The Cyclin-dependent Kinase-activating Kinase (CAK) Assembly Factor, MAT1, Targets and Enhances CAK Activity on the POU Domains of Octamer Transcription Factors*
- From the ‡Laboratory of Biochemistry and Molecular Biology and the ¶Laboratory of Molecular Biology, The Rockefeller University, New York, New York 10021 and the **Derald H. Ruttenberg Cancer Center, Mount Sinai Medical Center, New York, New York 10029-6574
Abstract
Octamer binding transcription factors (Oct factors) play important roles in activation of transcription of various genes but, in some cases, require cofactors that interact with the DNA binding (POU) domain. In the present study, a yeast two-hybrid screen with the Oct-1 POU domain as a bait identified MAT1 as a POU domain-binding protein. MAT1 is known to be required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK), which is functionally associated with the general transcription factor IIH (TFIIH). Further analyses showed that MAT1 interacts with POU domains of Oct-1, Oct-2, and Oct-3 in vitro in a DNA-independent manner. MAT1-containing TFIIH was also shown to interact with POU domains of Oct-1 and Oct-2. MAT1 is shown to enhance the ability of a recombinant CDK7-cyclin H complex (bipartite CAK) to phosphorylate isolated POU domains, intact Oct-1, and the C-terminal domain of RNA polymerase II, but not the originally defined substrate, CDK2. Phosphopeptide mapping indicates that the site (Ser385) of a mitosis-specific phosphorylation that inhibits Oct-1 binding to DNA is not phosphorylated by CAK. However, one CAK-phosphorylated phosphopeptide comigrates with a Cdc2-phosphorylated phosphopeptide previously shown to be mitosis-specific, suggesting that, in vitro, CAK is able to phosphorylate at least one site that is also phosphorylated in vivo. These results suggest (i) that interactions between POU domains and MAT1 can target CAK to Oct factors and result in their phosphorylation, (ii) that MAT1 not only functions as a CAK assembly factor but also acts to alter the spectrum of CAK substrates, and (iii) that a POU-MAT1 interaction may play a role in the recruitment of TFIIH to the preinitiation complex or in subsequent initiation and elongation reactions.
Footnotes
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↵* This work was supported by U.S. Public Health Service Grants CA42567 and AI2732 (to R. G. R.), the Life and Health Insurance Medical Research Fund (to Z.-Q. P.), and the New York Community Trust (to Z.-Q. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Supported in part by a fellowship from the Toyobo Biotechnology Foundation, in part by a fellowship from the International Human Frontier Science Program, and in part by a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad. Present address: Dept. of Microbiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.
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↵‖ Supported in part by the Charles Revson Foundation. Present address: Dept. of Molecular and Cellular Biology, House Ear Institute, 2100 W. Third St., Los Angeles, CA 90057.
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↵‡ Supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Present address: Dept. of Molecular Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411, Japan.
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↵§§ To whom correspondence should be addressed. Tel.: 212-327-7600; Fax: 212-327-7949. E-mail: roeder{at}rockvax.rockefeller.edu.
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↵1 The abbreviations used are: HSV, herpes simplex virus; TFIIE, TFIIF, and TFIIH, transcription factor IIE, IIF, and IIH, respectively; CDK, cyclin-dependent kinase; CAK, CDK-activating kinase; CTD, C-terminal domain; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis: PMSF, phenylmethylsulfonyl fluoride; TBP, TATA box-binding protein; BSA, bovine serum albumin; PTF, proximal sequence element-binding transcription factor.
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- Received June 6, 1997.
- Revision received August 24, 1997.
