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The Protein SET Binds the Neuronal Cdk5 Activator p35 nck5a and Modulates Cdk5/p35 nck5a Activity*

Open AccessPublished:December 10, 2001DOI:https://doi.org/10.1074/jbc.M107270200
      The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35 nck5a or its isoform, p39 nck5ai . To identify novel p35 nck5a - and p39 nck5ai -binding proteins, fragments of p35 nck5a and p39 nck5ai were utilized in affinity isolation of binding proteins from rat brain homogenates, and the isolated proteins were identified using mass spectrometry. With this approach, the nuclear protein SET was shown to interact with the N-terminal regions of p35 nck5a and p39 nck5ai . Our detailed characterization showed that the SET protein formed a complex with Cdk5/p35 nck5a through its binding to p35 nck5a . The p35 nck5a -interacting region was mapped to a predicted α-helix in SET. When cotransfected into COS-7 cells, SET and p35 nck5a displayed overlapping intracellular distribution in the nucleus. The nuclear co-localization was corroborated by immunostaining data of endogenous SET and Cdk5/p35 nck5a from cultured cortical neurons. Finally, we demonstrated that the activity of Cdk5/p35 nck5a, but not that of Cdk5/p25 nck5a, was enhanced upon binding to the SET protein. The tail region of SET, which is rich in acidic residues, is required for the stimulatory effect on Cdk5/p35 nck5a .
      Cdk5 is distinct from other cyclin-dependent kinases by virtue of its functions in post-mitotic neurons, but not in proliferating cells. Although Cdk5 is ubiquitously expressed, Cdk5-associated kinase activity has been primarily demonstrated in central nervous system neurons. In such neurons, Cdk5 is associated with p35 nck5a or a p35 nck5a isoform (p39 nck5ai) , two Cdk5 activators with restricted expression in central nervous system neurons (
      • Lew J.
      • Huang Q.Q., Qi, Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tang D.
      • Yeung J.
      • Lee K.Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S.J.
      • Chae T.
      • Harlow E.
      ). In a recent report, p35 nck5a was also found in muscle cells at the neuromuscular junction (
      • Fu A.K., Fu, W.Y.
      • Cheung J.
      • Tsim K.W., Ip, F.C.
      • Wang J.H.
      • Ip N.Y.
      ). Besides the full-length protein of p35 nck5a, a proteolytic fragment called p25 nck5a exists in central nervous system neurons. The p25 nck5a protein is generated when the N-terminal 98 amino acids are removed from p35 nck5a (
      • Lew J.
      • Huang Q.Q., Qi, Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      ,
      • Lee M.S.
      • Kwon Y.T., Li, M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      ,
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ). Moreover, p25 nck5a is fully functional in terms of Cdk5 activation (
      • Qi Z.
      • Huang Q.Q.
      • Lee K.Y.
      • Lew J.
      • Wang J.H.
      ). In association with p35 nck5a /p25 nck5a and p39 nck5ai, Cdk5 exhibits a variety of functions in neuronal differentiation and neurocytoskeleton dynamics as well as neuronal degeneration and cell death (
      • Smith D.S.
      • Greer P.L.
      • Tsai L.H.
      ,
      • Grant P.
      • Sharma P.
      • Pant H.C.
      ,
      • Paglini G.
      • Caceres A.
      ,
      • Maccioni R.B.
      • Otth C.
      • Concha I.I.
      • Munoz J.P.
      ,
      • Homayouni R.
      • Curran T.
      ).
      Despite little homology between p35 nck5a and cyclins at the primary sequence level, it was proposed that p35 nck5a forms a core structure similar to that of cyclins to support Cdk5 enzyme activity (
      • Chou K.C.
      • Watenpaugh K.D.
      • Heinrikson R.L.
      ,
      • Lim H.-Y.
      • Seow K.T., Li, Q.
      • Kesuma D.
      • Wang J.H.
      • Qi R.Z.
      ,
      • Tang D.
      • Chun A.S.
      • Zhang M.
      • Wang J.H.
      ). The minimal region required for Cdk5 binding and activation was localized to a region in the C-terminal half of p35 nck5a as well as in p25 nck5a (
      • Tang D.
      • Chun A.S.
      • Zhang M.
      • Wang J.H.
      ,
      • Poon R.Y.
      • Lew J.
      • Hunter T.
      ). Moreover, Cdk5/p35 nck5a shows many distinct regulatory properties. Cdk5 is highly activated upon association with p35 nck5a /p25 nck5a, and the activation process is not regulated by the cyclin-dependent kinase-activating kinase Cdk7/cyclin H (
      • Qi Z.
      • Huang Q.Q.
      • Lee K.Y.
      • Lew J.
      • Wang J.H.
      ,
      • Tang D.
      • Chun A.S.
      • Zhang M.
      • Wang J.H.
      ). Moreover, up-regulation of Cdk5/p35 nck5a activity was observed when Cdk5 was phosphorylated at Tyr-15 by the cellular tyrosine kinase c-Abl in complex with a bridging protein called Cables (
      • 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.
      ). In contrast, phosphorylation of Cdk2 Tyr-15 is inhibitory to Cdk2 activity (
      • Morgan D.O.
      ). It is believed that these distinct regulatory properties are due to unique features of the Cdk5/p35 nck5a structure.
      There are a few lines of evidence implying that Cdk5/p35 nck5a is a multifunctional enzyme existing in many protein complexes in cells. It was revealed by a column chromatographic procedure that Cdk5/p35 nck5a exists as a large molecular complex of >670 kDa in brain tissue extracts (
      • Lee K.Y.
      • Rosales J.L.
      • Tang D.
      • Wang J.H.
      ). Consistently, a number of proteins have been reported to associate with Cdk5/p35 nck5a with important functional implications, e.g. tau, neurofilaments M and H, amphiphysin-1, the Rac·p21-activated kinase-1 complex, and the β-catenin·N-cadherin complex (
      • Sobue K.
      • Agarwal-Mawal A., Li, W.
      • Sun W.
      • Miura Y.
      • Paudel H.K.
      ,
      • Qi Z.
      • Tang D.
      • Zhu X.
      • Fujita D.J.
      • Wang J.H.
      ,
      • Floyd S.R.
      • Porro E.B.
      • Slepnev V.I.
      • Ochoa G.C.
      • Tsai L.H.
      • De Camilli P.
      ,
      • Nikolic M.
      • Chou M.M., Lu, W.
      • Mayer B.J.
      • Tsai L.H.
      ,
      • Kwon Y.T.
      • Gupta A.
      • Zhou Y.
      • Nikolic M.
      • Tsai L.H.
      ). To learn more about the functional and regulatory properties of Cdk5/p35 nck5a, we have employed a biochemical approach to isolate proteins from brain lysates using the N- and C-terminal fragments derived from p35 nck5a and p39 nck5ai . The isolated proteins were identified using highly sensitive mass spectrometry. The nuclear protein SET was found specifically in the precipitates of the N-terminal constructs of p35 nck5a and p39 nck5ai . Detailed characterization was carried out on the interaction between SET and p35 nck5a . We demonstrate that SET further enhances the activity of Cdk5/p35 nck5a, but not that of Cdk5/p25 nck5a, through its association with p35 nck5a .

      DISCUSSION

      Although D-type cyclins have been shown to bind Cdk5 in fibroblasts and other cell lines, Cdk5/cyclin D displays no kinase activity (
      • Xiong Y.
      • Zhang H.
      • Beach D.
      ,
      • Zhang H.
      • Xiong Y.
      • Beach D.
      ). Restriction of Cdk5 activity to central nervous system neurons is apparently dictated by its specific activators, p35 nck5a and p39 nck5ai (
      • Lew J.
      • Huang Q.Q., Qi, Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tang D.
      • Yeung J.
      • Lee K.Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S.J.
      • Chae T.
      • Harlow E.
      ). Under certain cellular conditions, p35 nck5a is proteolytically cleaved to yield an N-terminally truncated species, p25 nck5a (
      • Lew J.
      • Huang Q.Q., Qi, Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      ,
      • Lee M.S.
      • Kwon Y.T., Li, M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      ,
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ,
      • Ishiguro K.
      • Kobayashi S.
      • Omori A.
      • Takamatsu M.
      • Yonekura S.
      • Anzai K.
      • Imahori K.
      • Uchida T.
      ). Although both p25 nck5a and p35 nck5a can sustain Cdk5 activity, they have many different biological properties. For example, p35 nck5a and p25 nck5a have different subcellular distribution patterns (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ). p35 nck5a has a much shorter lifetime than p25 nck5a in cells, and p25 nck5a, but not p35 nck5a, promotes neuronal apoptosis (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ,
      • Patrick G.N.
      • Zhou P.
      • Kwon Y.T.
      • Howley P.M.
      • Tsai L.H.
      ). A previous study showed that Cdk5/p35 nck5a exists as macromolecular complexes, whereas Cdk5/p25 nck5a exists as a heterodimeric complex (
      • Lee K.Y.
      • Rosales J.L.
      • Tang D.
      • Wang J.H.
      ). Therefore, p35 nck5a and p25 nck5a seem to be engaged in different cellular functions in neurons. We have performed affinity isolation of proteins interacting with various portions of p35 nck5a and p39 nck5ai were also used to isolate p39 nck5ai -binding proteins. To minimize elution of nonspecifically absorbed proteins, thrombin cleavage was used to retrieve the p35 nck5a or p39 nck5ai fragments and their associated proteins from the beads. Under the experimental conditions applied in this study, a few protein bands were found to coprecipitate with the N-terminal fragments of p35 nck5a (p10 nck5a and p16 nck5a) and p39 nck5ai (p19 nck5ai) , but not with p25 nck5a or p30 nck5ai, suggesting that the N-terminal portions of p35 nck5a and p39 nck5ai are regions that mediate protein-protein interaction.
      One of the proteins that specifically coprecipitated with the N-terminal regions of p35 nck5a and p39 nck5ai was identified as the protein SET. The SET gene was originally identified in a patient with acute undifferentiated leukemia as part of the SET-CAN fusion gene resulting from translocation (
      • von Lindern M.
      • van Baal S.
      • Wiegant J.
      • Raap A.
      • Hagemeijer A.
      • Grosveld G.
      ). The SET protein was also purified from HeLa cells as a chromatin-remodeling factor and called template-activating factor Iβ (
      • Nagata K.
      • Kawase H.
      • Handa H.
      • Yano K.
      • Yamasaki M.
      • Ishimi Y.
      • Okuda A.
      • Kikuchi A.
      • Matsumoto K.
      ). Our study demonstrates the physical association of SET with p35 nck5a and p39 nck5ai, but not p25 nck5a . The binding region in SET was mapped to an α-helical stretch adjacent to the N terminus. Moreover, the binding of SET modulates the kinase activity of Cdk5/p35 nck5a .
      SET expression has been widely detected in human tissues and cell lines predominantly as a nuclear protein (
      • Adachi Y.
      • Pavlakis G.N.
      • Copeland T.D.
      ,
      • Nagata K.
      • Saito S.
      • Okuwaki M.
      • Kawase H.
      • Furuya A.
      • Kusano A.
      • Hanai N.
      • Okuda A.
      • Kikuchi A.
      ). In mouse embryos, SET is highly expressed in developing neural tubes, and the expression is largely reduced after birth, implying its involvement in development of the central nervous system (
      • Compagnone N.A.
      • Zhang P.
      • Vigne J.L.
      • Mellon S.H.
      ). Cdk5/p35 nck5a has been demonstrated to play an essential role in neuronal differentiation (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ,
      • 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.
      ). In this study, Cdk5 and p35 nck5a were observed in both the cytoplasm and nucleus, in agreement with observations from early reports obtained by immunohistochemistry and immunocytochemistry (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ,
      • Ino H.
      • Chiba T.
      ). Moreover, Cdk5 and p35 nck5a were clearly found to co-localize with SET in the nuclei of the cultured neurons, supporting the in vivo association of SET and Cdk5/p35 nck5a . This implies coordinated actions of SET and Cdk5/p35 nck5a on neuronal maturation.
      Apparently, SET is a multifunctional protein, although its cellular role is not clearly defined. SET displays potent inhibitory activity on protein phosphatase 2A (
      • Li M.
      • Makkinje A.
      • Damuni Z.
      ,
      • Saito S.
      • Miyaji-Yamaguchi M.
      • Shimoyama T.
      • Nagata K.
      ). A few recent studies have also suggested a role of SET in transcription regulation (
      • Compagnone N.A.
      • Zhang P.
      • Vigne J.L.
      • Mellon S.H.
      ,
      • Seo S.B.
      • McNamara P.
      • Heo S.
      • Turner A.
      • Lane W.S.
      • Chakravarti D.
      ). In addition, a number of biochemical studies have demonstrated direct connections between the SET protein and the cyclin-dependent kinase regulatory machinery. SET was identified to bind specifically to cyclin B, but not to cyclin A (
      • Kellogg D.R.
      • Kikuchi A.
      • Fujii-Nakata T.
      • Turck C.W.
      • Murray A.W.
      ). However, the functional significance of its association with cyclin B is not understood. In a more recent report, SET was isolated as a binding protein of the cyclin-dependent kinase inhibitor p21 cip1 (
      • Estanyol J.M.
      • Jaumot M.
      • Casanovas O.
      • Rodriguez-Vilarrupla A.
      • Agell N.
      • Bachs O.
      ). This binding blocks the p21 cip1 inhibitory activity on Cdk2/cyclin E. In contrast, it has no effect on p21 cip1 inhibition of Cdk2/cyclin A, suggesting that the SET effect on p21 cip1 is cyclin-dependent. p35 nck5a and p39 nck5ai function as cyclins in neurons to support Cdk5 activity (
      • Lew J.
      • Huang Q.Q., Qi, Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tang D.
      • Yeung J.
      • Lee K.Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S.J.
      • Chae T.
      • Harlow E.
      ). It has been postulated that the core structure of p35 nck5a is very similar to that of cyclins even though they have only scarce homology at the primary sequence level (
      • Chou K.C.
      • Watenpaugh K.D.
      • Heinrikson R.L.
      ,
      • Lim H.-Y.
      • Seow K.T., Li, Q.
      • Kesuma D.
      • Wang J.H.
      • Qi R.Z.
      ). The fact that cyclin B shares a common binding protein (SET) with p35 nck5a and p39 nck5ai suggests that p35 nck5a or p39 nck5ai and cyclin have more in common than their primary function of cyclin-dependent kinase activation.
      There is a long acidic stretch in the tail of the SET protein. Similar acidic regions can be found in nucleosome assembly protein-1 and some other chromosomal proteins. In these proteins, the acidic stretch is thought to be responsible for chromatin binding and nucleosome assembly (
      • Ishimi Y.
      • Kikuchi A.
      ,
      • Earnshaw W.C.
      ). In this study, we have shown that the acidic tail of SET is required for its stimulatory effect on Cdk5/p35 nck5a, suggesting that the stimulation is due to an electrostatic effect on the enzyme. This stimulatory effect is reminiscent of the heparin effect on Cdk5/p25 nck5a activity. In the presence of heparin, Cdk5/p25 exhibits up-regulated phosphorylation of -(Ser/Thr)-Pro- peptides without Lys/Arg at position +3 (
      • Qi Z.
      • Zhu X.
      • Goedert M.
      • Fujita D.J.
      • Wang J.H.
      ). The heparin effect was, at least in part, attributed to its physical binding to the Cdk5/p25 nck5a enzyme (
      • Qi Z.
      • Zhu X.
      • Goedert M.
      • Fujita D.J.
      • Wang J.H.
      ). It seems that the phosphorylating efficiencies of Cdk5/p35 nck5a and Cdk5/p25 nck5a could be altered by bound anionic stretches. The fact that SET selectively enhances the activity of Cdk5/p35 nck5a, but not that of Cdk5/p25 nck5a, indicates that the stimulatory effect of SET depends on its specific association with the targeted enzyme and therefore is enzyme-specific. It is likely that the association of SET and Cdk5/p35 nck5a may facilitate substrate phosphorylation by Cdk5/p35 nck5a both by affecting enzyme activity and by anchoring the enzyme to the substrate.
      SET is a specific and noncompetitive inhibitor of protein phosphatase 2A, which is involved in a variety of cellular activities, including cell growth and differentiation (
      • Li M.
      • Makkinje A.
      • Damuni Z.
      ,
      • Saito S.
      • Miyaji-Yamaguchi M.
      • Shimoyama T.
      • Nagata K.
      ). Protein phosphatase 2A is also known as a major phosphatase in removal of phosphate groups from Cdk5 target proteins in brains (
      • Goedert M.
      • Jakes R., Qi, Z.
      • Wang J.H.
      • Cohen P.
      ,
      • Veeranna
      • Shetty K.T.
      • Link W.T.
      • Jaffe H.
      • Wang J.
      • Pant H.C.
      ). In in vitro assays, the presence of Cdk5/p35 nck5a did not show any detectable effect on the SET inhibition of protein phosphatase 2A (data not shown). Moreover, SET does not contain any proline-directed Ser/Thr motif, which is the potential phosphorylation sequence of Cdk5 (
      • Beaudette K.N.
      • Lew J.
      • Wang J.H.
      ). Consistently, phosphorylation of the SET protein was not detected with the active Cdk5/p35 nck5a kinase in an in vitroexperiment (data not shown). Considering the observation that SET has dual activity to elevate Cdk5/p35 nck5a activity and to block protein phosphatase 2A activity, it is tempting to suggest a concerted regulation by SET of protein phosphorylation/dephosphorylation in the nucleus during cellular processes requiring the kinase activity of Cdk5/p35 nck5a .
      The results of this study are the first to demonstrate the association of the SET protein with Cdk5/p35 nck5a and Cdk5/p39 nck5ai and the stimulatory effect of SET on Cdk5/p35 nck5a activity. Although its functional significancein vivo remains unclear, we suggest that SET and Cdk5/p35 nck5a have interrelated physiological functions in cells. It would be reasonable to postulate that, in addition to alteration of chromatin structure and protein phosphatase 2A inhibition, SET may exert its effect in developing central nervous systems through mediating phosphorylation of certain protein substrates by Cdk5/p35 nck5a .

      ACKNOWLEDGEMENTS

      We thank Dr. Terry D. Copeland (National Cancer Institute) for anti-SET antibodies and Dr. C. J. Pallen (Institute of Molecular and Cell Biology) for the anti-HA antibody.

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