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The von Hippel-Lindau Tumor Suppressor Protein and Egl-9-Type Proline Hydroxylases Regulate the Large Subunit of RNA Polymerase II in Response to Oxidative Stress

Olga Mikhaylova,^1,# Monika L. Ignacak,^1,# Teresa J. Barankiewicz,^1, Svetlana V. Harbaugh,^1, Ying Yi,¹ Patrick H. Maxwell,³ Martin Schneider,⁴ Katie Van Geyte,⁴ Peter Carmeliet,⁴ Monica P. Revelo,^5, Michael Wyder,¹ Kenneth D. Greis,¹ Jarek Meller,² and Maria F. Czyzyk-Krzeska^1*

Department of Molecular Oncogenesis, Genome Research Institute, University of Cincinnati College of Medicine, Cincinnati, Ohio 45237-0505,¹ Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0056,² Renal Section, Hammersmith Campus, Imperial College London, London W12 0NN, United Kingdom,³ Department for Transgene Technology and Gene Therapy, VIB, and The Center for Transgene Technology and Gene Therapy, K.U. Leuven, 3000 Leuven, Belgium,⁴ Department of Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45237⁵

Received 10 July 2007/ Returned for modification 10 August 2007/ Accepted 30 January 2008

Human renal clear cell carcinoma (RCC) is frequently associated with loss of the von Hippel-Lindau (VHL) tumor suppressor (pVHL), which inhibits ubiquitylation and degradation of the alpha subunits of hypoxia-inducible transcription factor. pVHL also ubiquitylates the large subunit of RNA polymerase II, Rpb1, phosphorylated on serine 5 (Ser5) within the C-terminal domain (CTD). A hydroxylated proline 1465 within an LXXLAP motif located N-terminal to the CTD allows the interaction of Rpb1 with pVHL. Here we report that in RCC cells, pVHL regulates expression of Rpb1 and is necessary for low-grade oxidative-stress-induced recruitment of Rpb1 to the DNA-engaged fraction and for its P1465 hydroxylation, phosphorylation, and nondegradative ubiquitylation. Egln-9-type prolyl hydroxylases, PHD1 and PHD2, coimmunoprecipitated with Rpb1 in the chromatin fraction of VHL⁺ RCC cells in response to oxidative stress, and PHD1 was necessary for P1465 hydroxylation while PHD2 had an inhibitory effect. P1465 hydroxylation was required for oxidative-stress-induced Ser5 phosphorylation of Rpb1. Importantly, overexpression of wild-type Rpb1 stimulated formation of kidney tumors by VHL⁺ cells, and this effect was abolished by P1465A mutation of Rpb1. These data indicate that through this novel pathway involving P1465 hydroxylation and Ser5 phosphorylation of Rbp1, pVHL may regulate tumor growth.

Published ahead of print on 19 February 2008.

^# Both authors contributed equally to this work.

Present address: Cinna Health Products, Molecular Research Center, Inc., 5645 Montgomery Rd., Cincinnati, OH 45212.

Present address: Air Force Research Laboratory/Human Effectiveness Biosciences & Protection Division, Applied Biotechnology Branch, 2729 R Street, Bldg. 837, Wright-Patterson AFB, Dayton, OH 45433-5707.

Present address: Department of Pathology, University of Utah, 15 North Medical Drive East, Salt Lake City, UT 84112.