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

Abstract

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 hypoxiainducible 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 oxidativestress-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.pVHL is the main tumor suppressor for which loss of activity is causatively linked to renal clear cell carcinoma (RCC), the most malignant and common form of kidney cancer. The VHL gene is mutated or hypermethylated in about 40 to 70% of sporadic RCC. Hereditary loss of pVHL function in von Hippel-Lindau (VHL) disease also results in highly vascularized RCC and capillary tumors of other organs, such as hemangioblastoma of the central nervous system and retinal angioma (19,20). A body of experimental evidence, based on a subcutaneous xenograft model system, supports the idea that accumulation of the alpha subunit of the hypoxia-inducible transcription factor (HIF) HIF-2␣ and induction of HIF target gene products, resulting from the loss of pVHL-mediated ubiquitylation, are necessary and sufficient to promote growth of RCC tumors (22,23). HIF activation has also been demonstrated as an early tumorigenesis event in kidneys from VHL patients (32). Biochemically, pVHL is the substrate-recognizing component of a multiprotein E3 ubiquitin ligase complex containing elongins C and B, Cullin 2, and the RING-H2 finger protein Rbx-1 (for a review, see reference 19). pVHL-dependent ubiquitylation of HIF-␣s is preceded by hydroxylation of conserved proline residues located within LXXLAP motifs (16,17) by the O 2 -, Fe(II)-, and oxyglutarate-regulated Egl-9-type proline hydroxylases (PHDs) (7). Thus, an important aspect of pVHL's tumor suppressing activity is the prevention of HIF-␣ accumulation, which in turn suppresses induction of the HIF target genes. Clearly, however, pVHL activity is not limited to regulation of HIFs. Other targets of pVHL-associated E...

Natural Product Triptolide Mediates Cancer Cell Death by Triggering CDK7-Dependent Degradation of RNA Polymerase II

“…2D). The von Hippel-Lindau tumor suppressor protein VHL and the tumor suppressor BRCA1 have been reported to ubiquitinate Rpb1 (11,24). However, the triptolideinduced degradation of Rpb1 seemed to be independent of either of them because Rpb1 degraded apparently in VHLdeficient 786-O renal cancer cells and BRCA1-deficient MDA-MB-436 and HCC1937 breast cancer cells when exposed to triptolide (Supplementary Fig.…”

Involvement of Net and Hif1α in Distinct yet Intricately Linked Hypoxia-induced Signaling Pathways

“…PHDs hydroxylate a number of proteins. PHD1 is involved in proline hydroxylation of RbpI, the large subunit of RNA polymerase II (28). The kinase activity of IB kinase ␤ may be inhibited by hydroxylation by PHD1 and PHD2 (29).…”

“…PHD3 binds to myogenin and increases its stability (31) and also induces oxygen-dependent degradation of ATF4 (30). PHD1 and PHD2 co-immunoprecipitate with Rpb1 in response to oxidative stress, but PHD1 is necessary for RbpI hydroxylation and consequent degradation, whereas PHD2 has an inhibitory effect on this process (28).…”

HIF Prolyl-4-hydroxylase Interacting Proteins: Consequences for Drug Targeting

“…Upon hyperphosphorylation, Rpb1 is bound by VHL and targeted for ubiquitinylation and subsequent proteasomal destruction [100]. The PHD1 and PHD2 oxygen sensors have been shown to bind Rpb1 [101]. As demonstrated by immunoblotting using an antibody derived against a synthetic hydroxylated Rpb1 peptide, but not by mass spectrometry, PHD1 hydroxylates the prolyl residue of the LXXLAP motif of Rpb1 under oxidative stress conditions.…”