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p35, the Neuronal-specific Activator of Cyclin-dependent Kinase 5 (Cdk5) Is Degraded by the Ubiquitin-Proteasome Pathway*

Open AccessPublished:September 11, 1998DOI:https://doi.org/10.1074/jbc.273.37.24057
      Cyclin-dependent kinase 5 (Cdk5) was originally isolated by its close homology to the human CDC2gene, which is a key regulator of cell cycle progression. However, unlike other Cdks, the activity of Cdk5 is required in post-mitotic neurons. The neuronal-specific p35 protein, which shares no homology to cyclins, was identified by virtue of its association and activation of Cdk5. Gene targeting studies in mice have shown that the p35/Cdk5 kinase is required for the proper neuronal migration and development of the mammalian cortex. We have investigated the regulation of the p35/Cdk5 kinase. Here we show that p35, the activator of Cdk5, is a short-lived protein with a half-life (t 1/2) of 20 to 30 min. Specific proteasome inhibitors such as lactacystin greatly stabilize p35 in vivo. Ubiquitination of p35 can be readily demonstrated in vitro and in vivo. Inhibition of Cdk5 activity by a specific Cdk inhibitor, roscovitine, or by overexpression of a dominant negative mutant of Cdk5 increases the stability of p35 by 2- to 3-fold. Furthermore, phosphorylation mutants of p35 also stabilize p35 2- to 3-fold. Together, these observations demonstrate that the p35/Cdk5 kinase can be subject to rapid turnover in vivo and suggest that phosphorylation of p35 upon Cdk5 kinase activation plays a autoregulatory role in p35 degradation mediated by ubiquitin-mediated proteolysis.
      Cdk5
      cyclin-dependent kinase 5
      LLnL
      N-acetyl-l-leucyl-l-leucyl-l-norleucinal
      Z-L3V5
      carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone
      CMV
      cytomegalovirus
      GST
      glutathione S- transferase.
      Cyclin-dependent kinase 5 (Cdk5)1 is a small serine/threonine kinase that shares a high degree of homology to the cell cycle regulators Cdc2 and Cdk2 kinases (
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.H.
      ). Despite this homology, a role for Cdk5 in cell cycle regulation has not been observed. Rather, expression of Cdk5 is most abundant in the adult nervous system (
      • Hellmich M.R.
      • Pant H.C.
      • Wada E.
      • Battey J.F.
      ,
      • Tsai L.H.
      • Takahashi T.
      • Caviness Jr., V.S.
      • Harlow E.
      ), which contains primarily post-mitotic neurons and glial cells. In addition, high levels of Cdk5 kinase activity are only present in brain lysates when compared with other tissues (
      • Tsai L.H.
      • Takahashi T.
      • Caviness Jr., V.S.
      • Harlow E.
      ,
      • Lew J.
      • Winkfein R.J.
      • Paudel H.K.
      • Wang J.H.
      ).
      The D-type cyclins have been shown to bind to Cdk5 in fibroblasts and other tissue culture cell lines (
      • Lee M.H.
      • Nikolic M.
      • Baptista C.A.
      • Lai E.
      • Tsai L.H.
      • Massague J.
      ,
      • Xiong Y.
      • Zhang H.
      • Beach D.
      ). However, this association does not result in an active kinase. In an effort to identify the Cdk5 activator in brain, a 35-kilodalton protein species (p35) was found to associate with Cdk5 in cultured primary neurons of embryonic rat cortices whose abundance correlates with the level of Cdk5 kinase activity (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ). Purified Cdk5 kinase activity from brain lysates contains two protein entities, Cdk5 and a partial fragment of p35 (
      • Lew J.
      • Winkfein R.J.
      • Paudel H.K.
      • Wang J.H.
      ,
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Ishiguro K.
      • Kobayashi S.
      • Omori A.
      • Takamatsu M.
      • Yonekura S.
      • Anzai K.
      • Imahori K.
      • Uchida T.
      ). Finally, recombinant p35 could activate Cdk5 kinase when mixed in vitro, formally establishing the regulatory role of p35 (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ,
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ). Despite the fact that p35 serves as a regulatory partner for Cdk5, it does not display any primary sequence homology to members of the cyclin family of proteins. However, the predicted tertiary structure of p35 is similar to cyclins (
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ,
      • Brown N.R.
      • Noble M.E.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ), suggesting that p35 binds and activates Cdk5 similar to other cyclin-Cdk complexes.
      The substrate specificity of the p35/Cdk5 kinase is similar to that of the Cdc2 and Cdk2 kinases, phosphorylating the K(S/T)PX(K/R) consensus sequence motif (
      • Beaudette K.N.
      • Lew J.
      • Wang J.H.
      ,
      • 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.
      ). The neuronal-specific intermediate filaments neurofilaments and the microtubule-associated proteins τ and mitogen-activated protein 2 have been shown to be substrates of the p35/Cdk5 kinase (
      • Lee K.Y.
      • Qi Z., Yu, Y.P.
      • Wang J.H.
      ).
      L. H. Tsai and K. S. Kosik, unpublished data.
      2L. H. Tsai and K. S. Kosik, unpublished data.
      In addition to these structural proteins, proteins associated with neural transmitter release such as synapsin I and Munc18 have also been shown to be phosphorylated by the p35/Cdk5 kinase (
      • Matsubara M.
      • Kusubata M.
      • Ishiguro K.
      • Uchida T.
      • Titani K.
      • Taniguchi H.
      ,
      • Shuang R.
      • Zhang L.
      • Fletcher A.
      • Groblewski G.E.
      • Pevsner J.
      • Stuenkel E.L.
      ).
      The biological function of the p35/Cdk5 kinase has been addressedin vivo and in vitro. Indeed, when a dominant negative Cdk5 mutant (DNK5) or an antisense p35 construct was introduced into cultured primary cortical neurons, neurite outgrowth was severely inhibited (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ). We also created a mouse strain lacking p35 (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ). These animals are viable and fertile but display seizures and sporadic lethality that is likely to be the result of terminal seizures. Histological examination reveals that the lamination pattern of the cerebral cortex is disrupted in p35-deficient animals, which results from an inverse packing order of post-mitotic cortical neurons (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ,
      • Kwon Y.T.
      • Tsai L.H.
      ). Cdk5-deficient animals die before or around birth with defects in the development of the cortex, cerebellum, and other compartments of the central nervous system (
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ). The cortex of these Cdk5−/− mice display a lamination defect reminiscent of p35.
      E. Gilmore, personal communication.
      3E. Gilmore, personal communication.
      Together, these results underscore the integral role of the p35/Cdk5 kinase in the migratory behavior of post-mitotic neurons.
      Cdk5 activity has recently been shown to be required for muscle development in Xenopus. Homologues of Cdk5 and p35 have also been isolated in Xenopus. One of the p35 homologues in Xenopus, Xp35.1, is highly expressed in developing somites where Cdk5 is also expressed. Overexpression of Xp35.1 or DNK5 disrupts muscle organization, and expression of MyoD and MRF4 is suppressed in the presence of DNK5 (
      • Philpott A.
      • Porro E.B.
      • Kirschner M.W.
      • Tsai L.H.
      ).
      In light of the essential function of the p35/Cdk5 kinase during mouse corticogenesis, the regulatory mechanism of this kinase is clearly part of the circuitry that underlies the development of the central nervous system. In addition to cyclin binding, Cdks can be regulated by post-translational phosphorylation and dephosphorylation events (
      • Morgan D.O.
      ). Cyclin degradation is also used as a unidirectional form of regulation. The fact that the Cdk5 kinase activity can be reconstituted in vitro using purified recombinant Cdk5 and p35 proteins suggests that binding to p35 is the rate-limiting step in kinase activation. Thus, the availability of p35 appears to be the primary determinant for Cdk5 activation. During neurogenesis, the expression of p35 is stringently regulated at the transcriptional level (
      • Delalle I.
      • Bhide P.G.
      • Caviness Jr., V.S.
      • Tsai L.H.
      ). Spatially, it is restricted to cells of the neuronal lineage. Temporally, p35 mRNA peaks around birth and quickly declines afterward (
      • Delalle I.
      • Bhide P.G.
      • Caviness Jr., V.S.
      • Tsai L.H.
      ,
      • Tomizawa K.
      • Matsui H.
      • Matsushita M.
      • Lew J.
      • Tokuda M.
      • Itano T.
      • Konishi R.
      • Wang J.H.
      • Hatase O.
      ). In addition, p35 mRNA is not present in dividing neuroblasts but is expressed when the neuroblasts exit the cell cycle and migrate out of the germinal zone during development of the cerebral cortex (
      • Delalle I.
      • Bhide P.G.
      • Caviness Jr., V.S.
      • Tsai L.H.
      ). In the adult, p35 is only present in certain areas of the forebrain including the hippocampal formation, the pyriform cortex, and layers 2, 3, 4, and 5 of the neocortex, structures that maintain high levels of plasticity (
      • Delalle I.
      • Bhide P.G.
      • Caviness Jr., V.S.
      • Tsai L.H.
      ).
      In this study, we show that p35 is regulated at the post-translational level based on the short half-life (t 1/2) of p35 protein. Our results indicate that the rapid turnover of p35 is mediated at least in part via the ubiquitin-dependent proteasome pathway. Ubiquitin is first activated in an ATP-dependent manner by the ubiquitin-activating enzyme (E1). It is then transferred to a ubiquitin-conjugating enzyme (E2) and then maybe transferred to a ubiquitin ligase (E3) involved in the recognition of substrate and transfer of ubiquitin molecule to the substrate. A multi-ubiquitinated protein can then be recognized by the proteasome, where it is hydrolyzed in a ATP-dependent fashion (
      • Coux O.
      • Tanaka K.
      • Goldberg A.L.
      ). It has been shown previously that important cell cycle regulators including the cyclins, Cdk inhibitors such as p21, p27, and p40SIC1, and p53 are all degraded via ubiquitin-mediated proteolysis (
      • Maki C.G.
      • Huibregtse J.M.
      • Howley P.M.
      ,
      • Maki C.G.
      • Howley P.M.
      ,
      • Pagano M.
      • Tam S.W.
      • Theodoras A.M.
      • Beer-Romero P.
      • Del Sal G.
      • Chau V.
      • Yew P.R.
      • Draetta G.F.
      • Rolfe M.
      ,
      • Sheaff R.J.
      • Roberts J.M.
      ). It is thought that periodic degradation of these proteins is essential for cell cycle progression. Our study thus demonstrates an involvement of ubiquitin-mediated proteolysis in the development of the nervous system. We further show that the active Cdk5 kinase stimulates the degradation of p35. These observations suggest that the active p35/Cdk5 kinase complex is subject to rapid inactivation mediated in part by its own activity leading to the autophosphorylation and degradation of the activator, p35, providing a negative feedback loop.

      DISCUSSION

      The biological function of the p35/Cdk5 kinase has been implicated in the histogenesis of the central nervous system. The adult mammalian cortex is characterized by a distinct laminar structure generated through a well defined pattern of neuronal migration whereby neurons born later in corticogenesis migrate through and past the older neurons to occupy a more superficial layer of the cortical plate (
      • Luskin M.B.
      • Shatz C.J.
      ,
      • Angevine J.B.
      • Sidman R.L.
      ). Mice with targeted mutations in either p35 or Cdk5 have a striking disruption of these cortical layers. The earlier born neurons reside in more superficial layers, and the latter born cells reside in more deep layers (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ,
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ).3 Furthermore, when primary cortical cultures were transfected with either an antisense construct of p35 or with dominant negative constructs of Cdk5 (Cdk5N144 and Cdk5T33), neurite outgrowth was inhibited. In contrast, when wild type p35 and Cdk5 were ectopically expressed, the neurite length was longer (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ). Taken together, these observations indicate that the p35/Cdk5 kinase is clearly involved in dynamic events of neuronal migration and neurite outgrowth.
      We have begun to look at the regulation of the p35/Cdk5 kinase. The high homology to cdc2 (
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.H.
      ) provides a strong precedence for Cdk5 to be regulated in similar manners as other Cdks. The requirement of an associated protein for kinase activity holds true, but the fact that the activator p35 and the kinase activity is only present in post-mitotic neurons suggests differences. Previous observations clearly show that there are at least some differences regarding activation. Phosphorylation of S159 by Cdk-activating kinase does not appear to be required for activation of Cdk5 despite the high homology to Cdc2/Cdk2 (
      • Poon R.Y.
      • Lew J.
      • Hunter T.
      ).
      Y. Ramos and L. H. Tsai, unpublished data.
      This is also corroborated by the fact that Cdk5 kinase activity can be reconstituted in vitro by mixing purified Cdk5 and p35 bacterial-expressed protein (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ,
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ), suggesting that the rate-limiting step for activation of Cdk5 is association with p35. Interestingly, p35 associations with Cdk5 stimulates p35 degradation, and thus, a negative feedback for the activity of the kinase is generated.
      We first established that p35 is a very unstable protein with at 1/2 of ∼20 to 30 min in vivo. This indicates that the active kinase complex is also unstable despite the fact that the t 1/2 of Cdk5 is greater than 90 min. The proteasome has been implicated in the degradation of many proteins such as cyclins in cell cycle regulation. This form of unidirectional post-translational regulation is widely used in processes in the cell. Lactacystin, a proteasome inhibitor, was identified by virtue of its ability to induce neurite outgrowth in neuro 2A neuroblastoma cells (
      • Omura S.
      • Matsuzaki K.
      • Fujimoto T.
      • Kosuge K.
      • Furuya T.
      • Fujita S.
      • Nakagawa A.
      ,
      • Omura S.
      • Fujimoto T.
      • Otoguro K.
      • Matsuzaki K.
      • Moriguchi R.
      • Tanaka H.
      • Sasaki Y.
      ), and it is the most specific inhibitor of the proteasome (
      • Fenteany G.
      • Standaert R.F.
      • Lane W.S.
      • Choi S.
      • Corey E.J.
      • Schreiber S.L.
      ). Using lactacystin and other inhibitors of the proteasome we were able to stabilize p35 in primary cortical neurons and transfected cell lines. E64, a cysteine protease inhibitor (
      • Mehdi S.
      ), does increase the overall levels of p35 as seen with the rest of the inhibitors; however, the turnover rate is unchanged, which indicates that there may be a population of p35 degraded by cysteine proteases in neurons, or the effect of E64 is indirect. Nevertheless degradation through the proteasome pathway appears to be the major route for degradation of p35. We also observed the accumulation of higher molecular weight species that were specifically recognized by p35 antibodies when p35-transfected C33A cells were treated with LLnL or MG132. In His6-myc-ubiquitin/p35 co-transfection experiments, we were able to confirm that higher molecular weight species of p35 were indeed p35-ubiquitin conjugates. Polyubiquitination of p35 was blocked by a K48R mutant version of the His6-myc-ubiquitin construct, which inhibits chain elongation. Taken together, these results suggest that p35 is a target for degradation by the proteasome in a ubiquitin-mediated fashion.
      The stability of p35, when transfected in COS7 or C33A cells, was similar to that seen in neurons, which provided an amenable system to study the mechanism of p35 degradation. Co-transfection of p35 with Cdk5 drastically reduced the steady state levels of p35 compared with transfection of p35 alone. Co-transfecting the catalytically inactive Cdk5, DNK5 (Cdk5 K33T), stabilized p35 by about 2- to 3-fold. Furthermore, inhibition of the endogenous p35/Cdk5 kinase activity in neurons by roscovitine also increased the stability of p35. These observations suggest that kinase activation stimulates p35 degradation. The fact that p35 becomes autophosphorylated by the p35/Cdk5 kinase indicates that phosphorylation of p35 plays a regulatory role in its degradation. When serine or threonine residues in the minimal Cdk phosphorylation consensus sites of p35 were mutated to alanine, the stability of these p35 mutants were increased 2- to 3-fold (p35 T138A and p35 QUAD, respectively). These results were similar to the stabilizing effect of p35 by DNK5 co-transfection and roscovitine treatment experiments. Taken together this data supports the hypothesis that phosphorylation stimulates p35 degradation. It is possible that phosphorylation may create an entrance point for the degradation machinery to bind and ubiquitinate p35, which awaits to be tested.
      Our initial deletion mutagenesis studies suggest that the p10 NH2-terminal region of p35 may be required for degradation, as the p25 fragment of p35 is a much more stable protein even in the presence of overexpressed Cdk5. Purification of the Cdk5 kinase from brain resulted in Cdk5 and the p25 NH2-terminal deletion fragment (
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Ishiguro K.
      • Kobayashi S.
      • Omori A.
      • Takamatsu M.
      • Yonekura S.
      • Anzai K.
      • Imahori K.
      • Uchida T.
      ). Additionally, p25 does appear to be physiologically produced.
      G. Patrick and L. H. Tsai, unpublished data.
      Results presented in this study not only demonstrate that the ubiquitin proteasome pathway plays a role in the fast turnover of p35 but also provide a model for the negative feedback regulation of the p35/Cdk5 kinase. Upon activation of Cdk5 by p35 association, the kinase phosphorylates p35 concurrently or soon after it phosphorylates its substrates. Phosphorylation further stimulates p35 degradation by the proteasome in a ubiquitin-mediated fashion, possibly by disassociation of p35 and Cdk5 or by creating an entrance point for ubiquitin-conjugating enzymes, which in turn ubiquitinate and target p35 for degradation. Thus, the Cdk5 kinase is quickly turned off once it is activated. It is likely that the proteasome pathway is broadly involved in the development of the nervous system in light of the regulatory role of this pathway in p35 protein levels and, thus, p35/Cdk5 kinase activity. As the p35/Cdk5 kinase plays a critical role in neurite outgrowth and neuronal migration, it is conceivable that to accommodate these processes, dynamic kinase activation-deactivation is required. The fact that Cdk5 can be activated by p35 association solely in the absence of other phosphorylation events is consistent with this notion, provided that there is intricate regulation of p35 synthesis and degradation.

      ACKNOWLEDGEMENTS

      We are indebted to Hidde Ploegh for the kind gift of proteasome inhibitor Z-L3VS, Ron Kopito for the kind gift of tagged-ubiquitin constructs, Jan Peters, Anna Philpott, Daniel Finley, Matthew Bogyo, Phil Hinds, and members of the Howley and Tsai laboratories for invaluable discussions and technical advice, Yolanda Ramos and Pamela Cabahug for technical assistance, and Keith Blackwell, Magareta Nikolic, Elena Porro, Deanna Smith, Maria Morabito, and Lawrence Zukerberg for reading and discussion of this manuscript.

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