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• W1970863847 abstract "Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35 nck5a and other protein components (Lee, K.-Y., Rosales, J. L., Tang, D., and Wang, J. H. (1996) J. Biol. Chem. 271, 1538–1543). The yeast two-hybrid system was employed to identify p35 nck5a -interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment revealed significant homology between the p35 nck5a -binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35 nck5a and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35 nck5a binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M. A region immediately amino-terminal to the kinase-activating domain in p35 nck5a is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35 nck5a and the Cdk5/p35 nck5a complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35 nck5a . Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35 nck5a and other protein components (Lee, K.-Y., Rosales, J. L., Tang, D., and Wang, J. H. (1996) J. Biol. Chem. 271, 1538–1543). The yeast two-hybrid system was employed to identify p35 nck5a -interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment revealed significant homology between the p35 nck5a -binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35 nck5a and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35 nck5a binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M. A region immediately amino-terminal to the kinase-activating domain in p35 nck5a is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35 nck5a and the Cdk5/p35 nck5a complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35 nck5a . Neuronal Cdc2-like kinase (Nclk) 1The abbreviations used are: Nclk, neuronal Cdc2-like kinase; Nck5a, neuronal Cdk5 activator; Nck5ai, neuronal Cdk5 activator isoform; NF-H, -M, and -H, neurofilament high, medium, and low molecular weight subunits; GST, glutathioneS-transferase; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; GFAP, glial fibrillary acidic protein.1The abbreviations used are: Nclk, neuronal Cdc2-like kinase; Nck5a, neuronal Cdk5 activator; Nck5ai, neuronal Cdk5 activator isoform; NF-H, -M, and -H, neurofilament high, medium, and low molecular weight subunits; GST, glutathioneS-transferase; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; GFAP, glial fibrillary acidic protein. was originally purified from bovine brains on the basis of its phosphorylating activity toward serine or threonine within the sequence motif Ser/Thr-Pro-X-Lys/Arg (X for any amino acid) (1Lew J. Beaudette K. Litwin C.M. Wang J.H. J. Biol. Chem. 1992; 267: 13383-13390Abstract Full Text PDF PubMed Google Scholar). In an independent study, Nclk was also isolated as a tau protein kinase since it catalyzed in vitro phosphorylation of tau, which is a brain microtubule-associated protein (2Ishiguro K. Takamatsu M. Tomizawa K. Omori A. Takahashi M. Arioka M. Uchida T. Imahori K. J. Biol. Chem. 1992; 267: 10897-10901Abstract Full Text PDF PubMed Google Scholar, 3Hisanaga S. Ishiguro K. Uchida T. Okumura E. Okano T. Kishimoto T. J. Biol. Chem. 1993; 268: 15056-15060Abstract Full Text PDF PubMed Google Scholar). Purified Nclk is a heterodimer of Cdk5 and a 25-kDa regulatory subunit (p25), which is derived proteolytically from a brain and neuron-specific 35-kDa protein (p35) (4Ishiguro K. Kobayashi S. Omori A. Takamatsu M. Yonekura S. Anzai K. Imahori K. Uchida T. FEBS Lett. 1994; 342: 203-208Crossref PubMed Scopus (147) Google Scholar, 5Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (805) Google Scholar, 6Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (537) Google Scholar). In view of its specific Cdk5 activating activity, p25/p35 is called neuronal Cdk5 activator (Nck5a). In addition, mammalian brains also contain a neuron-specific 39-kDa isoform of Nck5a, called neuronal Cdk5 activator isoform (Nck5ai) (7Tang 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). Nck5a and Nck5ai display a high degree of protein sequence identity, but share little sequence homology with members of the cyclin family, thus suggesting that they represent a novel family of Cdk-activating proteins.Along with its unique structure, Nck5a is endowed with unique regulatory properties distinct from those of cyclins. While the activation of Cdc2 by cyclin depends on phosphorylation of Cdc2 by a Cdk-activating kinase at a specific threonine residue (Thr-161), the activation of Cdk5 by Nck5a is independent of Cdk5 phosphorylation (8Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2923) Google Scholar, 9Lees E. Curr. Opin. Cell Biol. 1995; 7: 773-780Crossref PubMed Scopus (265) Google Scholar, 10Qi 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, 11Qi Z. Tang D. Matsuura I. Lee K.Y. Zhu X. Huang Q.Q. Wang J.H. Mol. Cell. Biochem. 1995; 149–150: 35-39Crossref PubMed Scopus (6) Google Scholar). Also, several Cdc2-like kinases have been shown to be inactivated readily upon tyrosine phosphorylation by a Wee1 kinase (8Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2923) Google Scholar,9Lees E. Curr. Opin. Cell Biol. 1995; 7: 773-780Crossref PubMed Scopus (265) Google Scholar). However, Cdk5/Nck5a is only sluggishly phosphorylated and inactivated by Wee1 (12Poon R.Y.C. Lew J. Hunter T. J. Biol. Chem. 1997; 272: 5703-5708Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Tang D. Lee K.Y. Qi Z. Matsuura I. Wang J.H. Biochem. Cell Biol. 1996; 74: 419-429Crossref PubMed Scopus (28) Google Scholar). In addition, Cdk-cyclin complexes are susceptible to inhibitory activities of specific inhibitory proteins, such as p21 cip1/WAF1 and p27 kip1 (8Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2923) Google Scholar, 9Lees E. Curr. Opin. Cell Biol. 1995; 7: 773-780Crossref PubMed Scopus (265) Google Scholar, 14Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3205) Google Scholar). In contrast, the kinase activity of Nclk is resistant to these proteins (15Harper J.W. Elledge S.J. Keyomarsi K. Dynlacht B. Tsai L.H. Zhang P. Dobrowolski S. Bai C. Connell-Crowley L. Swindell E. et al.Mol. Biol. Cell. 1995; 6: 387-400Crossref PubMed Scopus (856) Google Scholar, 16Lee M.H. Nikolic M. Baptista C.A. Lai E. Tsai L.H. Massague J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3259-3263Crossref PubMed Scopus (116) Google Scholar).Developmental, cell biological, and transgenic mouse studies have implicated Nclk as an important regulator in development of the central nervous system in mammals and in normal functions of terminally differentiated neurons (5Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (805) Google Scholar, 6Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (537) Google Scholar, 7Tang 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, 17Tsai L.H. Takahashi T. Caviness Jr., V.S. Harlow E. Development. 1993; 119: 1029-1040Crossref PubMed Google Scholar, 18Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (529) Google Scholar, 19Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 20Matsushita M. Matsui H. Itano T. Tomizawa K. Tokuda M. Suwaki H. Wang J.H. Hatase O. NeuroReport. 1995; 6: 1267-1270Crossref PubMed Scopus (25) Google Scholar, 21Matsushita M. Tomizawa K. Lu Y.-F. Moriwaki A. Tokuda M. Itano T. Wang J.H. Hatase O. Matsui H. Brain Res. 1996; 734: 319-322Crossref PubMed Scopus (25) Google Scholar, 22Hirooka K. Tomizawa K. Matsui H. Tokuda M. Itano T. Hasegawa E. Wang J.H. Hatase O. J. Neurochem. 1996; 67: 2478-2483Crossref PubMed Scopus (22) Google Scholar, 23Tomizawa K. Matsui H. Matsushita M. Lew J. Tokuda M. Itano T. Konishi R. Wang J.H. Hatase O. Neuroscience. 1996; 74: 519-529Crossref PubMed Scopus (46) Google Scholar). The apparent involvement of Nclk in neuronal differentiation and brain functions may be attributed to the roles of this enzyme in neurocytoskeletal dynamics. Among in vitro Nclk substrates are neuron-specific cytoskeletal proteins such as tau and neurofilaments (3Hisanaga S. Ishiguro K. Uchida T. Okumura E. Okano T. Kishimoto T. J. Biol. Chem. 1993; 268: 15056-15060Abstract Full Text PDF PubMed Google Scholar, 24Lew J. Winkfein R.J. Paudel H.K. Wang J.H. J. Biol. Chem. 1992; 267: 25922-25926Abstract Full Text PDF PubMed Google Scholar, 25Sun D. Leung C.L. Liem R.K.H. J. Biol. Chem. 1996; 271: 14245-14251Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 26Pant H.C. Veeranna Biochem. Cell Biol. 1995; 73: 575-592Crossref PubMed Scopus (171) Google Scholar, 27Guidato S. Tsai L.H. Woodgett J. Miller C.C. J. Neurochem. 1996; 66: 1698-1706Crossref PubMed Scopus (109) Google Scholar). In Alzheimer patients, tau from neurofibrillary tangles is hyperphosphorylated on many proline-directed Ser/Thr sites (28Vulliet R. Halloran S.M. Braun R.K. Smith A.J. Lee G. J. Biol. Chem. 1992; 267: 22570-22574Abstract Full Text PDF PubMed Google Scholar). A number of studies have suggested that Nclk is a major kinase involved in catalyzing the proline-directed tau phosphorylation (2Ishiguro K. Takamatsu M. Tomizawa K. Omori A. Takahashi M. Arioka M. Uchida T. Imahori K. J. Biol. Chem. 1992; 267: 10897-10901Abstract Full Text PDF PubMed Google Scholar, 29Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar). Mammalian neurofilaments consist of three protein components, neurofilament high (NF-H), medium (NF-M), and low (NF-L) molecular weight subunits, which are assembled into neurofilaments and transported down axons as discrete cytological structures (30Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1119) Google Scholar, 31Liem R.K.H. Curr. Opin. Cell Biol. 1993; 5: 12-16Crossref PubMed Scopus (105) Google Scholar, 32Fliegner K.H. Liem R.K.H. Int. Rev. Cytol. 1991; 131: 109-167Crossref PubMed Scopus (91) Google Scholar). During this transport, NF-H and NF-M are heavily phosphorylated on their tail domains at many proline-directed Ser/Thr sites (33Elhanany E. Jaffe H. Link W.T. Sheeley D.M. Gainer H. Pant H.C. J. Neurochem. 1994; 63: 2324-2335Crossref PubMed Scopus (69) Google Scholar, 34Betts J.C. Blackstock W.P. Ward M.A. Anderton B.H. J. Biol. Chem. 1997; 272: 12922-12927Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Cdk5 has been suggested to participate in this axonal transport-dependent phosphorylation of the neurofilament proteins (24Lew J. Winkfein R.J. Paudel H.K. Wang J.H. J. Biol. Chem. 1992; 267: 25922-25926Abstract Full Text PDF PubMed Google Scholar, 25Sun D. Leung C.L. Liem R.K.H. J. Biol. Chem. 1996; 271: 14245-14251Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 26Pant H.C. Veeranna Biochem. Cell Biol. 1995; 73: 575-592Crossref PubMed Scopus (171) Google Scholar, 27Guidato S. Tsai L.H. Woodgett J. Miller C.C. J. Neurochem. 1996; 66: 1698-1706Crossref PubMed Scopus (109) Google Scholar). This suggestion is supported by the observation that, in cultured neuronal cells, both Cdk5 and Nck5a are present throughout the axons where neurofilaments represent the bulk of the cytoskeletal proteins (18Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (529) Google Scholar). In the present study, we show that Nck5a and Nclk associate specifically with the neurofilament proteins, an observation that may be the molecular basis of the colocalization and possible co-transportation of Nclk and neurofilaments in the axons. Our characterization of the interaction between Nck5a and neurofilaments is compatible with the notion that, in addition to kinase activation, Nck5a functions in anchoring Cdk5 to its kinase substrates.RESULTSIn a previous study, we showed that Nck5a existed in bovine brain extracts either as a 25-kDa fragment in a heterodimeric complex of Cdk5/p25 nck5a, or as the full-length protein (p35 nck5a ) in a macromolecular complex of over 600 kDa, which also contained Cdk5 as a constituent protein (38Lee 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). This observation has led to the suggestion that p35 nck5a has the ability to undergo high affinity association with certain cellular proteins. The yeast two-hybrid system was therefore employed to screen a human brain cDNA library to identify the putative p35 nck5a -binding proteins. About 3.5 × 105 cotransformants were analyzed. A total of 108 positive clones were isolated and introduced into another yeast strain to test binding specificities. 14 clones from the primary isolates displayed specific interaction with the original bait p35 nck5a /pAS2. Upon sequence analysis, these 14 clones were revealed to originate from seven different cDNA sequences. One of the isolated sequences, which represents three individual clones, encodes a carboxyl-terminal fragment of glial fibrillary acidic protein (GFAP).Since Nck5a is a neuron-specific protein, whereas the expression of GFAP is restricted to glial cells, it is unlikely that these two proteins associate in brains under normal physiological conditions. However, the association of Nck5a with GFAP may reflect the ability of Nck5a to undergo association with certain neuronal proteins containing protein sequences similar to that of the Nck5a-binding region of GFAP. GFAP is a member of the intermediate filament family (30Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1119) Google Scholar). All intermediate filaments share the basic architecture of a conserved central rod domain flanked by variable head and tail domains (30Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1119) Google Scholar). The isolated Nck5a-binding fragment consists of a carboxyl-terminal portion of the rod domain and the entire tail region of GFAP. Among the intermediate filament family members are the neuron-specific intermediate filaments, NF-L, NF-M and NF-H (30Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1119) Google Scholar, 31Liem R.K.H. Curr. Opin. Cell Biol. 1993; 5: 12-16Crossref PubMed Scopus (105) Google Scholar, 32Fliegner K.H. Liem R.K.H. Int. Rev. Cytol. 1991; 131: 109-167Crossref PubMed Scopus (91) Google Scholar). NF-M and NF-H have been suggested as in vivo substrates of Cdk5 (3Hisanaga S. Ishiguro K. Uchida T. Okumura E. Okano T. Kishimoto T. J. Biol. Chem. 1993; 268: 15056-15060Abstract Full Text PDF PubMed Google Scholar,24Lew J. Winkfein R.J. Paudel H.K. Wang J.H. J. Biol. Chem. 1992; 267: 25922-25926Abstract Full Text PDF PubMed Google Scholar, 25Sun D. Leung C.L. Liem R.K.H. J. Biol. Chem. 1996; 271: 14245-14251Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 26Pant H.C. Veeranna Biochem. Cell Biol. 1995; 73: 575-592Crossref PubMed Scopus (171) Google Scholar, 27Guidato S. Tsai L.H. Woodgett J. Miller C.C. J. Neurochem. 1996; 66: 1698-1706Crossref PubMed Scopus (109) Google Scholar). Sequence alignment displays high degrees of protein sequence identity between the Nck5a-binding region of GFAP and the corresponding rod regions in neurofilaments (Fig. 1), suggesting that Nck5a may bind neurofilaments in cells.To test whether neurofilaments are indeed capable of association with Nck5a, a fragment of NF-M, NF-M346–467, corresponding to the Nck5a-binding fragment of GFAP, was engineered into the plasmidpACT2 for the two-hybrid assay. As shown in Fig.2, the reporter activity was detected when NF-M 346–467 /pACT2 was cotransformed with p35 nck5a /pAS2 into yeast cells, whereas cotransformation of a control plasmid lamin C/pAS2 with theNF-M 346–467 /pACT2 displayed no reporter activity. The result suggests that NF-M could indeed associate with Nck5a in the yeast. The fragment NF-M346–467 is composed of a carboxyl-terminal region of the conserved core domain and an amino-terminal region of the tail domain of NF-M. To localize more precisely the p35 nck5a -binding region in NF-M, thepACT2 constructs of NF-M346–413 and NF-M411–467 were generated, corresponding to the rod domain and the tail domain fragments, respectively. As shown in Fig. 2, cotransformation of the rod domain fragment (NF-M346–413) with Nck5a strongly activated the reporter activity, whereas no detectable reporter activity appeared in the cotransformants of the tail fragment constructNF-M 411–467 /pACT2 andp35 nck5a /pAS2. This observation indicates that the carboxyl-terminal portion of the NF-M rod domain, which is no more than 16% of the rod domain, is sufficient for the binding with Nck5a, and the tail region is not required for this interaction. Since Cdk5 phosphorylation sites in NF-M and NF-H are located at the carboxyl-terminal tails, the localization of the Nck5a-binding site immediately amino-terminal to the tail region may be significant for the kinase reaction. Mammalian brains contain an isoform of Nck5a, p39 nck5i (7Tang 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). Fig. 3 shows that p39 nck5i did not give rise to positive response when tested for its ability to bind NF-M using the yeast two-hybrid system. The result demonstrates specificity of the interaction between Nck5a and NF-M.Figure 2Interaction of Nck5a with NF-M fragments in the two-hybrid system. The recombinant pACT2 plasmids were introduced into yeast Y190 withp35 nck5a /pAS2 or lamin C/pAS. Cotransformants were selected on a synthetic dextrose medium without leucine and tryptophan. The colonies were then patched on the medium without leucine, tryptophan, and histidine, but containing 15 mm 3-amino-1,2,4-triazole. The cloned NF-M fragments are schematically shown beside the yeast growth pattern.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3The two-hybrid assay of interaction between NF-M and Nck5a or Nck5ai. The pACT2 constructs containing NF-M346–467 or NF-M346–413 were introduced into yeast Y190 with the pAS2constructs of p351–98, p25 nck5a (p3599–307), and p39 nck5ai individually. After colonies appeared on synthetic dextrose plates lacking leucine and tryptophan, the cotransformants were patched on the medium without leucine, tryptophan and histidine, but containing 15 mm 3-amino-1,2,4-triazole.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nck5a exists in bovine brain extracts either as a full-length protein of 307 amino acids or an amino-terminal-truncated form (p25 nck5a ), which consists of residues 99–307 of p35 nck5a (38Lee 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). To determine whether the interaction between NF-M and Nck5a requires the amino-terminal region of Nck5a, we dissected p35 nck5a into two fragments: the amino-terminal fragment (p351–98) and the fragment of p25 nck5a (p3599–307). These two fragments as well as the full-length Nck5a were tested for their abilities to bind NF-M in the yeast two-hybrid system. Figs. 2 and 3 show that NF-M interacts with both full-length Nck5a and p25 nck5a, but not with the amino-terminal fragment (p351–98). This observation suggests that the NF-M binding site is located within residues 99–307 of Nck5a. To define more precisely the region required for the NF-M binding, various amino-terminal-truncated forms of Nck5a were constructed and tested for their interactions with NF-M by the yeast two-hybrid assay. The results were summarized in Fig.4. It indicates that a short stretch of protein sequence spanning residues 144 to 150 is required for the NF-M binding. Recently, the Cdk5-activating domain in Nck5a has been mapped to a region of amino acid residues 150–291 (12Poon R.Y.C. Lew J. Hunter T. J. Biol. Chem. 1997; 272: 5703-5708Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 39Tang D. Chun A.C.S. Zhang M. Wang J.H. J. Biol. Chem. 1997; 272: 12318-12327Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). It should be noted that the region immediately amino-terminal to the minimal Cdk5-activating domain in Nck5a is essential for its interaction with NF-M.Figure 4Association of NF-M with Nck5a fragments in the two-hybrid system. NF-M 346–467 /pACT2 andNF-M 346–413 /pACT2 were transformed into yeast Y190 with the pAS2 constructs containing various truncated fragments of Nck5a. Cotransformants were selected on synthetic dextrose plates lacking leucine and tryptophan. After colonies appeared, β-galactosidase activity was tested by a filter lift assay using X-gal. Plus (+) means that the colonies turned blue, and minus (−) means that the colonies remained white in the assay of β-galactosidase activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To provide direct proof for the specific association between NF-M and Nck5a, this protein-protein interaction was carried out and characterized in vitro using bacterially expressed proteins. GST-fusion proteins of two NF-M carboxyl-terminal fragments: NF-M346–915 and NF-M411–915, were expressed in and isolated from bacteria (Figs. 5,A and B). The results from the yeast two-hybrid assay suggested that NF-M346–915 contains the Nck5a-binding site, whereas NF-M411–915 does not. To demonstrate protein associations, the GST-NF-M proteins were incubated with (His)6-tagged p35 nck5a and then affinity-precipitated using GSH beads. The precipitates were then analyzed on an immunoblot using an anti-Nck5a antibody to determine whether Nck5a had co-precipitated with the NF-M fragments. Fig.5 C shows that the bacteria-expressed and affinity-purified p35 nck5a -His contains intact p35 nck5a and two partially proteolyzed fragments showing immunoreactive protein bands of approximately 30 and 28 kDa. The p35 nck5a -His proteins were present in the precipitate of GST-NF-M346–915 but not in the precipitates of GST and GST-NF-M411–915 (Fig.5 C). It supports the results from the yeast two-hybrid analysis suggesting that Nck5a undergoes specific association with NF-M and that the Nck5a-binding site is localized at the carboxyl-terminal region of the NF-M rod domain.Figure 5In vitro binding of p35 nck5a with NF-M fragments. A, a schematic diagram of NF-M and expressed NF-M fragments. The shaded region is NF-M346–413. B, SDS-PAGE patterns of the expressed NF-M fragments. 5 μg of GST-NF-M411–915 was applied in lane 1, and 3.2 μg of GST-NF-M346–915 in lane 2. The gel was stained with Coomassie Blue. C, association of p35 nck5a with the NF-M fragments. 8 μg of p35 nck5a -His was incubated with 10 μg of GST (lane 2), GST-NF-M411–915(lane 3), GST-NF-M346–915 (lane 4) at 30 °C for 30 min. The GST-fusion proteins were then precipitated with GSH-Sepharose 4B. Proteins bound to the beads were dissolved in SDS-PAGE sample buffer. p35 nck5a -His was detected on the immunoblot with an anti-Nck5a antibody. 1 μg of p35 nck5a -His was applied in lane 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further characterize the interaction between NF-M and Nck5a, the interaction was examined with the heterodimer of Cdk5/p25 nck5a, which is the active form of Nclk existing in bovine brain extracts. Nclk was prepared by reconstitution from bacterially expressed Cdk5 and p25 nck5a as described previously (10Qi 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). Samples of Nclk were incubated with GST-NF-M346–915, GST-NF-M411–915, and GST separately. The GST fusion proteins were then affinity-precipitated using GSH beads. After being washed extensively, the precipitates were subjected to immunoblot analysis using antibodies against Cdk5 and Nck5a. As shown in Fig.6, both Cdk5 and Nck5a could be detected in the precipitated sample containing GST-NF-M346–915 but not in those of GST-NF-M411–915 and GST. The binding of Nclk and NF-M346–915 is essentially quantitative under the experimental conditions. The results indicate that NF-M binds both Nck5a monomer and the Cdk5/Nck5a heterodimer, and the same site in NF-M may be involved in the binding of the two protein species.Figure 6In vitro binding of NF-M fragments with the Cdk5/p25 nck5a complex. 5 μl of Cdk5/p25 nck5a were incubated with 10 μg of GST (lane 1), GST-NF-M411–915 (lane 2), GST-NF-M346–915 (lane 3), at 30 °C for 30 min. The GST fusion proteins were recovered with GSH-Sepharose 4B. Proteins bound to the beads were dissolved in SDS-PAGE sample buffer. p25 nck5a and Cdk5 were detected on the immunoblots with antibodies against Nck5a (A) and Cdk5 (B), respectively. 1 μl of Cdk5/p25 nck5a was applied in lane 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further address the question of potential physiological significance of the interaction between NF-M and Nck5a, we examined any effect on the Cdk5-activating activity of Nck5a by interaction with NF-M. In one experiment, the activation of Cdk5 by a suboptimal amount of p25 nck5a was determined in the presence of NF-M346–915. It was found that NF-M346–915, over a large concentration range, could neither enhance nor impede the Cdk5-activating activity of Nck5a (Fig.7 A). All three proteins used in the experiment of Fig. 7 A were bacterially expressed GST fusion proteins. However, similar results were obtained if the proteins were pretreated by thrombin to remove the GST moiety (data not shown). To ensure that the kinase activity measured was indeed that of NF-M-bound Nclk, samples of reconstituted Cdk5/p25 nck5a were incubated with varying amounts of GST-NF-M346–915. GST-NF-M346–915 along with its bound-Nclk was then affinity-precipitated using GSH beads and the Cdk5 kinase activity in the precipitates was then assayed. As shown in Fig. 7 B, the Cdk5 kinase activity could be recovered in the precipitates in a NF-M346–915 concentration-dependent manner. The kinase in the precipitate could also phosphorylate NF-M as shown in Fig. 7 C. In a separate experiment, the Cdk5-catalyzed phosphorylation of NF-M346–915 was compared with that of NF-M411–915, and they were about the same (data not shown). NF-M346–915 is capable of binding to Nck5a, but NF-M411–915 is not. Thus, under various conditions, NF-M-bound Nclk and free Nclk appeared to have the same kinase activity.Figure 7Effect of the NF-M binding on the kinase activity of Cdk5/Nck5a. A, effect of GST-NF-M346–915 on Cdk5 activation by p25 nck5a . 0.6 μg of GST-Cdk5 was mixed with 0.1 μg of GST-p25 nck5a and different amounts of GST-NF-M346–915 as indicated. After incubation at 30 °C for 30 min, the Cdk5 kinase activity was measured by phosphorylation of the HS(9–18) peptide at 30 °C for 20 min. B, the kinase activity of NF-M-bound Cdk5/p25 nck5a . 7 μl of Cdk5/p25 nck5a were incubated with indicated amounts of GST-NF-M346–915 at 30 °C for 30 min. The GST fusion proteins were" @default.
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• W1970863847 title "Association of Neurofilament Proteins with Neuronal Cdk5 Activator" @default.
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