ). On the other hand, it exhibits several features that are distinct from other members of the Cdk family. For example, the size of Pctaire1 is twice that of other Cdks (i.e. ∼60 kDa compared with ∼30 kDa) because of the presence of the N-terminal and C-terminal extensions. Unlike other Cdks, and similar to Cdk5, Pctaire1 is not involved in the regulation of cell cycle progression. The expression of Pctaire1 is ubiquitous, and is prominently expressed in highly differentiated tissues, including brain, eye, skeletal muscle, and heart (
      • Besset V.
      • Rhee K.
      • Wolgemoth D.J.
      ). In particular, Pctaire1 is abundant in the cytoplasm of terminally differentiated cells, such as elongated spermatids in testis, and postmitotic neurons in brain.
      Although Pctaire1 is expressed in both neuronal and non-neuronal cells, their precise functions are still unknown. Kinase activity has been detected in both brain and testis (
      • Besset V.
      • Rhee K.
      • Wolgemoth D.J.
      ,
      • Le Bouffant F.
      • Capdevielle J.
      • Guilemot J.C.
      • Sladeczek F.
      ), suggesting a role for Pctaire1 in the process of differentiation in these tissues. However, purified Pctaire1 does not exhibit kinase activity,
      K. Cheng and N. Y. Ip, unpublished observation.
      (
      • Charrasse S.
      • Carena I.
      • Hagmann J.
      • Wood-Cook K.
      • Ferrari S.
      ), suggesting that Pctaire1 activation requires binding to a regulatory partner. We report here that the kinase activity of Pctaire1 depends on a brain-specific activator, consistent with the observation that Pctaire1 activity is much higher in adult brain than in adult muscle. Thus, like the other members of the Cdk family, such as p35 for Cdk5 or cyclins for other Cdks, Pctaire1 needs to associate with specific activator(s) to trigger its activity. Whereas p35 is not the activator of Pctaire1 (
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S.
      • Chae T.
      • Harlow E.
      ),2 we have provided several lines of evidence to demonstrate that Cdk5/p35 is involved in the regulation of Pctaire1 activity. We found that Pctaire1 is phosphorylated by Cdk5/p25 and that its kinase activity is significantly reduced in brain and muscle of Cdk5 null mice. In addition, Pctaire1 activity can be further enhanced by incubation with Cdk5/p25. Taken together, our findings suggest that Pctaire1 activity is regulated by Cdk5 in vivo. It is possible that p35 may act as a substrate-targeting protein for Cdk5 to phosphorylate Pctaire1, which in turn modulates the activity of Pctaire1. The identification of the brain-specific activator of Pctaire1 will be critical to delineate the biological functions of Pctaire1 in Cdk5 signaling and to facilitate our understanding of the regulation of this protein.

      ACKNOWLEDGEMENTS

      We thank Drs. J. Chamberlain and L. Mei for the muscle cDNA library, and Drs. A. B. Kulkarni and T. Curran for the Cdk5 null mice. We are grateful to Dr. F. C. F. Ip, K.-C. Lok, and J. Cheung for expert technical assistance.

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