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Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5*

Open AccessPublished:May 28, 2014DOI:https://doi.org/10.1074/jbc.M113.501148
      Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.

      Introduction

      Cyclin-dependent kinases (Cdks)
      The abbreviations used are: Cdk
      cyclin-dependent kinase
      SFK
      Src family kinase
      kn
      kinase-negative
      AD
      activation domain
      ca
      constitutively active
      DIV
      days in vitro.
      are a family of serine/threonine kinases that mainly function in cell cycle progression. Cell cycle-associated Cdks, which are expressed in proliferating cells, require cyclin binding for activation (
      • Morgan D.O.
      Principles of CDK regulation.
      ). In addition to cyclin binding, Cdk activity is regulated by multiple phosphorylation on the Cdk itself. The activation mechanism of Cdk1-cyclin B (CycB) has been investigated extensively. Cdk1 is phosphorylated at Thr-161 in the activation loop by Cdk activation kinase upon binding to CycB. This phosphorylation is a prerequisite for activation (
      • Ducommun B.
      • Brambilla P.
      • Félix M.A.
      • Franza Jr., B.R.
      • Karsenti E.
      • Draetta G.
      Cdc2 phosphorylation is required for its interaction with cyclin.
      ). Concomitantly, Cdk1 is phosphorylated at Thr-14 and Tyr-15 in the ATP-binding G-loop by Wee1 or Myt1 kinase (
      • Atherton-Fessler S.
      • Liu F.
      • Gabrielli B.
      • Lee M.S.
      • Peng C.Y.
      • Piwnica-Worms H.
      Cell cycle regulation of the p34cdc2 inhibitory kinases.
      ). The latter phosphorylation keeps the Cdk1-CycB complex in an inactive state until the onset of M phase, when these sites are dephosphorylated by the phosphatase Cdc25 (
      • Strausfeld U.
      • Labbé J.C.
      • Fesquet D.
      • Cavadore J.C.
      • Picard A.
      • Sadhu K.
      • Russell P.
      • Dorée M.
      Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein.
      ). Although many Cdks have the conserved TY sequence in the N-terminal region (
      • Malumbres M.
      • Harlow E.
      • Hunt T.
      • Hunter T.
      • Lahti J.M.
      • Manning G.
      • Morgan D.O.
      • Tsai L.H.
      • Wolgemuth D.J.
      Cyclin-dependent kinases: a family portrait.
      ), the role of this residue has not been investigated for other Cdks except Cdk1 and Cdk2 and Cdk4-Cdk6.
      Cdk5 is a unique Cdk that plays a role in various neuronal activities unrelated to the cell cycle (
      • Barnett D.G.
      • Bibb J.A.
      The role of Cdk5 in cognition and neuropsychiatric and neurological pathology.
      ,
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      ,
      • Cheung Z.H.
      • Ip N.Y.
      The roles of cyclin-dependent kinase 5 in dendrite and synapse development.
      ) because the Cdk5 activation subunit, p35 or p39, is expressed predominantly in postmitotic neurons (
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      A brain-specific activator of cyclin-dependent kinase 5.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5.
      ,
      • Uchida T.
      • Ishiguro K.
      • Ohnuma J.
      • Takamatsu M.
      • Yonekura S.
      • Imahori K.
      Precursor of cdk5 activator, the 23 kDa subunit of Tau protein kinase II: its sequence and developmental change in brain.
      ). The activation mechanism also differs from that of Cdks involved in the cell cycle (Cdk1, Cdk2, Cdk4, and Cdk6). Cdk5 can be activated only by binding to p35 or p39 without phosphorylation in the activation T-loop. Furthermore, in contrast to Cdks involved in the cell cycle, Cdk5 activity has been reported to be stimulated by phosphorylation at Tyr-15 by non-receptor-type tyrosine kinases such as c-Abl and Fyn or receptor-type tyrosine kinases such as TrkB and EphA4 (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ,
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ,
      • Fu W.Y.
      • Chen Y.
      • Sahin M.
      • Zhao X.S.
      • Shi L.
      • Bikoff J.B.
      • Lai K.O.
      • Yung W.H.
      • Fu A.K.
      • Greenberg M.E.
      • Ip N.Y.
      Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism.
      ,
      • Cheung Z.H.
      • Chin W.H.
      • Chen Y.
      • Ng Y.P.
      • Ip N.Y.
      Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.
      ,
      • Lin H.
      • Lin T.Y.
      • Juang J.L.
      Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration.
      ). Activation of Cdk5-p35 by Tyr-15 phosphorylation may play a role in neurite and spine retraction, dendrite outgrowth, and neuron death (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ,
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ,
      • Fu W.Y.
      • Chen Y.
      • Sahin M.
      • Zhao X.S.
      • Shi L.
      • Bikoff J.B.
      • Lai K.O.
      • Yung W.H.
      • Fu A.K.
      • Greenberg M.E.
      • Ip N.Y.
      Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism.
      ,
      • Cheung Z.H.
      • Chin W.H.
      • Chen Y.
      • Ng Y.P.
      • Ip N.Y.
      Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.
      ,
      • Lin H.
      • Lin T.Y.
      • Juang J.L.
      Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration.
      ). Thus, phosphorylation of Tyr-15 appears to be important for various Cdk5 functions. However, it is not known how Tyr-15 phosphorylation of Cdk5 is regulated. Cdk5 has been shown to be phosphorylated by c-Abl and then activated by binding p35 (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ). However, whether Cdk5 is activated similarly by other tyrosine kinases has not been investigated. Cdk5 has a ternary structure similar to Cdk2, with an amino acid sequence identity of about 60% (
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.H.
      A family of human Cdc2-related protein kinases.
      ,
      • De Bondt H.L.
      • Rosenblatt J.
      • Jancarik J.
      • Jones H.D.
      • Morgan D.O.
      • Kim S.H.
      Crystal structure of cyclin-dependent kinase 2.
      ,
      • Tarricone C.
      • Dhavan R.
      • Peng J.
      • Areces L.B.
      • Tsai L.H.
      • Musacchio A.
      Structure and regulation of the CDK5-p25(nck5a) complex.
      ,
      • Mapelli M.
      • Massimiliano L.
      • Crovace C.
      • Seeliger M.A.
      • Tsai L.H.
      • Meijer L.
      • Musacchio A.
      Mechanism of CDK5/p25 binding by CDK inhibitors.
      ). It would be interesting, therefore, to understand why Cdks involved in the cell cycle and neuronal Cdk5 are oppositely regulated by phosphorylation at Tyr-15. To address this issue, we investigated the effect of Tyr-15 phosphorylation on Cdk5 activation using the COS-7 cell overexpression system and cultured neurons.

      DISCUSSION

      All Cdks in the human and mouse genomes have been recently grouped and numbered on the basis of the amino acid sequence around the N-terminal cyclin-binding region (
      • Malumbres M.
      • Harlow E.
      • Hunt T.
      • Hunter T.
      • Lahti J.M.
      • Manning G.
      • Morgan D.O.
      • Tsai L.H.
      • Wolgemuth D.J.
      Cyclin-dependent kinases: a family portrait.
      ). There are 20 Cdks in the mouse genome and 21 Cdks in the human genome. Tyr-15 is ∼30 residues upstream of the cyclin-binding region in the small N-terminal lobe of Cdk1, Cdk2, and Cdk5. Tyr-15 constitutes the roof of the ATP binding pocket, which is in the cleft between the N-terminal and C-terminal lobes (
      • Morgan D.O.
      Principles of CDK regulation.
      ,
      • Malumbres M.
      • Harlow E.
      • Hunt T.
      • Hunter T.
      • Lahti J.M.
      • Manning G.
      • Morgan D.O.
      • Tsai L.H.
      • Wolgemuth D.J.
      Cyclin-dependent kinases: a family portrait.
      ). The equivalent Tyr residue is found in 17 of 20 mouse Cdks (
      • Malumbres M.
      • Harlow E.
      • Hunt T.
      • Hunter T.
      • Lahti J.M.
      • Manning G.
      • Morgan D.O.
      • Tsai L.H.
      • Wolgemuth D.J.
      Cyclin-dependent kinases: a family portrait.
      ), indicating the importance of Tyr at this position for kinase activity. Phosphorylation at Tyr-15 acts oppositely on the kinase activity of Cdk1 and Cdk5: inhibition for Cdk1 and Cdk2 and activation for Cdk5. In this study, contrary to previous studies, we found that Tyr-15 phosphorylation is not involved in Cdk5 activation. It is very important to clarify the role of Tyr-15 phosphorylation for the activation mechanism of Cdk5 as well as other members of Cdks, which will be investigated in the future. Here, we discuss why Tyr-15 phosphorylation was interpreted to activate Cdk5.
      p35 binding is necessary and sufficient for Cdk5 activation. If Tyr-15 phosphorylation induces further stimulation of the kinase activity, Cdk5 complexed with p35 should be phosphorylated at Tyr-15. However, we could not detect Tyr-15 phosphorylation of Cdk5 in the Cdk5-p35 complex under our experimental conditions. By contrast, p35 coexpression inhibited Tyr-15 phosphorylation of Cdk5. Phosphorylation at Tyr-15 occurred on inactive Cdk5, in agreement with the first report by Zukerberg et al. (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ). They showed that Cdk5, in a trimeric complex with c-Abl and Cables but not p35, is phosphorylated by c-Abl in cells (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ). However, subsequent studies demonstrated stimulation of Cdk5 by tyrosine kinases under various cellular circumstances (
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ,
      • Fu W.Y.
      • Chen Y.
      • Sahin M.
      • Zhao X.S.
      • Shi L.
      • Bikoff J.B.
      • Lai K.O.
      • Yung W.H.
      • Fu A.K.
      • Greenberg M.E.
      • Ip N.Y.
      Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism.
      ,
      • Cheung Z.H.
      • Chin W.H.
      • Chen Y.
      • Ng Y.P.
      • Ip N.Y.
      Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.
      ,
      • Lin H.
      • Lin T.Y.
      • Juang J.L.
      Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration.
      ) but have not shown how Cdk5 is phosphorylated and how Cdk5 activity is stimulated. Moreover, many recent reports demonstrate Tyr-15 phosphorylation-induced Cdk5 activation only by immunoblotting or immunostaining of brain or neurons with anti-phospho-Tyr-15 antibodies, which are not sensitive enough to detect phospho-Tyr-15 Cdk5 without immunoprecipitation with anti-Cdk5 antibody.
      We examined the results of early several reports (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ,
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ,
      • Fu W.Y.
      • Chen Y.
      • Sahin M.
      • Zhao X.S.
      • Shi L.
      • Bikoff J.B.
      • Lai K.O.
      • Yung W.H.
      • Fu A.K.
      • Greenberg M.E.
      • Ip N.Y.
      Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism.
      ,
      • Cheung Z.H.
      • Chin W.H.
      • Chen Y.
      • Ng Y.P.
      • Ip N.Y.
      Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.
      ,
      • Lin H.
      • Lin T.Y.
      • Juang J.L.
      Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration.
      ) that analyzed the role of Tyr-15 phosphorylation on Cdk5 activation and found no substantial discrepancies between their results and our results using cultured cell lines. For example, Cdk5 is phosphorylated at Tyr-15 by c-Abl or Fyn, even in the presence of p35 (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ,
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ), which was also seen in our case. Cdk5 is usually expressed at higher levels than p35 because of a labile property of p35 (
      • Lee K.Y.
      • Rosales J.L.
      • Tang D.
      • Wang J.H.
      Interaction of cyclin-dependent kinase 5 (Cdk5) and neuronal Cdk5 activator in bovine brain.
      ,
      • Zhu Y.S.
      • Saito T.
      • Asada A.
      • Maekawa S.
      • Hisanaga S.
      Activation of latent cyclin-dependent kinase 5 (Cdk5)-p35 complexes by membrane dissociation.
      ,
      • Patrick G.N.
      • Zhou P.
      • Kwon Y.T.
      • Howley P.M.
      • Tsai L.H.
      p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) is degraded by the ubiquitin-proteasome pathway.
      ). Therefore, the amounts of Cdk5 protein are more abundant than that of p35 when overexpressed in cultured cells. The ratio of Cdk5 and p35 is much greater in neurons (Fig. 6G). There are two pools of Cdk5 in neurons, Cdk5 complexed with p35 and free monomeric Cdk5 (
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.H.
      The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity.
      ). Only a proportion of monomeric Cdk5 was phosphorylated at Tyr-15. Previous data showing Tyr-15 phosphorylation were obtained by immunoblotting of Cdk5 immunoprecipitates with anti-phospho-Tyr-15 (
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ,
      • Cheung Z.H.
      • Chin W.H.
      • Chen Y.
      • Ng Y.P.
      • Ip N.Y.
      Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.
      ,
      • Lin H.
      • Lin T.Y.
      • Juang J.L.
      Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration.
      ). Because Cdk5 immunoprecipitates contain both free Cdk5 and Cdk5 bound to p35, it is hard to determine which of them is phosphorylated. Thus, in a strict sense, Tyr-15 phosphorylation of Cdk5 bound to p35 has not been shown previously. In contrast, all of our current results indicate that Tyr-15 phosphorylation occurs only on free Cdk5 and not on Cdk5 complexed with p35.
      Another issue is whether Tyr-15-phosphorylated Cdk5 can bind p35. Zukerberg et al. (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ) proposed a model in which Cdk5 complexed with c-Abl and Cables is phosphorylated by c-Abl, and then phospho-Cdk5 exchanges its binding partner from c-Abl and Cables to p35 for activation. This was on the basis of several results, such as lower kinase activity of Cdk5 Y15F and higher activity and higher phospho-Tyr-15 phosphorylation of Cdk5 in COS-7 cells when cotransfected with c-Abl and Cables. Although the experiments were performed carefully, those are correlative lines of evidence, and one cannot definitively conclude that Cdk5 phosphorylated at Tyr-15 forms a complex with p35 that has higher kinase activity. Zukerberg et al. (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ) also showed the presence of phospho-Tyr-15 Cdk5 in the p35 immunoprecipitate from E15 brain lysates, but its amount was considerably less than that found in Cdk5 immunoprecipitates. The latter results can also be interpreted to mean that most of phospho-Tyr-15 Cdk5 is monomeric. In fact, we found marginal phospho-Tyr-15 Cdk5 in the anti-p35 immunoprecipitates. These results suggest that the binding affinity of phospho-Tyr-15 Cdk5 for p35, if any, is weaker than unphosphorylated Cdk5 in cells.
      The important question is the activation of Cdk5. Previously, Cdk5 activation has been demonstrated mainly by coexpression with tyrosine kinases in cultured cell lines (
      • 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.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ,
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ). Our results are in agreement with this. Cdk5 activity was stimulated more than 2-fold in COS-7 cells by coexpression with active Fyn. The difference is interpretation. The previous reports concluded that activation of Cdk5 was due to Tyr-15 phosphorylation, whereas we demonstrate increased p35 expression. The previous reports assessed Tyr-15 phosphorylation and Cdk5 activation in separate experiments. Tyr-15 phosphorylation of Cdk5 was not compared directly in the presence or absence of p35, and expression levels of p35 were not measured. In contrast, we found that coexpression of active Fyn increased the levels of p35 in COS-7 cells (Fig. 4B). Increased expression of p35 can explain the Fyn or Abl-induced Cdk5 activation reported previously.
      We think that the similar mechanism takes place in neurons, although the situation is not clear. We could not detect the activation of Cdk5 in cortical neurons treated with Sema3A. To our knowledge, there is only one report that describes the activation of Cdk5 in cortical neurons treated by Sema3A (
      • Chen G.
      • Sima J.
      • Jin M.
      • Wang K.Y.
      • Xue X.J.
      • Zheng W.
      • Ding Y.Q.
      • Yuan X.B.
      Semaphorin-3A guides radial migration of cortical neurons during development.
      ), but it is a statement under discussion without data. However, we could not detect an increased expression of p35 by immunoblotting of total cell lysates after treatment with Sema3A, Ephrin-1A, or BDNF. In contrast, p35 was increased in neurons when caFyn was cotransfected. These results may suggest that Cdk5 is activated by stabilization of p35 downstream of tyrosine kinase signals, but because these signals may function locally at the region where the receptors accumulate, the activation of Cdk5 or increase in p35 expression by physiological ligands has not been reported.
      Previous studies indicated that Cdk5 mediates Sema3A signaling in growth cone retraction, Ephrin-A1 signaling in dendritic spine retraction, and BDNF signaling in dendritic growth (
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ,
      • Fu W.Y.
      • Chen Y.
      • Sahin M.
      • Zhao X.S.
      • Shi L.
      • Bikoff J.B.
      • Lai K.O.
      • Yung W.H.
      • Fu A.K.
      • Greenberg M.E.
      • Ip N.Y.
      Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism.
      ,
      • Cheung Z.H.
      • Chin W.H.
      • Chen Y.
      • Ng Y.P.
      • Ip N.Y.
      Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.
      ). Our results do not necessarily rule out the possibility that Cdk5-p35 is downstream of Src family kinases or receptor-type tyrosine kinases, but only demonstrate that Tyr-15 phosphorylation is not its activation mechanism. There could be cross-talk at several levels of those signaling pathways. One possible mechanism may keep active Cdk5 complexes in the cellular region stimulated, otherwise, Cdk5 activity would be down-regulated by degradation of p35. Alternatively, tyrosine kinase signals may recruit the active Cdk5-p35 complex to the stimulated region. Because of the lack of an appropriate antibody for detailed cellular localization of endogenous p35, however, we cannot currently examine this possibility. In any case, further analysis is required to clarify the activation mechanism of Cdk5 in neurons, an important issue concerning tyrosine kinase-mediated signaling pathways.

      Acknowledgments

      We thank Drs. T. Tezuka and M. Yamamoto for Fyn and Src expression plasmids and Govinda Sharma for reading the manuscript.

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