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Phosphorylation of Pak1 by the p35/Cdk5 Kinase Affects Neuronal Morphology*

  • Tahira Rashid
    Footnotes
    Affiliations
    Molecular and Developmental Neurobiology Medical Research Council Centre, New Hunt's House, King's College London, London, SE1 1UL, United Kingdom
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  • Monisha Banerjee
    Footnotes
    Affiliations
    Molecular and Developmental Neurobiology Medical Research Council Centre, New Hunt's House, King's College London, London, SE1 1UL, United Kingdom
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  • Margareta Nikolic
    Footnotes
    Affiliations
    Molecular and Developmental Neurobiology Medical Research Council Centre, New Hunt's House, King's College London, London, SE1 1UL, United Kingdom
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  • Author Footnotes
    * This work was supported by the Wellcome Trust BBSRC and GlaxoSmith Kline.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 the work.
    § Recipient of a Wellcome Trust career development award. To whom correspondence should be sent. Tel.: 44-20-7848-6802; Fax: 44-20-7848-6816; E-mail: [email protected]
Open AccessPublished:December 28, 2001DOI:https://doi.org/10.1074/jbc.M105599200
      The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.
      CNS
      central nervous system
      GST
      glutathione S-transferase
      The development of the mammalian central nervous system (CNS)1 is determined by the correct migration of immature neurons and their ability to extend neurites and form synapses. In the cortex, proliferating neuronal precursors move while passing through different stages of the cell cycle. At set times, these precursors arrest and embark on a long distance migration to form distinct cortical layers where they pass through several stages of neurite remodeling, pathfinding, and synaptogenesis (
      • Hatten M.E.
      ). A fundamental role in all of these processes is played by the continuous restructuring of the neuronal cytoskeleton.
      The p35/Cdk5 kinase is enriched in the developing CNS with the highest levels seen when neurons are migrating and remodeling (
      • Kwon Y.T.
      • Tsai L.H.
      ,
      • Grant P.
      • Sharma P.
      • Pant H.C.
      ,
      • Paglini G.
      • Caceres A.
      ). It is a proline-directed serine/threonine kinase previously shown to promote neurite outgrowth and co-localize in axonal growth cones with F-actin (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ,
      • Xiong W.
      • Pestell R.
      • Rosner M.R.
      ,
      • Pigino G.
      • Paglini G.
      • Ulloa L.
      • Avila J.
      • Cáceres A.
      ,
      • Paglini G.
      • Pigino G.
      • Kunda P.
      • Morfini G.
      • Maccioni R.
      • Quiroga S.
      • Ferreira A.
      • Cáceres A.
      ,
      • Li B.S.
      • Zhang L.
      • Gu J.
      • Amin N.D.
      • Pant H.C.
      ). Mice that lack Cdk5 or p35 exhibit a severely disrupted laminar structure of the cerebral cortex proposed to result from the inability of cortical neurons to migrate past each other (
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ,
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ,
      • Gilmore E.C.
      • Ohshima T.
      • Goffinet A.M.
      • Kulkarni A.B.
      • Herrup K.
      ,
      • Kwon Y.T.
      • Tsai L.H.
      ,
      • Ohshima T.
      • Gilmore E.C.
      • Longenecker G.
      • Jacobowitz D.M.
      • Brady R.O.
      • Herrup K.
      • Kulkarni A.B.
      ). Axon guidance and fasciculation defects have also been observed in p35−/− and Cdk5−/− mice (
      • Gilmore E.C.
      • Ohshima T.
      • Goffinet A.M.
      • Kulkarni A.B.
      • Herrup K.
      ,
      • Kwon Y.T.
      • Tsai L.H.
      ). Both p35 and Cdk5 are essential for the proper development of theDrosophila and Xenopus CNS in which alterations in kinase activity affect differentiation and the accuracy of axonal patterning (
      • Philpott A.
      • Tsai L.
      • Kirschner M.W.
      ,
      • Connell-Crowley L.
      • Le Gall M.
      • Vo D.J.
      • Giniger E.
      ). A role for the p35/Cdk5 kinase has also been suggested in regulating synapses (
      • Shuang R.
      • Zhang L.
      • Fletcher A.
      • Groblewski G.E.
      • Pevsner J.
      • Stuenkel E.L.
      ,
      • Bibb J.A.
      • Snyder G.L.
      • Nishi A.
      • Yan Z.
      • Meijer L.
      • Fienberg A.A.
      • Tsai L.H.
      • Kwon Y.T.
      • Girault J.A.
      • Czernik A.J.
      • Huganir R.L.
      • Hemmings Jr., H.C.
      • Nairn A.C.
      • Greengard P.
      ,
      • Fletcher A.I.
      • Shuang R.
      • Giovannucci D.R.
      • Zhang L.
      • Bittner M.A.
      • Stuenkel E.L.
      ,
      • Humbert S.
      • Lanier L.M.
      • Tsai L.H.
      ,
      • Rosales J.L.
      • Nodwell M.J.
      • Johnston R.N.
      • Lee K.Y.
      ), whereas deregulation of its activity may play a part in several neuropathologies, especially Alzheimer's (
      • Gleeson J.G.
      • Walsh C.A.
      ,
      • Maccioni R.B.
      • Otth C.
      • Concha I.I.
      • Munoz J.P.
      ).
      The p35/Cdk5 kinase is a downstream effector of the small GTPase Rac1 (subsequently referred to as Rac) (
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.H.
      ). In fibroblasts, Rac was first shown to regulate the formation of lamellipodia (
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ). In neurons, the function of Rac has been associated with dendritic remodeling, neurite branching, neurite outgrowth, synaptogenesis, and growth cone collapse (
      • Luo L.
      ,
      • Dickson B.J.
      ,
      • Redmond L.
      • Ghosh A.
      ). We have demonstrated a GTP-dependent association between p35 and Rac, making p35/Cdk5 the first known neuronal-specific Rac effector (
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.H.
      ). In Xenopus retinal ganglion cells, loss of p35/Cdk5 kinase activity can partially rescue defective axonogenesis induced by constitutively active Rac mutants, showing a conservation of this signaling pathway in vertebrates (
      • Ruchhoeft M.L.
      • Ohnuma S.
      • McNeill L.
      • Holt C.E.
      • Harris W.A.
      ). In transfected cell lines or primary neurons, the p35/Cdk5 kinase is found in association with Rac and the serine/threonine kinase Pak1, a well characterized Rac and Cdc42 effector (
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.H.
      ). Pak kinases are known to play an important role in regulating polarity, migration, and adhesion (
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Daniels R.H.
      • Bokoch G.M.
      ). InDrosophila retinal ganglion cells, axonal outgrowth and pathfinding is dependent on interactions between DPak and the adapter protein Dock, the kinase activity of DPak, and signals received from Trio, a Rac exchange factor (
      • Hing H.
      • Xiao J.
      • Harden N.
      • Lim L.
      • Zipursky S.L.
      ,
      • Newsome T.P.
      • Schmidt S.
      • Dietzl G.
      • Keleman K.
      • Asling B.
      • Debant A.
      • Dickson B.J.
      ). In mammals, Pak1 is highly enriched in the brain, specifically the cortex, at developmental stages when the p35/Cdk5 kinase is induced (
      • Tsai L.H.
      • Takahashi T.
      • Caviness V.S.
      • Harlow E.
      ,
      • Manser E.
      • Chong C.
      • Zhao Z.S.
      • Leung T.
      • Michael G.
      • Hall C.
      • Lim L.
      ,
      • Tomizawa K.
      • Matsui H.
      • Matsushita M.
      • Lew J.
      • Tokuda M.
      • Itano T.
      • Konishi R.
      • Wang J.H.
      • Hatase O.
      ). In cultured rat cortical neurons, Pak1 co-localizes with p35 and Cdk5 at the distal tips of axonal growth cones, whereas over-expression of a membrane-targeted Pak1 in PC12 cells induces neurite outgrowth (
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.H.
      ,
      • Daniels R.H.
      • Hall P.S.
      • Bokoch G.M.
      ).
      Our previous work revealed Pak1 phosphorylation and subsequent down-regulation of its kinase activity in the presence of p35/Cdk5 (
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.H.
      ). We have now observed that the p35/Cdk5 kinase associates with a large portion of the Pak1 kinase domain and directly phosphorylates Pak1 on a unique site. This phosphorylation is crucial for the proper regulation of the cytoskeleton during neurite outgrowth. Together our data reveal the complexity of p35/Cdk5-mediated regulation of Pak1 in differentiating neurons.

      DISCUSSION

      The importance of signals that emanate from Rac and regulate the actin cytoskeleton in differentiating neurons has become increasingly apparent. It is therefore not surprising that two Rac effectors, the neuronal-specific p35/Cdk5 and neuronal-enriched Pak1 kinase, have also been shown to be essential for the proper functioning of CNS neurons. Work using Drosophila, Xenopus, and mammalian systems has indicated that functional alterations in p35, Cdk5, Pak1, or Rac have many overlapping but also distinct features. Overall, the common biological consequences can be summed up as defects in neuronal migration and axon and dendrite remodeling (
      • Grant P.
      • Sharma P.
      • Pant H.C.
      ,
      • Paglini G.
      • Caceres A.
      ,
      • Luo L.
      ,
      • Dickson B.J.
      ,
      • Redmond L.
      • Ghosh A.
      ). The complexity of these phenotypes has suggested that the function of the p35/Cdk5 kinase in vivo is not simply to promote neuronal migration and neurite outgrowth but to provide a signaling mechanism to tightly regulate the accuracy of these events. We propose that this is achieved, at least in part, by the p35/Cdk5 kinase acting downstream of Rac and regulating the membrane-associated functions of the Pak1 kinase. To date it has been shown that localization and activity of the Pak1 kinase can have profound and varying consequences in different experimental systems, probably reflecting the dynamic complexity of its in vivo regulation (
      • Daniels R.H.
      • Hall P.S.
      • Bokoch G.M.
      ,
      • Symons M.
      ,
      • Kiosses W.B.
      • Daniels R.H.
      • Otey C.
      • Bokoch G.M.
      • Schwartz M.A.
      ,
      • Sells M.A.
      • Boyd J.T.
      • Chernoff J.
      ,
      • Sells M.A.
      • Knaus U.G.
      • Bagrodia S.
      • Ambrose D.M.
      • Bokoch G.M.
      • Chernoff J.
      ). Our work is the first to demonstrate the functional consequences of Pak1 regulation at the membranes of differentiating mammalian CNS neurons.
      The structural features of Pak1 allow for several levels of regulation. In unstimulated cells Pak1 resides in the cytoplasm in an autoinhibited conformation achieved by virtue of interactions between an N-terminal region (amino acids 75–132) with the C-terminal kinase domain. Association with RacGTP or Cdc42GTP at the CRIB/PBD domain, which partially overlaps with the autoinhibitory sequences, induces conformational changes in the Pak1 protein coupled with autophosphorylation, which results in its activation (
      • Zhao Z.S.
      • Manser E.
      • Chen X.Q.
      • Chong C.
      • Leung T.
      • Lim L.
      ,
      • Zenke F.T.
      • King C.C.
      • Bohl B.P.
      • Bokoch G.M.
      ,
      • Tu H.
      • Wigler M.
      ,
      • Lei M.
      • Lu W.
      • Meng W.
      • Parrini M.C.
      • Eck M.J.
      • Mayer B.J.
      • Harrison S.C.
      ). How active Pak1 is subsequently down-regulated is largely unknown. The p35/Cdk5 kinase associates with and phosphorylates Pak1 in a RacGTP-dependent manner, suggesting that p35 binds active Pak1. We therefore propose that in neurons, the p35/Cdk5 kinase modulates the function of Pak1 subsequent to its activation.
      We have demonstrated that phosphorylation of Pak1 by the p35/Cdk5 kinase takes place in vitro and in vivo on Thr-212. In Swiss 3T3 fibroblasts, co-expression of p35/Cdk5, Pak1, and constitutively active Rac results in extensive Pak1 phosphorylation on Thr-212, which localizes to the membrane and cytoplasm of these cells. In addition, a population of endogenous Pak1 is phosphorylated on Thr-212 in mouse and rat cortical and hippocampal neurons. In growth cones, Thr-212-phosphorylated Pak1 appears to outline distally extending microtubule bundles and co-localizes with F-actin. P35/Cdk5 phosphorylation of Pak1 may therefore affect the cross-talk between microtubules and microfilaments during the forward movement and turning of neuronal growth cones. The defects we observed upon over-expression of a membrane-targeted non-phosphorylatable Pak1 mutant further support this hypothesis. The Pak1A212 mutant remains down-regulatable by p35/Cdk5, suggesting that phosphorylation of Pak1 is not sufficient for p35/Cdk5-induced inhibition. The Pak1A212 mutant also shows no detectable impairment for Pak1/Pak1 association.
      Margareta Nikolic, unpublished data.
      The crystal structure of Pak1 domains revealed that Thr-212 resides in a flexible region for which a structure could not be predicted (
      • Lei M.
      • Lu W.
      • Meng W.
      • Parrini M.C.
      • Eck M.J.
      • Mayer B.J.
      • Harrison S.C.
      ). It is conserved in mouse, human, and rat orthologues. Two other closely related members of the Pak family of kinases, Pak2 and Pak3, do not have a p35/Cdk5 phosphorylation site at the position equivalent to Thr-212. Therefore, in neurons, phosphorylation of Pak1 on Thr-212 may regulate a specific signaling pathway, distinguishing it from other members of this family of kinases.
      Phosphorylation of Pak1 by a Cdk and association of the two kinases is evolutionarily conserved. The Saccharomyces cerevisiaeCdc28/Cln1 and Cdc28/Cln2 kinases have been shown to extensively phosphorylate Ste20, a member of the Pak family, predominantly at the late G1 and S phases of the cell cycle (
      • Oehlen L.J.
      • Cross F.R.
      ,
      • Wu C.
      • Leeuw T.
      • Leberer E.
      • Thomas D.Y.
      • Whiteway M.
      ,
      • Oda Y.
      • Huang K.
      • Cross F.R.
      • Cowburn D.
      • Chait B.T.
      ). The Cln2-dependent repression of the pheromone signaling pathway was shown to take place by affecting the function of Ste20 through direct interaction (
      • Oehlen L.J.
      • Cross F.R.
      ,
      • Oda Y.
      • Huang K.
      • Cross F.R.
      • Cowburn D.
      • Chait B.T.
      ). Cdc28/Cln2 also acts to abolish binding between Ste20 and the Gβ protein Ste4. In mammalian cells, Cdc2 was found to phosphorylate Pak1 on Thr-212 during mitosis.
      Jonathan Chernoff, personal communication.
      Cdk5, Cdk2, and Cdc2 are highly homologous and have a similar substrate specificity (
      • Beaudette K.N.
      • Lew J.
      • Wang J.H.
      ,
      • Shetty K.T.
      • Link W.T.
      • Pant H.C.
      ,
      • Songyang Z.
      • Lu K.P.
      • Kwon Y.T.
      • Tsai L.H.
      • Filhol O.
      • Cochet C.
      • Brickey D.A.
      • Soderling T.R.
      • Bartleson C.
      • Graves D.J.
      • DeMaggio A.J.
      • Hoekstra M.F.
      • Blenis J.
      • Hunter T.
      • Cantley L.C.
      ). However, in postmitotic neurons, Cdk2 and Cdc2 are absent and/or inhibited, whereas Cdk5 is highly active (
      • Tsai L.H.
      • Takahashi T.
      • Caviness V.S.
      • Harlow E.
      ,
      • Lee M.H.
      • Nikolic M.
      • Baptista C.A.
      • Lai E.
      • Tsai L.H.
      • Massague J.
      ). It is therefore likely that Cdk5 is the sole kinase responsible for phosphorylating Pak1 on Thr-212 in differentiating neurons of the CNS.
      Because Pak1A212 is inhibited by p35/Cdk5, the cytoskeletal defects we observed in neurons over-expressing this mutant are most likely not due to alterations in the activity of Pak1 at the membrane. Rather we speculate that phosphorylation of Thr-212 regulates an interaction between Pak1 and another cellular protein. Interestingly, Thr-212 is part of a putative SH3 binding PXXP domain. Mutation of this and another downstream PXXP site was shown to reduce the ability of activated Pak1 to disassemble focal adhesions and cause the collapse of cell peripheries (
      • Frost J.A.
      • Khokhlatchev A.
      • Stippec S.
      • White M.A.
      • Cobb M.H.
      ). It had no effect on the ability of Pak1 to associate with the PIX/Cool/p85 exchange factor, which binds a non-conventional PXXP motif in close proximity (
      • Frost J.A.
      • Khokhlatchev A.
      • Stippec S.
      • White M.A.
      • Cobb M.H.
      ,
      • Bagrodia S.
      • Taylor S.J.
      • Jordon K.A.
      • Van Aelst L.
      • Cerione R.A.
      ,
      • Manser E.
      • Loo T.H.
      • Koh C.G.
      • Zhao Z.S.
      • Chen X.Q.
      • Tan L.
      • Tan I.
      • Leung T.
      • Lim L.
      ). Likewise, we have not observed any differences in the ability of Pak1A212 or Glu-212 mutants to associate with PIX/Cool/p85 or Nck.2
      Fig. 9 depicts our proposed model of p35/Cdk5-mediated Pak1 regulation. Inactive auto-inhibited Pak1 predominates in the cytoplasm. Upon stimulation by extracellular signals, Pak1 is recruited to the membrane by molecules such as Nck (
      • Lu W.
      • Mayer B.J.
      ) in which association with the membrane-bound RacGTP or Cdc42GTP followed by sequential autophosphorylation activates the kinase. We propose that RacGTP-mediated Pak1 activation makes Pak1 a target of the p35/Cdk5 kinase. Phosphorylation of Pak1 on Thr-212 regulates an interaction between Pak1 and an as yet unidentified signaling molecule. In addition, association between p35/Cdk5 and the Pak1 C-terminal domain may alter the Pak1 structure to resemble its inhibited state. Because p35 is membrane-associated and short-lived, these events are highly localized and transient. This is particularly important in growth cones in which frequently opposing effects take place simultaneously in areas of close proximity. In conclusion we believe that a subpopulation of Pak1 is regulated by the p35/Cdk5 kinase in the developing CNS, thus providing a rapid and localized effect on the cytoskeleton of postmitotic neurons.
      Figure thumbnail gr9
      Figure 9A proposed model for p35/Cdk5-mediated regulation of Pak1 in neurons. At the membrane, Pak1 cycles between an active and inactive state. Autoinhibited Pak1, Cdk5 monomer, and RacGDP predominate in the neuronal cytoplasm where they are largely inactive (1). Upon receptor-mediated stimulation, Pak1 is recruited to the membrane via proteins such as Nck, where it associates with GTP-bound Rac (which is membrane-associated via C-terminal geranyl-geranylation) and a putative interactor (X) through the 220PVTP213 domain (2). Cdk5 is indirectly associated with the membrane via newly synthesized p35, which is myristoylated and also associates with RacGTP. The p35/Cdk5 kinase phosphorylates Pak1 on Thr-212, thus preventing association with the unknown protein X (3). In addition, p35/Cdk5 associates with Pak1 extensively over its C-terminal kinase domain and more weakly with the N terminus. We propose that this interaction, in combination with Pak1 phosphorylation, acts to regulate the downstream signaling on the neuronal cytoskeleton. Because p35/Cdk5 is short-lived, its effects on membrane-bound Pak1 are transient, bringing the signaling pathway back to stage 1.

      Acknowledgments

      We thank Prof. Patrick Doherty for providing a supportive working environment and Prof. Gary Bokoch and Prof. Jonathan Chernoff for supplying Pak1 reagents and sharing unpublished results. We thank Dr. Dorit Zharhary, Dr. Eli Kopf, and Sigma for producing the phospho-Pak1 antibody, Prof. Roger Morris for use of the deconvoluting microscope, Dr. John Dunnlop for confocal assistance, Dr. Li-Huei Tsai for support, Prof. Bryan Anderton and Dr. Graham Gibbs for help with two-dimensional electrophoresis, Dr. Margaret Chou for help with in-gel kinase assays, and Dr. David Prowse for continuous support, technical advice, and help with the manuscript.

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