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.
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.
Mikhaylova
Olga
O
Department of Molecular Oncogenesis, Genome Research Institute, University of Cincinnati, Cincinnati, OH 45237-0505, USA. maria.czyzykkrzeska@uc.edu
Ignacak
Monika L
ML
Barankiewicz
Teresa J
TJ
Harbaugh
Svetlana V
SV
Yi
Ying
Y
Maxwell
Patrick H
PH
Schneider
Martin
M
Van Geyte
Katie
K
Carmeliet
Peter
P
Revelo
Monica P
MP
Wyder
Michael
M
Greis
Kenneth D
KD
Meller
Jarek
J
Czyzyk-Krzeska
Maria F
MF
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D017874:Immediate-Early Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018384:Oxidative Stress; D010768:Phosphoserine / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D054840:Retinol-Binding Proteins, Cellular / Q000378:metabolism; D054875:Ubiquitination; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D021122:Protein Subunits; C518456:RBP1 protein, human; D054840:Retinol-Binding Proteins, Cellular; D010768:Phosphoserine; C522721:PHD1 protein, mouse; D011393:Procollagen-Proline Dioxygenase; C490189:Egln1 protein, mouse; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D012319:RNA Polymerase II