Nuclear Receptor Coactivator Thyroid Hormone Receptor-binding Protein (TRBP) Interacts with and Stimulates Its Associated DNA-dependent Protein Kinase

Nuclear receptors mediate gene activation through ligand-dependent interaction with coactivators. We previously cloned and characterized thyroid hormone receptor-binding protein, TRBP (NcoA6: AIB3/ASC-2/RAP250/PRIP/TRBP/NRC), as an LXXLL-containing coactivator that associates with coactivator complexes through its C terminus. To search for protein factors involved in TRBP action, we identified a distinct set of proteins from HeLa nuclear extract that interacts with the C terminus of TRBP. Analysis by mass spectrometric protein sequencing revealed a DNA-dependent protein kinase (DNA-PK) complex including its catalytic subunit and regulatory subunits, Ku70 and Ku86. DNA-PK is a heterotrimeric nuclear phosphatidylinositol 3-kinase that functions in DNA repair, recombination, and transcriptional regulation. DNA-PK phosphorylates TRBP at its C-terminal region, which directly interacts with Ku70 but not Ku86 in vitro. In addition, in the absence of DNA, TRBP itself activates DNA-PK, and the TRBP-stimulated DNA-PK activity has an altered phosphorylation pattern from DNA-stimulated activity. An anti-TRBP antibody inhibits TRBP-induced kinase activity, suggesting that protein content of TRBP is responsible for the stimulation of DNA-independent kinase activity. Furthermore, in DNA-PK-deficient scid cells, TRBP-mediated transactivation is significantly impaired, and nuclear localization of TRBP is altered. The activation of DNA-PK in the absence of DNA ends by the coactivator TRBP suggests a novel mechanism of coactivator-stimulated DNA-PK phosphorylation in transcriptional regulation. Nuclear receptors mediate gene activation through ligand-dependent interaction with coactivators. We previously cloned and characterized thyroid hormone receptor-binding protein, TRBP (NcoA6: AIB3/ASC-2/RAP250/PRIP/TRBP/NRC), as an LXXLL-containing coactivator that associates with coactivator complexes through its C terminus. To search for protein factors involved in TRBP action, we identified a distinct set of proteins from HeLa nuclear extract that interacts with the C terminus of TRBP. Analysis by mass spectrometric protein sequencing revealed a DNA-dependent protein kinase (DNA-PK) complex including its catalytic subunit and regulatory subunits, Ku70 and Ku86. DNA-PK is a heterotrimeric nuclear phosphatidylinositol 3-kinase that functions in DNA repair, recombination, and transcriptional regulation. DNA-PK phosphorylates TRBP at its C-terminal region, which directly interacts with Ku70 but not Ku86 in vitro. In addition, in the absence of DNA, TRBP itself activates DNA-PK, and the TRBP-stimulated DNA-PK activity has an altered phosphorylation pattern from DNA-stimulated activity. An anti-TRBP antibody inhibits TRBP-induced kinase activity, suggesting that protein content of TRBP is responsible for the stimulation of DNA-independent kinase activity. Furthermore, in DNA-PK-deficient scid cells, TRBP-mediated transactivation is significantly impaired, and nuclear localization of TRBP is altered. The activation of DNA-PK in the absence of DNA ends by the coactivator TRBP suggests a novel mechanism of coactivator-stimulated DNA-PK phosphorylation in transcriptional regulation. CREB-binding protein (where CREB is cAMP-response element-binding protein) thyroid hormone receptor-binding protein coactivator activator DNA-dependent protein kinase DNA-PK catalytic subunit poly(ADP-ribose) polymerase glutathioneS-transferase C-terminal domain vitamin D receptor-interacting protein severe combined immune deficiency amino acid antibody mouse mammary tumor virus glucocorticoid receptor dithiothreitol phosphatidylinositol 3-kinase double strand Nuclear receptor coactivators mediate gene activation though interactions with nuclear receptors and components in the transcriptional apparatus. The interactions subsequently permit the RNA polymerase II complexes to access target genes (1Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Google Scholar, 2McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Google Scholar). Studies of coactivators in the past several years have significantly refined our model of ligand-induced interaction between the receptor ligand-binding domains and the coactivator LXXLL motifs. However, the role of receptor-bound coactivators in their interactions with the transcriptional complex is less clear, and it remains a current research focus. Coactivator actions appear to involve multiple cooperative mechanisms. The functional properties of coactivators include the following: direct protein-protein interactions with transcriptional complexes (1Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Google Scholar); enzymatic activities of certain coactivators, such as histone acetyltransferase in CBP1 and steroid receptor coactivator-1 (SRC-1) family or arginine transmethylase in coactivator-associated arginine methyltransferase 1 (CARM1) (3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholar, 4Chen D. Ma H. Hong H. Koh S.S. Huang S.M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2177Google Scholar); RNA interactions, with RNA recognition motifs such as in peroxisome proliferator-activated receptor-γ-coactivator-1 (PGC-1), CoAA, and SMRT/HDAC1-associated repressor protein (SHARP); and RNA alone such as steroid receptor RNA activator (SRA) (5Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 20: 829-839Google Scholar, 6Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Google Scholar, 7Shi Y. Downes M. Xie W. Kao H.Y. Ordentlich P. Tsai C.C. Hon M. Evans R.M. Genes Dev. 2001; 15: 1140-1151Google Scholar, 8Lanz R.B. McKenna N.J. Onate S.A. Albrecht U. Wong J. Tsai S.Y. Tsai M.J. O'Malley B.W. Cell. 1999; 97: 17-27Google Scholar). It is becoming increasingly important to understand how coactivator-targeted molecules, such as interacting proteins or enzyme substrates, are regulated by coactivators. We previously cloned and characterized thyroid hormone receptor-binding protein (TRBP) as a nuclear receptor coactivator (9Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Google Scholar). TRBP, designated as NcoA6 by the National Center for Biotechnology Information (NCBI) nomenclature committee, was concurrently identified by several groups as AIB3/ASC-2/RAP250/PRIP/TRBP/NRC (1Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Google Scholar, 2McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Google Scholar). TRBP is a high molecular weight, ubiquitously expressed coactivator. A single LXXLL motif is required for the ligand-dependent interaction with a number of nuclear receptors and subsequent transcriptional activation. The Ser-884 residue adjacent to the TRBP LXXLL motif was shown to regulate the selectivity of TRBP for different nuclear receptors (10Ko L. Cardona G.R. Iwasaki T. Bramlett K.S. Burris T.P. Chin W.W. Mol. Endocrinol. 2002; 16: 128-140Google Scholar). TRBP also coactivates multiple transcriptional factors including AP-1 and NF-κB (1Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Google Scholar). In addition, gene amplification was observed for TRBP in human breast cancers (11Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Google Scholar). Furthermore, the C terminus of TRBP was shown to interact with coactivator CoAA (6Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Google Scholar), CBP/p300, and DRIP complexes (9Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Google Scholar). In a search for additional nuclear factors that might be targeted by TRBP, we identified a distinct set of TRBP-bound proteins using a mass spectrometric protein sequencing approach. Proteins identified include the DNA-dependent protein kinase (DNA-PK) components. DNA-PK is a nuclear serine/threonine protein kinase that belongs to the PI3K family (12Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 503-512Google Scholar, 13Dynan W.S. Yoo S. Nucleic Acids Res. 1998; 26: 1551-1559Google Scholar, 14Fugmann S.D. Nature. 2002; 416: 691-694Google Scholar, 15Jeggo P.A. Mutat. Res. 1997; 384: 1-14Google Scholar, 16Smith G.C.M. Jackson S.P. Genes Dev. 1999; 13: 916-934Google Scholar). Previous biochemical and genetic studies revealed DNA-PK to be a heterotrimeric enzyme composed of a catalytic subunit, DNA-PKcs, and two regulatory subunits, Ku86 and Ku70. Although DNA-PK is known to be activated by DNA ends, recent compelling evidence suggests that its kinase activity can also be stimulated by protein interactions (17Schild-Poulter C. Pope L. Giffin W. Kochan J.C. Ngsee J.K. Traykova-Andonova M. Hache R.J. J. Biol. Chem. 2001; 276: 16848-16856Google Scholar, 18Yavuzer U. Smith G.C.M. Bliss T. Werner D. Jackson S.P. Genes Dev. 1998; 12: 2188-2199Google Scholar, 19Willis D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Google Scholar). Supported by a large body of evidence, DNA-PK has been shown to be involved in transcriptional regulation as well as in recombination and DNA repair (12Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 503-512Google Scholar, 13Dynan W.S. Yoo S. Nucleic Acids Res. 1998; 26: 1551-1559Google Scholar, 19Willis D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Google Scholar, 20Woodard R.L. Anderson M.G. Dynan W.S. J. Biol. Chem. 1999; 274: 478-485Google Scholar, 21Sheppard H.M. Liu X. Biochim. Biophys. Acta. 2000; 1493: 41-47Google Scholar). DNA-PK is unique among nuclear protein kinases because it associates with DNA templates and phosphorylates a variety of protein factors that are important for transcriptional regulation. The identified in vitrosubstrates of DNA-PK include DNA-binding transcriptional factors such as c-Myc, c-Jun, Sp1, Oct-1, and nuclear receptors GR, progesterone receptor, tumor suppressor p53, HMG proteins, Ku subunits, as well as DNA-PKcs itself through autophosphorylation (12Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 503-512Google Scholar). The close of DNA-PK with transcriptional regulation is also by its to the C-terminal domain of RNA polymerase II A. R.L. Dynan W.S. J. Biol. Chem. Scholar, S.A. A. Wu C. Dynan W.S. J. Biol. Chem. Scholar, R. M. H. R. P. Anderson C.W. S. D. Nature. 1996; Scholar), which might be important for and G. D. Cell. 2002; 108: Scholar). In addition, mouse with severe combined immune deficiency that functional DNA-PK the not in DNA recombination and DNA repair S.S. Mutat. Res. but also in Ku phosphorylation and transcriptional activation H.M. Liu X. Biochim. Biophys. Acta. 2000; 1493: 41-47Google Scholar, Mol. Cell. Biol. 15: Scholar). DNA-PK kinase activity might be for transcriptional We the of DNA-PK components as its C-terminal region, TRBP interacts with DNA-PK a direct interaction with the regulatory subunit, Ku70. TRBP can be by DNA-PK in vitro. also that TRBP DNA-PK kinase activity in the absence of DNA DNA-independent activity in altered phosphorylation In addition, DNA-PK-deficient have altered TRBP nuclear localization and a in TRBP-mediated activation. studies a novel between coactivator-stimulated DNA-PK phosphorylation and transcriptional regulation. TRBP in and its have been previously (9Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Google Scholar). or of as in human Ku70 and cloned by and of mammary tumor virus was by and was by human the of as previously (6Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Google Scholar). and from anti-TRBP from two and using the as The TRBP antibody was by to the The and in and by Nuclear from a large of HeLa or cells, in A 1 1 1 with of and for The by at and with 1 1 1 with the for the nuclear with a to is important to protein in the subsequent The with of proteins of nuclear in of 1 1 and with at with and by by of identified by and with to the J.K. J. Scholar, W.S. 1998; Scholar). at the the of by high mass and mass which was with the and at the by to the known In by and in proteins by Proteins at for 1 in the 1 1 proteins with and to and with of HeLa or nuclear at in the as with additional proteins and by and from of TRBP in by DNA-PK was using the DNA-dependent from with DNA-PK enzyme from HeLa nuclear extract was with with or double strand DNA as and with proteins as proteins and by and from Biotechnology was as a for the DNA-dependent and alone as an protein was as a TRBP was for its activity, was as a The of DNA-PK in was and with a to DNA-PK from HeLa nuclear was and with HeLa nuclear and TRBP DNA-PK in the DNA-PK kinase activities using the in the or the absence of DNA, a was as by the and the and in a alone and as TRBP by to the and human and with anti-TRBP and and at a of using a HeLa cells, mouse and in with and in at in with and TRBP well using with the of and and activities of DNA for well by are shown as of of evidence the of the C terminus of coactivator TRBP as a domain for the interactions with multiple nuclear proteins, including coactivator CBP/p300, DRIP complex (9Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Google Scholar), and coactivator activator CoAA (6Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Google Scholar). In addition, the of the transcriptional activity of TRBP is through its C terminus (9Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Google Scholar). To in protein complexes in close with the TRBP C-terminal region, we mass spectrometric of nuclear proteins with TRBP HeLa nuclear with TRBP including its C and proteins identified by with different and in to the proteins in of that might be by A number of proteins from to However, the for The identified proteins are and proteins from the and their by sequencing among the proteins, of to the DNA-PK including DNA-PK catalytic subunit DNA-PK regulatory subunits, Ku and Ku and The two proteins DNA and The of proteins for and DNA-PKcs, Ku70 and Ku86. was with the is DNA-PK not to be with TRBP suggesting that the of proteins was to the C-terminal of TRBP The of Ku and interaction is high Ku might have TRBP. In the number of the to to a number with the of the of a protein also revealed the of which was shown with a number of It be that proteins in not be identified by sequencing to TRBP directly or such as the be for the of proteins (6Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Google Scholar). we from our that DNA-PK complexes interact with TRBP at its C interacting with the C terminus of from HeLa nuclear designated to their molecular of of that the proteins in catalytic of and Proteins from HeLa nuclear designated to their molecular of that the proteins in of and in a We to our current studies DNA-PK and its with TRBP because the role of DNA-PK in transcriptional regulation is becoming increasingly D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Google Scholar, R.L. Huang J. Dynan W.S. J. Biol. Chem. 2001; 276: Scholar). To the interaction of the DNA-PK complex with TRBP observed we using and shown in with HeLa nuclear and proteins with that DNA-PKcs and Ku70 interact with TRBP, but at its C terminus. with antibody and with regulatory Ku70 and Ku86 interact with proteins that regulate DNA-PK kinase activity (17Schild-Poulter C. Pope L. Giffin W. Kochan J.C. Ngsee J.K. Traykova-Andonova M. Hache R.J. J. Biol. Chem. 2001; 276: 16848-16856Google Scholar, 19Willis D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Google Scholar), an in was to the direct interactions of Ku with TRBP. as shown in interaction of TRBP with Ku was interaction with Ku86 was including and alone not the of TRBP was not to the Ku70 but to of Ku70. to a regulated of Ku70 with TRBP. that TRBP interacts with DNA-PK, through its Ku70 The that TRBP might have a close with To understand interaction we the that TRBP is a of the TRBP as in a DNA-PK kinase as shown in A. the alone was not C terminus was by The was as a for DNA-dependent the phosphorylation of or which at C was The TRBP can be but in a DNA-dependent TRBP not to be for DNA-PK not that can be by DNA-PK in a DNA-independent from the phosphorylation of or which is A of the TRBP phosphorylation revealed that the DNA-independent phosphorylation a DNA-dependent phosphorylation to the of which was for the we phosphorylation of and in shown in C. The DNA-independent phosphorylation of two phosphorylation phosphorylation can be by a PI3K that the kinase activity of DNA-PK, the phosphorylation was The that the TRBP C terminus by as an activator to DNA-independent kinase activity. In addition, the kinase TRBP at different the kinase to a with the that is a of DNA-PK the is TRBP or DNA with DNA ends, TRBP might be a unique of We by that might DNA-PK TRBP. TRBP itself not kinase activity, as a of DNA-PK enzyme was not to the to DNA, DNA not a suggesting that the phosphorylation might be different for the protein We also with high to protein in the In addition, we with of TRBP with or to acid the of In DNA not a are with our that TRBP protein is at in for the DNA-independent activation of To we evidence for TRBP-stimulated activity of In TRBP-stimulated kinase a was as a which can be from TRBP shown in the TRBP C including and was to kinase activities in the absence of DNA, the kinase was also stimulated in the of anti-TRBP but not or in the DNA-independent activities significantly However, the less or the DNA-dependent activities in the Furthermore, in the from the with two anti-TRBP and we that but not DNA-independent and DNA-dependent activities not that TRBP at its C-terminal a domain or which is to DNA-PK kinase activity by interacting with the DNA-PK complex in the absence of DNA as an the of studies with the that might DNA-PK, we that an antibody alone was to DNA-PK in the absence of that the of antibody and TRBP protein can a DNA-independent activity that is with the of DNA-stimulated activity. The anti-TRBP the kinase activity, and and or which as Although DNA-PK not the that a of protein interactions, TRBP and for might in a of DNA-PK and subsequently the kinase with activity with activity. that DNA-PK might be regulated in by interacting proteins as well as DNA TRBP an important role in the direct stimulation by TRBP, or combined with proteins, DNA-PK in the absence of DNA ends in transcriptional regulation. TRBP can the activity of DNA-PK in we TRBP might also DNA-PK in shown in DNA-PK from HeLa nuclear was using TRBP and the TRBP-bound DNA-PK activity was DNA-PK from HeLa nuclear extract DNA-dependent activity, the TRBP-bound DNA-PK activity in the absence of that TRBP not interacts with DNA-PK but is also to To the activation of DNA-PK is for the transcriptional activity of TRBP in we TRBP-mediated transcriptional activity in DNA-PK-deficient A mouse scid is in DNA-PK The human the human DNA-PK catalytic subunit and has DNA-PK phosphorylation S.S. Mutat. Res. Scholar, Mol. Cell. Biol. 15: Scholar). TRBP activity was in two TRBP activity was in the DNA-PK-deficient with that observed in cells, which has DNA-PK activity the that TRBP in its transcriptional activity in through the DNA-PK as DNA-PK a enzymatic activity in the transcriptional We also studies to the nuclear localization of TRBP in DNA-PK-deficient and and with anti-TRBP and It that the of TRBP as well as Ku complex in with cells, in which TRBP was in the However, the two of TRBP and Ku70 as shown in the of TRBP and Ku to be in in In DNA-PK-deficient cells, TRBP to have a which not the Ku70 TRBP less with Ku70 in DNA-PK-deficient Although complex in the in localization it is that a of DNA-PK activation to a of TRBP phosphorylation as well as Ku phosphorylation Mol. Cell. Biol. 15: Scholar). The absence of DNA-PK also the nuclear localization of its interaction In the of DNA-PK in TRBP in which suggests that DNA-PK be for TRBP activity in To DNA-PK TRBP transcriptional using HeLa cells, with a PI3K that inhibits that of TRBP-stimulated TRBP and TRBP the C terminus are Although also inhibits PI3K family in to DNA-PK, DNA-PK is of the in the in HeLa are with the that DNA-PK be involved in TRBP-mediated transcriptional activation. The of the interaction of TRBP with the DNA-PK complex was the of coactivator and interactions with TRBP a protein from nuclear revealed as DNA-PKcs by protein was to interact with the C terminus of TRBP. of additional interacting proteins, which identified components including the DNA-PK It the that nuclear as the also directly or with TRBP, proteins be to permit DNA-PK has been characterized as a nuclear PI3K involved in transcriptional DNA repair, recombination (12Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 503-512Google Scholar, 15Jeggo P.A. Mutat. Res. 1997; 384: 1-14Google Scholar, S.S. Mutat. Res. Scholar). The two regulatory of DNA-PK, Ku70 and identified as the in to are and to DNA Y. J. C. L. 1998; Scholar, N. L. J.M. P. 1997; Scholar, P. Cell. 1996; Scholar). Ku is a protein that DNA in a and DNA-PKcs catalytic activity. Although DNA was shown as a activator of DNA-PKcs, recent studies (17Schild-Poulter C. Pope L. Giffin W. Kochan J.C. Ngsee J.K. Traykova-Andonova M. Hache R.J. J. Biol. Chem. 2001; 276: 16848-16856Google Scholar, 19Willis D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Google that the multiple protein factors by the Ku subunit also as to DNA-PK activity. several proteins, including Oct-1, have been shown (17Schild-Poulter C. Pope L. Giffin W. Kochan J.C. Ngsee J.K. Traykova-Andonova M. Hache R.J. J. Biol. Chem. 2001; 276: 16848-16856Google to interact with Ku70 and DNA-PK with their TRBP interacts with Ku70 but not in with high interaction in stimulation of DNA-PK in the absence of DNA and The DNA-independent stimulation of DNA-PK by protein factors was also observed in proteins such as in U. Smith G.C.M. Bliss T. Werner D. Jackson S.P. Genes Dev. 1998; 12: 2188-2199Google Scholar, T. D. U. J. Cell Sci. 2002; Scholar). In addition, an antibody activates DNA-PK with TRBP that DNA-PK can be activated in the absence of It that DNA-PK can be stimulated by DNA ends O. G. J. 1999; Scholar), or by kinases S. P. S. S. A. R. D. D. Nature. 1997; Scholar, A. M. P. S. S.P. R. D. D. S. Mol. Cell. Biol. 1998; Scholar), protein interactions Ku70 be important but previously less mechanism for DNA-PK in transcriptional regulation. In to DNA-PK subunits, several proteins also in the including DNA and that have not been in factors also with TRBP directly or is a protein that the of from to nuclear is the enzyme that can be by DNA-PK T. J. J. Biol. Chem. 1998; in is in human J. J. J. Y. T. Res. 1996; Scholar, C. Smith L. Jackson S.P. 1997; and as a in D.M. M. L. A. 1998; Scholar). has also been shown to with DNA-PK and regulate including gene activation by nuclear receptors T. T. Mol. Cell. Biol. 1999; Scholar, S. T. J. Biol. Chem. 1999; 274: Scholar, M. G. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). It is that associates with TRBP through the DNA-PK In to the DNA-PK the C terminus of TRBP has been shown previously (6Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Google Scholar, L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Google Scholar), by different to interact with coactivators such as CBP/p300, DRIP and coactivator interactions a mechanism for the of TRBP as a coactivator in its C terminus. The of DNA-PK A number of phosphorylation including in the are at or by or J. Biol. Chem. 1999; 274: Scholar). However, of DNA-PK have phosphorylation RNA polymerase II can be in at that are not by (12Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 503-512Google Scholar, S.A. A. Wu C. Dynan W.S. J. Biol. Chem. Scholar). It is the of DNA-PK has with its stimulation phosphorylation in distinct we observed that the TRBP-stimulated DNA-independent phosphorylation an additional that is from of DNA-stimulated phosphorylation A was using RNA polymerase II as and as an activator S.A. A. Wu C. Dynan W.S. J. Biol. Chem. Scholar). The and phosphorylation in be and the phosphorylation might be in a regulation of DNA-PK by coactivators, which might be important for the in In to DNA repair and recombination, the of DNA-PK in is P.A. Mutat. Res. 1997; 384: 1-14Google Scholar, 19Willis D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Google Scholar, 21Sheppard H.M. Liu X. Biochim. Biophys. Acta. 2000; 1493: 41-47Google Scholar). DNA-PK phosphorylates transcriptional factors including glucocorticoid receptor and the W. H. Pope L. Hache R.J. Nature. 1996; Scholar). The or are in multiple in and the deficiency of Ku to severe in R.L. Anderson M.G. Dynan W.S. J. Biol. Chem. 1999; 274: 478-485Google Scholar). It is also that are protein factors are involved in the regulation of DNA-PK in A domain was previously shown to DNA-PK phosphorylation of RNA polymerase II A. R.L. Dynan W.S. J. Biol. Chem. S.A. A. Wu C. Dynan W.S. J. Biol. Chem. Scholar). proteins DNA-PK Ku70 (17Schild-Poulter C. Pope L. Giffin W. Kochan J.C. Ngsee J.K. Traykova-Andonova M. Hache R.J. J. Biol. Chem. 2001; 276: 16848-16856Google Scholar). A recent R.L. Huang J. Dynan W.S. J. Biol. Chem. 2001; 276: also that a protein which is not be responsible for the of with our current it that DNA-PK is targeted by multiple such as coactivators, and functions as a kinase for a variety of nuclear functions including with scid have a immune to a of recombination and double strand repair G.C.M. Jackson S.P. Genes Dev. 1999; 13: 916-934Google Scholar). revealed the of in the catalytic domain of scid are also in phosphorylation of DNA-PK such as Ku Mol. Cell. Biol. 15: Scholar). cells, TRBP phosphorylation by DNA-PK and TRBP-stimulated DNA-PK activation be the severe of TRBP-stimulated transactivation because DNA-PK be responsible for the regulation of with evidence has that of multiple genes is in scid H.M. Liu X. Biochim. Biophys. Acta. 2000; 1493: 41-47Google Scholar). It is also that the localization of nuclear including TRBP, be altered in scid DNA-PK activity might be required for the phosphorylation of a number of nuclear proteins including TRBP and Ku70. the phosphorylation nuclear in DNA-PK activity in cells, and the coactivator of TRBP. In as the for recombination, and DNA repair and factors Science. 1998; Scholar, Science. 1999; 284: Scholar), DNA-PK be of the and components. with TRBP DNA-PK in proteins in the transcriptional We are to S. at and Analysis for protein We the of mouse and from and at We Iwasaki and Cardona for