|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
Journal of Cell Science, Vol 107, Issue 11 3223-3233, Copyright © 1994 by Company of Biologists
JOURNAL ARTICLES |
J Wang, M Satoh, A Pierani, J Schmitt, CH Chou, HG Stunnenberg, RG Roeder and WH Reeves
Department of Medicine, University of North Carolina, Chapel Hill 27599-7280.
The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.
This article has been cited by other articles:
|
|
 |
|
  T. Mesplede, M.-L. Island, N. Christeff, F. Petek, J. Doly, and S. Navarro The POU Transcription Factor Oct-1 Represses Virus-Induced Interferon A Gene Expression Mol. Cell. Biol., October 1, 2005; 25(19): 8717 - 8731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Hammer, N. A. Compagnone, J.-L. Vigne, S. R. Bair, and S. H. Mellon Transcriptional Regulation of P450scc Gene Expression in the Embryonic Rodent Nervous System Endocrinology, February 1, 2004; 145(2): 901 - 912. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Schaffer, E. C. Kim, X. Wu, H. Zan, L. Testoni, S. Salamon, A. Cerutti, and P. Casali Selective Inhibition of Class Switching to IgG and IgE by Recruitment of the HoxC4 and Oct-1 Homeodomain Proteins and Ku70/Ku86 to Newly Identified ATTT cis-Elements J. Biol. Chem., June 13, 2003; 278(25): 23141 - 23150. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Willis, A. P. Loewy, N. Charlton-Kachigian, J.-S. Shao, D. M. Ornitz, and D. A. Towler Regulation of Osteocalcin Gene Expression by a Novel Ku Antigen Transcription Factor Complex J. Biol. Chem., September 27, 2002; 277(40): 37280 - 37291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Sobel, M. Satoh, Y. Chen, E. K. Wakeland, and L. Morel The Major Murine Systemic Lupus Erythematosus Susceptibility Locus Sle1 Results in Abnormal Functions of Both B and T Cells J. Immunol., September 1, 2002; 169(5): 2694 - 2700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Rodgers, S. J. Jordan, and J. D. Capra Transient Association of Ku with Nuclear Substrates Characterized Using Fluorescence Photobleaching J. Immunol., March 1, 2002; 168(5): 2348 - 2355. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J Bertinato, C Schild-Poulter, and R. Hache Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits J. Cell Sci., January 1, 2001; 114(1): 89 - 99. [Abstract] [PDF]
|
|
|
|
|
|
Y.-T. Tai, G. Teoh, B. Lin, F. E. Davies, D. Chauhan, S. P. Treon, N. Raje, T. Hideshima, Y. Shima, K. Podar, et al. Ku86 Variant Expression and Function in Multiple Myeloma Cells Is Associated with Increased Sensitivity to DNA Damage J. Immunol., December 1, 2000; 165(11): 6347 - 6355. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. N. Mahajan, L. Gangi-Peterson, D. H. Sorscher, J. Wang, K. N. Gathy, N. P. Mahajan, W. H. Reeves, and B. S. Mitchell Association of terminal deoxynucleotidyl transferase with Ku PNAS, November 23, 1999; 96(24): 13926 - 13931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Galande and T. Kohwi-Shigematsu Poly(ADP-ribose) Polymerase and Ku Autoantigen Form a Complex and Synergistically Bind to Matrix Attachment Sequences J. Biol. Chem., July 16, 1999; 274(29): 20521 - 20528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Shackelford, T. Tobaru, S. Zhang, and J. A. Zivin Changes in Expression of the DNA Repair Protein Complex DNA-Dependent Protein Kinase after Ischemia and Reperfusion J. Neurosci., June 15, 1999; 19(12): 4727 - 4738. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wang, X. Dong, and W. H. Reeves A Model for Ku Heterodimer Assembly and Interaction with DNA. IMPLICATIONS FOR THE FUNCTION OF Ku ANTIGEN J. Biol. Chem., November 20, 1998; 273(47): 31068 - 31074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Barlev, V. Poltoratsky, T. Owen-Hughes, C. Ying, L. Liu, J. L. Workman, and S. L. Berger Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku-DNA-Dependent Protein Kinase Complex Mol. Cell. Biol., March 1, 1998; 18(3): 1349 - 1358. [Abstract] [Full Text]
|
|
|
|
|
|
J. Wang, X. Dong, K. Myung, E. A. Hendrickson, and W. H. Reeves Identification of Two Domains of the p70 Ku Protein Mediating Dimerization with p80 and DNA Binding J. Biol. Chem., January 9, 1998; 273(2): 842 - 848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Osipovich, S. K. Durum, and K. Muegge Defining the Minimal Domain of Ku80 for Interaction with Ku70 J. Biol. Chem., October 24, 1997; 272(43): 27259 - 27265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Cortes, F. Weis-Garcia, Z. Misulovin, A. Nussenzweig, J.-S. Lai, G. Li, M. C. Nussenzweig, and D. Baltimore In vitro V(D)J recombination: Signal joint formation PNAS, November 26, 1996; 93(24): 14008 - 14013. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Han, N. Malik, T. Carter, W. H. Reeves, J. H. Wyche, and E. A. Hendrickson DNA-dependent Protein Kinase Is a Target for a CPP32-like Apoptotic Protease J. Biol. Chem., October 4, 1996; 271(40): 25035 - 25040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Han, C. Johnston, W. H. Reeves, T. Carter, J. H. Wyche, and E. A. Hendrickson Characterization of a Ku86 Variant Protein That Results in Altered DNA Binding and Diminished DNA-dependent Protein Kinase Activity J. Biol. Chem., June 14, 1996; 271(24): 14098 - 14104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Fewell and E. Kuff Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium J. Cell Sci., January 7, 1996; 109(7): 1937 - 1946. [Abstract] [PDF]
|
|
