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Structural Basis for the Different Stability and Activity between the Cdk5 Complexes with p35 and p39 Activators*

Open AccessPublished:September 30, 2013DOI:https://doi.org/10.1074/jbc.M113.512293
      Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane-bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35-Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminal AD than the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)-Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators.
      Background: Cdk5 activated by p39 has not been characterized, likely because of its instability.
      Results: Hydrogen bond interaction was reduced between p39 and Cdk5 and experimentally confirmed with amino acid substitution mutants of p35 and p39.
      Conclusion: Instability of p39-Cdk5 is caused by decreased hydrogen bond interaction.
      Significance: Structural basis of the instability of p39-Cdk5 was unveiled.

      Introduction

      Cyclin-dependent kinases (Cdks)
      The abbreviations used are: Cdk, cyclin-dependent kinase; AD, activation domain; TM, triple amino acid mutation.
      are Ser/Thr kinases that are activated by binding to a cyclin regulatory subunit (
      • Morgan D.O.
      Cyclin-dependent kinases. Engines, clocks, and microprocessors.
      ,
      • Murray A.W.
      Recycling the cell cycle. Cyclins revisited.
      ). The Cdk-cyclin complexes play a critical role in cell cycle progression at several cell cycle points, including the G1/S and G2/M checkpoints. In yeast, a single Cdk, Cdk1, is utilized throughout the entire cell cycle by activation with different cyclins at different cell cycle phases. Thus, Cdk1 mediates different cell cycle events in combination with different cyclins. In contrast, in mammalian cells, several Cdk family members are activated by their respective partner cyclins at specific cell cycle phases. Nevertheless, many mammalian cell cycle Cdks are still activated by multiple cyclins. Only a few cyclin-specific functions are known for particular Cdks, and the others remain to be investigated.
      Cdk5, a unique member of the Cdks, is activated in postmitotic neurons by the p35 and p39 activators, which share no amino acid sequence homology with cyclins. Cdk5 is a multifunctional protein kinase, playing an important role in neuronal development, survival, and synaptic activity (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ,
      • Su S.C.
      • Tsai L.-H.
      Cyclin-dependent kinases in brain development and disease.
      ,
      • Cheung Z.H.
      • Ip N.Y.
      Cdk5. A multifaceted kinase in neurodegenerative diseases.
      ). However, how p35 and p39 contribute to these various Cdk5 functions remains unknown. Mice lacking Cdk5 die perinatally with abnormal positioning of neurons in many brain regions, including the cerebral and cerebellar cortices (
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.
      ,
      • Gilmore E.C.
      • Ohshima T.
      • Goffinet A.M.
      • Kulkarni A.B.
      • Herrup K.
      Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex.
      ). p35 and p39 double-null mice show identical phenotypes to Cdk5−/− mice (
      • Ko J.
      • Humbert S.
      • Bronson R.T.
      • Takahashi S.
      • Kulkarni A.B.
      • Li E.
      • Tsai L.-H.
      p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.
      ). Mice deficient in p35 are viable but exhibit inverted layering of neurons in the cerebral cortex (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.-H.
      Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality.
      ). In contrast, p39−/− mice show no obvious defects (
      • Ko J.
      • Humbert S.
      • Bronson R.T.
      • Takahashi S.
      • Kulkarni A.B.
      • Li E.
      • Tsai L.-H.
      p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.
      ). These observations suggest that Cdk5-p39 may have a distinct physiological role, even if most p39 functions could be compensated by p35-Cdk5.
      In addition to the physiological functions described above, Cdk5 is involved in pathological neurodegeneration. Abnormal activation of Cdk5, which is induced by the cleavage of p35 to the p25 C-terminal activation domain (AD) by calpain (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.-H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      Calpain-dependent proteolytic cleavage of the p35 CDK5 activtor to p25.
      ,
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.-H.
      Neurotoxicity induces cleavage of p35 to p25 by calpain.
      ), is associated with Alzheimer and Parkinson diseases (
      • Cheung Z.H.
      • Ip N.Y.
      Cdk5. A multifaceted kinase in neurodegenerative diseases.
      ,
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.-H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Cruz J.C.
      • Tsai L.H.
      Cdk5 deregulation in the pathogenesis of Alzheimer's disease.
      ,
      • Shukla V.
      • Skuntz S.
      • Pant H.C.
      Deregulated Cdk5 activity is involved in inducing Alzheimer's disease.
      ). Calpain cleavage alters the cellular localization of active Cdk5 (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.-H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      Calpain-dependent proteolytic cleavage of the p35 CDK5 activtor to p25.
      ,
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.-H.
      Neurotoxicity induces cleavage of p35 to p25 by calpain.
      ,
      • Asada A.
      • Yamamoto N.
      • Gohda M.
      • Saito T.
      • Hayashi N.
      • Hisanaga S.
      Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cycline-dependent kinase 5 (Cdk5) complexes.
      ,
      • Asada A.
      • Saito T.
      • Hisanaga S.
      Subcellular localization of active Cdk5 is determined by its own kinase activity.
      ) and lengthens the half-life (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.-H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Minegishi S.
      • Asada A.
      • Miyauchi S.
      • Fuchigami T.
      • Saito T.
      • Hisanaga S.
      Membrane association facilitates degradation and cleavage of the cyclin-dependent kinase 5 activators p35 and p39.
      ). On the other hand, Cdk5 activity is shown to be protective against Huntingtin aggregation toxicity (
      • Luo S.
      • Vacher C.
      • Davies J.E.
      • Rubinsztein D.C.
      Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases. Implications for mutant huntingtin toxicity.
      ,
      • Schilling B.
      • Gafni J.
      • Torcassi C.
      • Cong X.
      • Row R.H.
      • LaFevre-Bernt M.A.
      • Cusack M.P.
      • Ratovitski T.
      • Hirschhorn R.
      • Ross C.A.
      • Gibson B.W.
      • Ellerby L.M.
      Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity.
      ,
      • Kaminosono S.
      • Saito T.
      • Oyama F.
      • Ohshima T.
      • Asada A.
      • Nagai Y.
      • Nukina N.
      • Hisanaga S.
      Suppression of mutant huntingtin aggregate formation by Cdk5/p35 through the effect on microtubule stability.
      ). Thus, regulation of Cdk5 kinase activity is critical for protection against these diseases.
      p39 is an isoform of p35 and was isolated from a cDNA library of the mouse brain hippocampus by PCR using the nucleotide sequence of p35 (
      • Tang D.
      • Yeung J.
      • Lee K.Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      An isoform of the neuronal cyclin-dependent kinase 5 (Cdk5) activator.
      ,
      • Zheng M.
      • Leung C.L.
      • Liem R.K.
      Region-specific expression of cyclin-dependent kinase 5 (cdk5) and its activators, p35 and p39, in the developing and adult rat central nervous system.
      ). p35 and p39 have 57% amino acid identity, with a particularly high homology of 72% in the C-terminal Cdk5 AD, which is also called the globular domain after similar folding to cyclin A (
      • Tarricone C.
      • Dhavan R.
      • Peng J.
      • Areces L.B.
      • Tsai L.H.
      • Musacchio A.
      Structure and regulation of the CDK5-p25(nck5a) complex.
      ). However, in contrast to p35-Cdk5, which has been studied intensively, little is known about p39-Cdk5. This is due to the difficulty of handling the p39-Cdk5 complex. p39-Cdk5 is inactivated by dissociation in the presence of nonionic detergent (
      • Yamada M.
      • Saito T.
      • Sato Y.
      • Kawai Y.
      • Sekigawa A.
      • Hamazumi Y.
      • Asada A.
      • Wada M.
      • Doi H.
      • Hisanaga S.
      Cdk5-p39 is a labile complex with the similar substrate specificity to Cdk5-p35.
      ), in contrast to Cdk5-p35, which is activated in these conditions. Nonetheless, nonionic detergent must be used for isolation of the active Cdk5 complex, because p35 and p39 associate with membranes through N-terminal myristoylation (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.-H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Asada A.
      • Yamamoto N.
      • Gohda M.
      • Saito T.
      • Hayashi N.
      • Hisanaga S.
      Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cycline-dependent kinase 5 (Cdk5) complexes.
      ).
      Here, we studied the structural basis of the different stabilities of Cdk5 complexed with p35 or p39. We found that three amino acids in α-helices 5 and 6 in the AD of p35 and p39 are the source for the different observed stabilities.

      DISCUSSION

      p39 is a dispensable Cdk5 activator (
      • Ko J.
      • Humbert S.
      • Bronson R.T.
      • Takahashi S.
      • Kulkarni A.B.
      • Li E.
      • Tsai L.-H.
      p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.
      ). However, it is likely that p39 plays an unidentified role in activating Cdk5, although currently only limited knowledge is available for p39. One major reason for the lack of data on p39 may be the difficulty in handling the p39-Cdk5 complex. We previously showed that p39-Cdk5 is a labile complex that dissociates and is inactivated in the presence of nonionic detergents, which is in contrast to p35-Cdk5 that is stable and activated under the same conditions (
      • Yamada M.
      • Saito T.
      • Sato Y.
      • Kawai Y.
      • Sekigawa A.
      • Hamazumi Y.
      • Asada A.
      • Wada M.
      • Doi H.
      • Hisanaga S.
      Cdk5-p39 is a labile complex with the similar substrate specificity to Cdk5-p35.
      ,
      • Zhu Y.-S.
      • Saito T.
      • Asada A.
      • Maekawa S.
      • Hisanaga S.
      Activation of latent cyclin-dependent kinase 5 (Cdk5)–p35 complexes by membrane dissociation.
      ). In this study, we used computer simulation to identify differences in p35-Cdk5 and p39-Cdk5 at the molecular level that could explain the apparent and different stability. Three amino acids in α-helices 5 and 6 in the globular AD of p35 that form the hydrogen bond network with Cdk5 were altered in p39, resulting in reduced hydrogen bond interactions. We experimentally confirmed this simulation using substitution mutants of these three amino acids in p35 and p39. The triple mutations destabilized p35-Cdk5 and increased the stability of p39-Cdk5.
      p39-Cdk5 and p35-Cdk5 are both membrane-associated protein kinases (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.-H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Asada A.
      • Yamamoto N.
      • Gohda M.
      • Saito T.
      • Hayashi N.
      • Hisanaga S.
      Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cycline-dependent kinase 5 (Cdk5) complexes.
      ,
      • Asada A.
      • Saito T.
      • Hisanaga S.
      Subcellular localization of active Cdk5 is determined by its own kinase activity.
      ,
      • Minegishi S.
      • Asada A.
      • Miyauchi S.
      • Fuchigami T.
      • Saito T.
      • Hisanaga S.
      Membrane association facilitates degradation and cleavage of the cyclin-dependent kinase 5 activators p35 and p39.
      ). Therefore, nonionic detergent is required for solubilization and isolation. Because p39-Cdk5 is dissociated in the presence of nonionic detergent, however, careful attention must be paid to the concentrations of nonionic detergent upon its preparation. For an example, p39-Cdk5 can be prepared from rat brain lysates by immunoprecipitation in reduced concentrations of nonionic detergent in isolation buffer (
      • Yamada M.
      • Saito T.
      • Sato Y.
      • Kawai Y.
      • Sekigawa A.
      • Hamazumi Y.
      • Asada A.
      • Wada M.
      • Doi H.
      • Hisanaga S.
      Cdk5-p39 is a labile complex with the similar substrate specificity to Cdk5-p35.
      ,
      • Takahashi S.
      • Saito T.
      • Hisanaga S.
      • Pant H.C.
      • Kulkarni A.B.
      Tau phosphorylation by cyclin-dependent kinase 5/p39 during brain development reduces its affinity for microtubules.
      ) and isolated p39-Cdk5 exhibited comparable kinase activity to p35-Cdk5 when the protein amounts of Cdk5 were adjusted (
      • Yamada M.
      • Saito T.
      • Sato Y.
      • Kawai Y.
      • Sekigawa A.
      • Hamazumi Y.
      • Asada A.
      • Wada M.
      • Doi H.
      • Hisanaga S.
      Cdk5-p39 is a labile complex with the similar substrate specificity to Cdk5-p35.
      ). However, the p39 immunoprecipitates displayed much less activity compared with the p35 immunoprecipitates when they were expressed in HEK293 cells (Fig. 1B). Because we used FLAG-tagged versions of p35 and p39 in this study, we could directly compare the abundance of p35 or p39 associated with Cdk5, in contrast to the previous report (
      • Yamada M.
      • Saito T.
      • Sato Y.
      • Kawai Y.
      • Sekigawa A.
      • Hamazumi Y.
      • Asada A.
      • Wada M.
      • Doi H.
      • Hisanaga S.
      Cdk5-p39 is a labile complex with the similar substrate specificity to Cdk5-p35.
      ), in which we used respective antibodies to p35 and p39 and could not directly compare their protein ratio. We found that a considerably large amount of p39 was required to obtain levels of Cdk5 comparable to that in p35 immunoprecipitates, indicating that p39 has weaker affinity to Cdk5 even in low concentrations of nonionic detergent. Nevertheless, comparable kinase activity was detected when the amount of Cdk5 was adjusted (Fig. 1B), suggesting that Cdk5 exhibits similar histone H1 kinase activity when bound to either p35 or p39.
      p39 has unique amino acid sequences in the regions flanking the globular Cdk5-AD (Fig. 1A). The N-terminal region is an extension of the proline-rich sequence, and the C-terminal sequence is an insertion, where muskelin is reported to bind (
      • Ledee D.R.
      • Gao C.Y.
      • Seth R.
      • Fariss R.N.
      • Tripathi B.K.
      • Zelenka P.S.
      A specific interaction between muskelin and the cyclin-dependent kinase 5 activator p39 promotes peripheral localization of muskelin.
      ). First we speculated that these flanking sequences might compromise the stability of p39-Cdk5. However, this was not the case. The activation domain of p39 itself was found to contain the sequences that contribute to instability, despite the high homology of AD between p35 and p39. Major differences are found in α-helices 5 and 6, which constitute the interface to Cdk5 (Fig. 3A). The hydrophobicity was one of the differences found in the regions. The lower hydrophobicity of p39 was attributed to Arg at amino acids 292, 299, 304, and 310, whereas the corresponding amino acids in p35 are Ala-256, Ser-262, Leu-267, and Gln-274. These Arg residues also contribute to the higher isoelectric point of 11.9 of p39, compared with 8.29 of p35. However, the 4R mutation did not change the stability of Cdk5-p35 AD and Cdk5-p35 in nonionic detergent (Fig. 2B). Arg residues are positioned in intervals of three or four amino acids, indicating that these Arg residues reside at one side of the α-helix (Fig. 2A) that is perpendicular to the side that binds to Cdk5 (Fig. 2D). Therefore, these Arg residues are not involved in the direct interaction with Cdk5, although it is still possible that the basic nature of p39 may modulate the stability of the p39-Cdk5 complex in cells by providing the site for interaction with other proteins.
      The three-dimensional structure modeling of p39 AD-Cdk5 based on five p25 (p35 AD)-Cdk5 complexes (Protein Data Bank codes 1UNG, 1UNH, 1UNL, 3O0G, and 1H4L) (
      • 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.
      ,
      • Ahn J.S.
      • Radhakrishnan M.L.
      • Mapelli M.
      • Choi S.
      • Tidor B.
      • Cuny G.D.
      • Musacchio A.
      • Yeh L.A.
      • Kosik K.S.
      Defining Cdk5 ligand chemical space with small molecule inhibitors of tau phosphorylation.
      ) pointed to the difference in the hydrogen bond network between p35-Cdk5 and p39-Cdk5, although their overall conformation is similar. The hydrogen bond network is formed between Asp-259, Asn-266, and Ser-270 of p35 and His-71, Lys-56, and Glu-57 of Cdk5 (Fig. 3B). In contrast, in p39, these amino acids are changed to Gln-295, Gln-302, and Pro-306, and they cannot form hydrogen bonds with Cdk5. The importance of these three amino acids in the interaction with Cdk5 was experimentally validated using substitution mutants; triple mutations in p35 dramatically reduced the stability of the complex with Cdk5 (Fig. 4), and triple mutations in p39 increased the stability of the complex with Cdk5 (Fig. 5). These results indicate the importance of the hydrogen bond network for the stability of Cdk5-activator complexes.
      The physiological significance of the different affinity of p35 and p39 to Cdk5 remains elusive. We used mouse cDNA of p35 or p39 to activate Cdk5 in HEK293 cells. The amino acid sequences of α5-α6 of these activators are completely conserved in the mammalian species sequenced thus far, such as human, mouse, rat, bovine, and porcine, suggesting that these two activators play a similar role with similar properties in mammals. In other vertebrates, Xenopus and zebrafish are also reported to have two Cdk5 activators, which were assigned to mammalian p35 and p39 based on amino acid sequence homology. The AD of Xenopus p35 and p39 shares 84.2 and 84.1% identities with mammalian p35 and p39, respectively, and those of zebrafish show 91.7 and 77.1% identities, respectively (Fig. 6A). In specific analysis of the α5-α6 region, however, the two activators of both Xenopus and zebrafish show greater similarity to mammalian p35 rather than p39 (Fig. 6B). Two of the three amino acids, Asp-259, Asn-266, and Ser-270, of mammalian p35 are conserved in p35 of Xenopus and zebrafish, suggesting that Xenopus or zebrafish p35 forms a stable complex with Cdk5, as does mammalian p35. In contrast, whereas Xenopus p39 has Gln at the position of Gln-295 of mammalian p39, all three amino acids of zebrafish p39 were different from mammalian p39 (Fig. 6B). Looking at the entire sequence of α5–6 (Fig. 6B), it is known that p39 of both Xenopus and zebrafish have a closer resemblance to p35 than p39 (Fig. 6B), suggesting diversity of the stability and kinase activity of the complex with Cdk5. It may be interesting to examine the stability and activity of not only Xenopus and zebrafish Cdk5-activator complexes, but also various combinations of Cdk and cyclin complexes.
      Figure thumbnail gr6
      FIGURE 6Amino acid comparisons of the AD of p35 and p39 among mouse, Xenopus and zebrafish. A, homology of p35 AD and p39 AD between mouse and Xenopus or zebrafish. Identity of amino acids of p35 AD and p39 AD between mouse and Xenopus or zebrafish is indicated as a percentage. B, amino acid alignment of α-helices 5 and 6 of p35 and p39 between mouse and Xenopus or zebrafish. Amino acids common to mouse p35 and p39 are represented by −. Amino acids identical to p35 but not p39 are indicated in yellow, whereas those identical to that of p39 are indicated in blue.

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