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• W1997923894 abstract "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 . 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 (1Lew J. Huang Q.Q., Qi, Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (539) Google Scholar, 2Tang D. Yeung J. Lee K.Y. Matsushita M. Matsui H. Tomizawa K. Hatase O. Wang J.H. J. Biol. Chem. 1995; 270: 26897-26903Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 3Tsai L.H. Delalle I. Caviness V.S.J. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (810) Google Scholar). In a recent report, p35 nck5a was also found in muscle cells at the neuromuscular junction (4Fu A.K., Fu, W.Y. Cheung J. Tsim K.W., Ip, F.C. Wang J.H. Ip N.Y. Nat. Neurosci. 2001; 4: 374-381Crossref PubMed Scopus (153) Google Scholar). 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 (1Lew J. Huang Q.Q., Qi, Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (539) Google Scholar, 5Kusakawa G. Saito T. Onuki R. Ishiguro K. Kishimoto T. Hisanaga S. J. Biol. Chem. 2000; 275: 17166-17172Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 6Lee M.S. Kwon Y.T., Li, M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (907) Google Scholar, 7Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1320) Google Scholar). Moreover, p25 nck5a is fully functional in terms of Cdk5 activation (8Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). 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 (9Smith D.S. Greer P.L. Tsai L.H. Cell Growth Differ. 2001; 12: 277-283PubMed Google Scholar, 10Grant P. Sharma P. Pant H.C. Eur. J. Biochem. 2001; 268: 1534-1546Crossref PubMed Scopus (102) Google Scholar, 11Paglini G. Caceres A. Eur. J. Biochem. 2001; 268: 1528-1533Crossref PubMed Scopus (68) Google Scholar, 12Maccioni R.B. Otth C. Concha I.I. Munoz J.P. Eur. J. Biochem. 2001; 268: 1518-1527Crossref PubMed Scopus (160) Google Scholar, 13Homayouni R. Curran T. Curr. Biol. 2000; 10: R331-R334Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). 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 (14Chou K.C. Watenpaugh K.D. Heinrikson R.L. Biochem. Biophys. Res. Commun. 1999; 259: 420-428Crossref PubMed Scopus (177) Google Scholar, 15Lim H.-Y. Seow K.T., Li, Q. Kesuma D. Wang J.H. Qi R.Z. Biochem. Biophys. Res. Commun. 2001; 285: 77-83Crossref PubMed Scopus (8) Google Scholar, 16Tang D. Chun A.S. Zhang M. Wang J.H. J. Biol. Chem. 1997; 272: 12318-12327Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). 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 (16Tang D. Chun A.S. Zhang M. Wang J.H. J. Biol. Chem. 1997; 272: 12318-12327Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Poon R.Y. Lew J. Hunter T. J. Biol. Chem. 1997; 272: 5703-5708Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). 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 (8Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 16Tang D. Chun A.S. Zhang M. Wang J.H. J. Biol. Chem. 1997; 272: 12318-12327Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). 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 (18Zukerberg 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. Neuron. 2000; 26: 633-646Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). In contrast, phosphorylation of Cdk2 Tyr-15 is inhibitory to Cdk2 activity (19Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1803) Google Scholar). 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 (20Lee K.Y. Rosales J.L. Tang D. Wang J.H. J. Biol. Chem. 1996; 271: 1538-1543Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). 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 (21Sobue K. Agarwal-Mawal A., Li, W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 22Qi Z. Tang D. Zhu X. Fujita D.J. Wang J.H. J. Biol. Chem. 1998; 273: 2329-2335Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Floyd S.R. Porro E.B. Slepnev V.I. Ochoa G.C. Tsai L.H. De Camilli P. J. Biol. Chem. 2001; 276: 8104-8110Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24Nikolic M. Chou M.M., Lu, W. Mayer B.J. Tsai L.H. Nature. 1998; 395: 194-198Crossref PubMed Scopus (352) Google Scholar, 25Kwon Y.T. Gupta A. Zhou Y. Nikolic M. Tsai L.H. Curr. Biol. 2000; 10: 363-372Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). 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 . Various fragments of p35 nck5a and p39 nck5ai were engineered by PCR into the vector pGEX2T for preparation of GST 1GSTglutathioneS-transferaseDTTdithiothreitolHAhemagglutininPBSphosphate-buffered salineMOPS4-morpholinepropanesulfonic acid fusion proteins: p10 nck5a (residues 1–98 of p35 nck5a) , p16 nck5a (residues 1–149 of p35 nck5a) , and p19 nck5ai (residues 1–184 of p39 nck5ai) . The GST-p25 nck5a (residues 99–307 of p35 nck5a) and GST-p30 nck5ai (residues 115–367 of p39 nck5ai) constructs were reported previously (2Tang D. Yeung J. Lee K.Y. Matsushita M. Matsui H. Tomizawa K. Hatase O. Wang J.H. J. Biol. Chem. 1995; 270: 26897-26903Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 8Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The p35 nck5a -His expression construct was generated by insertion of a PCR sequence of p35 nck5a into pQE12 (QIAGEN Inc.). The resulting plasmid, pQE12/p35 nck5a, allows expression of p35 nck5a with a C-terminal His tag. p35 nck5a and p39 nck5ai were also engineered into pcDNA3.1 (Invitrogen) and pCI-neo (Promega) for protein expression in mammalian cells. glutathioneS-transferase dithiothreitol hemagglutinin phosphate-buffered saline 4-morpholinepropanesulfonic acid The full-length encoding sequence of human SET was cloned by reverse transcription-PCR using total RNA from MCF12 cells and inserted into pcDNA3.1C, pXJ40-HA, pGEX-2TK, and pQE30. The SET fragments SET-(1–225), SET-(29–118) and SET-(29–94) were cloned into pcDNA3.1C by PCR using the full-length coding sequence of SET as a template. SET-(1–225) was also cloned into pQE30 (QIAGEN Inc.) for protein expression in bacteria. The GST fusion proteins GST-p10 nck5a, GST-p16 nck5a, GST-p25 nck5a, GST-p19 nck5ai, and GST-p30 nck5ai were prepared as described previously (2Tang D. Yeung J. Lee K.Y. Matsushita M. Matsui H. Tomizawa K. Hatase O. Wang J.H. J. Biol. Chem. 1995; 270: 26897-26903Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 8Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The GST-tagged proteins were expressed inEscherichia coli BL21(DE3) and purified using GSH-Sepharose (Amersham Biosciences, Inc.). Expression of the His-tagged proteins His-SET, His-SET-(1–225) and p35 nck5a -His in bacteria and purification with Ni2+-nitrilotriacetic acid beads (QIAGEN Inc.) were performed according to the manufacturer's instruction. The whole isolation procedure was carried out at 4 °C. Rat brains were homogenized in Buffer A (25 mm Tris-HCl (pH 7.5), 50 mm NaCl, 1 mm EDTA, 1 mm DTT, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 0.1 mmbenzamidine, and 3 μg/ml each leupeptin and pepstatin). The crude homogenate was centrifuged at 100,000 × g for 40 min, and the supernatant was carefully collected. GSH beads coated with 100 μg of GST, GST-p10 nck5a, GST-p16 nck5a, GST-p25 nck5a, GST-p19 nck5ai, or GST-p30 nck5ai were mixed with the lysate and incubated for 3 h with gentle shaking. The beads were then extensively washed with Buffer B (25 mm Tris-HCl (pH 7.5), 100 mm NaCl, 1 mm DTT, 1 mm EDTA, 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, and 0.1 mmbenzamidine). After briefly washing the beads with Buffer C (25 mm Tris-HCl (pH 7.5), 100 mm NaCl, 1 mm DTT, and 1 mm EDTA), the coupled proteins were eluted by incubation with 3 units of thrombin (Sigma) in Buffer C overnight. The beads were then washed twice with the cleavage buffer without thrombin. The washing and eluting solutions were combined, and 3 volumes of cold acetone were added to precipitate the proteins at −20 °C for 2 h. The precipitates were washed with cold 70% methanol and then dissolved in SDS-PAGE sample buffer. The proteins were resolved by SDS-PAGE and visualized following colloidal Coomassie Blue staining. Protein bands excised from SDS-polyacrylamide gels were destained, reduced, alkylated, and then in-gel digested with trypsin as described by Shevchenko et al. (26Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7820) Google Scholar). The extracted peptides were analyzed on a quadrupole/time-of-flight mass spectrometer (QSTAR, PE Sciex) equipped with a nanoelectrospray ion source. Protein identities were revealed by searching sequence data bases with peptide sequence tags generated by tandem mass spectrometry (27Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar, 28Mann M. Wilm M. Anal. Chem. 1994; 66: 4390-4399Crossref PubMed Scopus (1317) Google Scholar). 5 μg of GST or GST-tagged proteins was incubated with 5 μg of His-SET in 200 μl of Buffer B. After incubation at 4 °C for 2 h, the GST-tagged proteins were retrieved with GSH-Sepharose by further incubation for 1 h and then centrifugation. The beads were extensively washed with Buffer B, and the bound proteins were subsequently released by boiling with SDS-PAGE sample buffer for 5 min. The released proteins were resolved by SDS-PAGE and detected on Western blots. The anti-Cdk5 (C-8) and anti-p35 nck5a (C-19) antibodies were obtained from Santa Cruz Biotechnology. The antibody recognizing the hexahistidine tag in fusion proteins was a monoclonal antibody from Upstate Biotechnology, Inc. COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transient transfection was carried out using LipofectAMINE (Invitrogen). For immunoprecipitation, transfected cells were harvested after 48 h of culturing and lysed in Buffer A. The lysates were clarified by centrifugation and then used for immunoprecipitation with antibodies as indicated and protein A/protein G beads. After extensively washing the beads with Buffer B, the precipitated proteins were resolved by SDS-PAGE and detected by Western blotting with the indicated antibodies. The polyclonal anti-p39 nck5ai antibody was generated against a peptide derived from p39 nck5ai . The anti-HA antibody is a monoclonal antibody. The anti-SET antibodies used were SP-1, SP-2 and SP-3, recognizing SET peptides AQAAKVSKKELNSN, RLNEQASEEILKV, and RSSQTQNKASRKR, respectively (29Adachi Y. Pavlakis G.N. Copeland T.D. J. Biol. Chem. 1994; 269: 2258-2262Abstract Full Text PDF PubMed Google Scholar). For immunocytochemistry, cells were fixed in 4% paraformaldehyde for 10 min at room temperature. After washing with PBS containing 0.2% Tween 20 (PBST), the cells were permeabilized with PBST and then blocked in PBST supplemented with 10% goat serum. The cells were sequentially treated with primary and secondary antibodies diluted in the blocking solution. The secondary antibody was Texas Red-conjugated anti-rabbit antibody or fluorescein isothiocyanate-conjugated anti-mouse antibody. The stained cells were visualized under a confocal laser scanning microscope. Nuclei were also isolated using a nucleus isolation kit (Nuclei EZ prep, Sigma) for immunostaining. The isolated nuclei were fixed in cold methanol/acetone (1:1) for 5 min at −20 °C. The fixed nuclei were washed with PBS, blocked with 3% bovine serum albumin in PBS, and treated with the indicated primary and secondary antibodies. The stained nuclei were dropped onto a slide and then covered with a coverslip. Cultures of murine neocortical neurons (gestational days 15–16) were prepared as previously described (30Cheung N.S. Beart P.M. Pascoe C.J. John C.A. Bernard O. J. Neurochem. 2000; 74: 1613-1620Crossref PubMed Scopus (50) Google Scholar). In brief, cortices were microdissected from the brains and subjected to trypsin digestion and mechanical trituration. The dissociated cells were harvested by centrifugation (1000 ×g) and resuspended in neurobasal growth medium containing B27 supplement, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.5 mml-glutamax-I (Invitrogen), and 10% dialyzed fetal calf serum. Cells were seeded to a density of 2 × 105 cells/cm2 in six-well plates previously coated with poly-d-lysine (100 μg/ml) or coverslips coated with poly-d-lysine/laminin (BD Biosciences). After 24 h in vitro, the culture medium was replaced with serum-free growth medium. The cultures were maintained in a humidified 5% CO2 incubator at 37 °C. Immunocytochemical staining of the cultures for microtubule-associated protein-2 and glial fibrillary acidic protein indicated that >95% of the cells were neurons with minimal contamination by glia (31Cheung N.S. Pascoe C.J. Giardina S.F. John C.A. Beart P.M. Neuropharmacology. 1998; 37: 1419-1429Crossref PubMed Scopus (163) Google Scholar). The primary culture of mouse cortical neurons was washed three times with PBS and then with Buffer D (10 mm Tris (pH 7.4), 10 mm KCl, 2 mm MgCl2, 1 mm DTT, 30% sucrose, and Roche Molecular Biochemicals protease inhibitor mixture). After washing, the cells were suspended in Buffer D, and the suspension was kept on ice for 20 min. The cells were then lysed by gentle homogenization in a Dounce homogenizer. Nuclei were pelleted by centrifugation at 1000 × g for 10 min, and the pellet was washed twice with Buffer D and retrieved by centrifugation. The nuclear pellet was dissolved in SDS-PAGE sample buffer as the nuclear fraction. After precipitation of the nuclei, the supernatant of the lysate was collected and used as the cytoplasmic fraction. The nuclear and cytoplasmic fractions were then made up to the same volume for Western blot analysis. Preparation of in vitroreconstituted Cdk5/p35 nck5a and Cdk5/p25 nck5a kinases was carried out following the procedure described by Qi et al. (8Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Briefly, 1 μg of GST-Cdk5 was incubated with 1 μg of GST-p35 nck5a or GST-p25 nck5a in 30 μl of Buffer E (30 mm MOPS (pH 7.4), 1 mm DTT, and 10 mm MgCl2) at room temperature for 30 min. Cdk5 kinase activity was measured by catalysis of phosphate incorporation into the synthetic peptide HS-(9–18), derived from histone H1 (8Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). To assess the SET effect on Cdk5 activity, Cdk5/p35 nck5a or Cdk5/p25 nck5a was incubated with varying amounts of His-SET or His-SET-(1–225) in Buffer E at room temperature for 30 min. The kinase activities of Cdk5/p35 nck5a and Cdk5/p25 nck5a were then determined by phosphorylation of HS-(9–18), and the assay was carried out at 30 °C for 10 min. It has been shown that Cdk5/p35 nck5a, but not Cdk5/p25 nck5a, exists as macromolecular complexes in brain extracts, implying that the N-terminal region of 98 amino acids is important for protein binding (20Lee K.Y. Rosales J.L. Tang D. Wang J.H. J. Biol. Chem. 1996; 271: 1538-1543Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Therefore, p10 nck5a, comprising the N-terminal 98 residues of p35 nck5a, and the longer fragment p16 nck5a, comprising the N-terminal fragment of 149 residues before the Cdk5-binding and -activating domain, as well as GST-p25 nck5a were constructed and prepared as GST fusion proteins. Likewise, p19 nck5ai, comprising the N-terminal 184 amino acids of p39 nck5ai, and p30 nck5ai were prepared as the N- and C-terminal fragments of p39 nck5ai, respectively. To isolate proteins binding to p35 nck5a and p39 nck5ai, GST and GST-tagged p35 nck5a or p39 nck5ai fragments were utilized in pull-down experiments with rat brain extracts. The binding proteins were specifically eluted by thrombin cleavage to release the p35 nck5a or p39 nck5ai fragments from GST immobilized on the beads. After separation of the eluted proteins by SDS-PAGE, it was observed that a protein band of ∼39 kDa was clearly present in the pull-down experiments with p10 nck5a, p16 nck5a, and p19 nck5ai, but not visible in those with GST, GST-p25 nck5a, and GST-p30 nck5ai (Fig.1 A). To reveal the identity of this protein band, the excised gel bands were subjected to in-gel digestion and mass spectrometric analysis. Tandem mass spectrometric data were acquired from three prominent peptide signals. Searches of protein sequence data bases with sequence tags generated from the tandem mass spectrometric data revealed that all three peptides matched the sequences of the α- and β-isoforms of the rat protein SET, indicating that the SET protein was specifically isolated with the N-terminal fragments of p35 nck5a and p39 nck5ai (Fig.1 B). The protein-protein interaction between the protein SET and Cdk5/p35 nck5a was carried out and characterized using bacterially expressed proteins. SET was expressed in bacteria either with a GST tag or with a hexahistidine tag at its N terminus. In Fig.2 A, His-SET was individually incubated with GST, GST-p10 nck5a, and GST-p25 nck5a . The recombinant GST proteins were then retrieved using GSH affinity beads. The precipitated proteins were analyzed on an immunoblot with an antibody recognizing the His tag to determine whether SET had coprecipitated with the GST-tagged proteins. In Fig. 2 A, SET was present in the precipitated beads of p10 nck5a, but not in those of the other proteins, providing proof of specific and direct interaction between SET and the N-terminal fragment of p35 nck5a . In Fig. 2 B, the binding assay was carried out by incubating GST-SET or control GST with p35 nck5a -His, His-Cdk5, or a mixture of p35 nck5a -His and His-Cdk5. GST and GST-SET were then isolated using GSH beads, and the coprecipitated proteins were examined for the presence of p35 nck5a -His and His-Cdk5 using p35 nck5a - and Cdk5-specific antibodies, respectively. This showed clear binding of SET to full-length p35 nck5a, but no detectable binding to Cdk5 in the absence of p35 nck5a (Fig. 2 B). However, when SET was incubated with the mixture of p35 nck5a -His and His-Cdk5, both p35 nck5a and Cdk5 were found in the precipitate of SET (Fig.2 B). Clearly, these results indicate that SET forms a complex with Cdk5/p35 nck5a through its specific association with the N-terminal region of p35 nck5a . To demonstrate in vivo association of SET and Cdk5/p35 nck5a, the protein-protein interaction was probed in a transfected cell system. Cultured COS-7 cells were transiently transfected with the construct expressing SET with an N-terminal HA tag in various combinations of the p35 nck5a and Cdk5 plasmids. Protein expression of the introduced clones in COS-7 cells is shown in Western blots of the cell lysates in Fig.3 A (three upper panels). SET was immunoprecipitated by an antibody directed against the HA tag at its N terminus. In the vector control, the anti-HA antibody did not precipitate coexpressed p35 nck5a and Cdk5, indicating no detectable nonspecific reactivity between the anti-HA antibody and these proteins (Fig. 3 A). Because SET does not bind Cdk5 directly and COS-7 cells have no endogenously expressed p35 nck5a and p39 nck5ai, double transfection of SET and Cdk5 followed by their immunoprecipitation did not result in co-immunoprecipitation of Cdk5 (Fig. 3 A). In double transfection of SET and p35 nck5a plasmids as well as in triple transfection of SET, p35 nck5a, and Cdk5 plasmids in COS-7 cells, SET clearly formed complexes with p35 nck5a and Cdk5/p35 nck5a, respectively (Fig. 3 A). These results corroborate the in vitro binding data and indicate the existence of a trimolecular complex of SET, Cdk5, and p35 nck5a in the transfected cells. Similar results were obtained when p35 nck5a was replaced with p39 nck5ai using the COS-7 cell system (Fig. 3 B), indicating association of SET Cdk5/p39 nck5ai through its interaction with p39 nck5ai . According to the SET sequence, it has an acidic tail of ∼50 amino acids. The secondary structure of SET was predicted using a number of computer structural modeling programs. A long α-helical structure comprising residues 29–94 and a few short helical stretches were predicted to be present in SET (Fig. 4 A). Based on the predicted structure, three truncated SET constructs were made for mammalian expression with a His tag at the N terminus (Fig.4 A). The p35 nck5a plasmid was cotransfected with the plasmid expressing full-length SET or its truncated fragments in COS-7 cells. After affinity precipitation of the recombinant SET proteins from the cell lysates using Ni2+-coupled beads, p35 nck5a was detected in the precipitates of both intact SET and the three truncated fragments (Fig. 4 B). In contrast, p35 nck5a was not detected in the control vector lane (Fig.4 B). It appears that the α-helix composed of residues 29–94 of SET is sufficient for the association with p35 nck5a . Given the interaction between p35 nck5a and SET, we investigated thein vivo localization of SET and Cdk5/p35 nck5a . In COS-7 cells transfected with both SET and p35 nck5a constructs, the staining of p35 nck5a was observed throughout the cells, with lower intensity in the nucleus than in the cell peripheral and cytosolic regions (Fig. 5 A). In accordance with previous reports (29Adachi Y. Pavlakis G.N. Copeland T.D. J. Biol. Chem. 1994; 269: 2258-2262Abstract Full Text PDF PubMed Google Scholar, 32Nagata K. Saito S. Okuwaki M. Kawase H. Furuya A. Kusano A. Hanai N. Okuda A. Kikuchi A. Exp. Cell Res. 1998; 240: 274-281Crossref PubMed Scopus (106) Google Scholar), SET was primarily visualized in the nuclear region (Fig. 5 A). When the stained images of p35 nck5a and SET were overlaid, an overlapping localization of the two proteins in the nucleus was revealed (Fig.5 A). To provide more precise data on the nuclear localization of SET and p35 nck5a, intact nuclei were isolated from the transfected COS-7 cells for immunostaining of p35 nck5a and HA-tagged SET. In agreement with the observation from the whole cell staining, the co-localization of SET and p35 nck5a was clearly observed in the isolated nuclei (Fig.5 A). Both SET and Cdk5/p35 nck5a have been known to exist in neuronal cells (33Compagnone N.A. Zhang P. Vigne J.L. Mellon S.H. Mol. Endocrinol. 2000; 14: 875-888Crossref PubMed Scopus (60) Google Scholar, 34Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (530) Google Scholar). Their subcellular localizations were examined in cultured primary neurons derived from gestational day 15 mouse cortices by immunocytochemistry. p35 nck5a labeling was observed in the entire cell body and neurites (Fig. 5 B). Like the p35 nck5a -transfected COS cells, p35 nck5a was stained at a lower extent in the nuclear region (Fig. 5 B). SET appeared to localize primarily in the nuclei of the cultured neurons (Fig.5 B). Nuclear co-localization of SET and p35 nck5a was evident in the merged image of these two proteins (Fig. 5 B). On the other hand, the existence of SET and Cdk5/p35 nck5a in the nuclei of the cultured neurons was ascertained by subcellular fractionation and Western blotting (Fig. 5 C). In this experiment, lysates of the cultured neurons were fractionated into nuclear and cytoplasmic fractions for Western blotting with antibodies individually recognizing SET, Cdk5, and p35 nck5a . Cdk5 and p35 nck5a displayed similar distribution patterns, with the majority of the proteins stained in the cytoplasmic fraction (Fig.5 C). Moreover, Cdk5 and p35 nck5a were readily detected in the nuclear fraction (Fig. 5 C). SET was mostly stained in the nuclear fraction and barely detectable in the cytoplasmic fraction (Fig. 5 C). These results unambiguously show the coexistence of SET and Cdk5/p35 nck5a in the nuclear compartment of cultured neurons. Cdk5 can be activated by in vitro reconstitution with p25 nck5a or p35 nck5a (8Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). To examine the effect of SET on Cdk5/p35 nck5a, the reconstituted Cdk5/p35 nck5a kinase was incubated with increasing amounts of bacterially prep" @default.
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• W1997923894 date "2002-03-01" @default.
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• W1997923894 title "The Protein SET Binds the Neuronal Cdk5 Activator p35 and Modulates Cdk5/p35 Activity" @default.
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• W1997923894 doi "https://doi.org/10.1074/jbc.m107270200" @default.
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