The research presented here opens several avenues regarding novel activities of pVHL and Egl-9-type proline hydroxylases that may be fundamental to their function. On the basis of these results, we propose a mechanism whereby pVHL and PHDs modulate gene expression by regulating RNAPII activity. We further propose that this mechanism is independent of proline hydroxylation and pVHL-dependent ubiquitylation of HIF and that the effects of pVHL and PHDs on RNAPII are critical factors in the regulation of tumorigenesis.
We have shown that reconstitution of pVHL in RCC cells stimulates total steady-state levels of Rpb1 which is Ser5 phosphorylated and P1465 hydroxylated. Furthermore, we have demonstrated that pVHL is necessary for oxidative-stress-stimulated recruitment of Rpb1 to the DNA and P1465 hydroxylation, CTD Ser5 phosphorylation, and ubiquitylation of the fraction of Rpb1 that is engaged on the DNA. Interestingly, this ubiquitylation does not cause Rpb1 degradation but rather correlates with accumulation of engaged Rpb1. While the necessity of P1465 hydroxylation for the subsequent pVHL-dependent ubiquitylation of Rpb1 has been recognized before (25
), in the present work we have significantly expanded our understanding of the role of pVHL by showing that pVHL also regulates P1465 hydroxylation of the DNA-engaged Rpb1 and that this hydroxylation involves the PHDs. One of the most important and novel findings of our study is that this pVHL-dependent P1465 hydroxylation of Rpb1 is required for oxidative-stress-induced Ser5 phosphorylation of Rpb1 engaged on the DNA. Thus, it can be concluded that pVHL regulates the amount of Rpb1 phosphorylated on Ser5 engaged on the DNA under oxidative-stress conditions. The finding that a CTD-associated E3 ubiquitin ligase regulates CTD phosphorylation is independently supported by the recent demonstration that the yeast Rpb1 E3 ligase, Rsp5, stimulates CTD phosphorylation in an in vitro assay (35
). We also found that overexpression of wild-type Rpb1 stimulates the oncogenic potential of VHL+
cells and that this effect can be inhibited by P1465A mutation of Rpb1. This finding implicates the pVHL-mediated hydroxylation of Rpb1 as an oncogenic mechanism and is supported by the data from human RCCs. Although the number of samples studied is small and potential correlations with tumor grade or stage and the status of pVHL are not yet established, 50% of human RCCs demonstrated an increase in P1465. These data are consistent with results published by other laboratories in which under some circumstances, such as in teratomas or fibrosarcomas derived from VHL+/−
cells, the presence of pVHL has tumor-promoting activities (30
). The potential growth-promoting activity of pVHL is also supported by the role of pVHL in embryonic development (11
) and by clinical evidence that the prognosis is worse for RCC associated with a wild-type VHL gene than for tumors with mutated pVHL, although the mechanism of this phenomenon is not understood (44
A direct evaluation of the role of pVHL-dependent modifications of Rpb1 in the transcriptional or posttranscriptional regulation of specific genes is likely to be very complex and will require further studies. However, our analysis of protein changes occurring in response to H2
treatment, as the final readout correlating with the pVHL-dependent modification of Rpb1, indicates an overall stimulatory effect of oxidative stress on subsets of proteins in VHL+
cells, consistent with increased levels of a potentially active fraction of Rpb1 on the DNA. It is striking that the majority of proteins explicitly identified in our proteomic screen, including EIF5, CPNE1, and Caprin 1, play a role in the transport and regulation of translation of specific mRNAs. It is particularly interesting that Caprin 1 binds G3BP1 (RasGAP SH3 domain binding protein), which associates with mRNAs for two Rpb1 kinases, cdk7 and cdk9, increasing levels of the cdk7 protein while decreasing those of cdk9 (29
). Potential effects of pVHL in the regulation of protein translation have a precedent in the reported role of pVHL in the stimulation of p53 translation (9
). The effects regarding induction of cytoskeletal proteins are also consistent with the previously reported role of pVHL in binding and stabilizing microtubules (14
). Our study potentially expands this role into regulation of expression levels of vimentin and dynactin, at least in response to some stressors.
Perhaps one of our most interesting observations is that proline hydroxylases have a strong presence and activity within the chromatin fraction and that PHD1 and PHD2 bind to Rpb1 in response to oxidative stress in a pVHL-dependent manner. The biochemical nature of these interactions and the role of pVHL in the formation of such a complex will be determined in future studies. Because in the in vitro hydroxylation experiments using Rpb1 peptides and purified enzymes none of the PHDs hydroxylates Rpb1 (data not shown), it is very likely that PHDs and Rpb1 are part of a much bigger protein complex. The formation of protein complexes in which individual PHDs homodimerize and heterodimerize has been reported in the context of PHD3 (43
). We identified PHD1 as the enzyme necessary for hydroxylation of Rpb1 and found that its knockdown inhibited Rpb1 hydroxylation in response to oxidative stress. This is consistent with previous reports showing expression of PHD1 predominantly in the nuclei of cells in different organs (36
) and that PHD2 and PHD3 but not PHD1 are the main HIF-α hydroxylases (1
). The mechanism by which oxidative stress induces PHD1 activity is currently not understood but clearly must differ from the inhibition of PHD2 activity by oxidative stress reported in the case of HIF-α hydroxylation (3
). This difference is potentially due to oxidative-stress-induced PHD1 activity taking place in the context of a multiprotein complex associated with RNAPII.
Surprisingly, we found that a knockdown of PHD2 stimulated constitutive hydroxylation and Ser5 phosphorylation of Rpb1, which occurred in a pVHL-dependent manner. This was accompanied by a strong induction of PHD3 in the chromatin fraction, and indeed, Rpb1 hydroxylation under these conditions required PHD3 activity. It remains to be determined whether the effect of PHD2 knockout is mediated through the loss of physical interaction of PHD2 with PHD1 and Rpb1. The PHD2 knockout may allow more PHD1 and PHD3 binding to Rpb1 and stronger hydroxylating activity. In addition, knockout of PHD2 induces HIF-2α (1
), which in turn may strongly stimulate PHD3 expression and its chromatin association. This raises the interesting possibility that HIF might also regulate P1465 hydroxylation and Ser5 phosphorylation of Rpb1, thus controlling gene expression at a different level in addition to stimulating transcription initiation.
Several different kinases phosphorylate Ser5, of which cdk7, cdk8, cdk9, and ERKs are the most thoroughly investigated (15
). Ser5 is also subject to dephosphorylation by different phosphatases that when inhibited may affect steady-state levels of Ser5 phosphorylation (52
) and gene expression. The roles of these individual kinases in the pVHL-dependent phosphorylation of Rpb1 remain to be determined. We do not expect ERKs to be involved in this regulation, because the phosphorylation of Ser5 resulting from their activity occurs on soluble Rpb1 and functions as an adaptation to severe oxidative stress to prevent reentry of Rpb1 molecules into transcription (47
). However, it is important to note that with our experimental model system, the effect of oxidative stress on the pVHL-dependent induction of P1465 hydroxylation, Ser5 phosphorylation, and ubiquitylation of Rpb1 occurs on engaged Rpb1 and is long lasting, starting immediately after exposure to H2
but reaching a plateau at approximately 4 h after stimulation and then persisting for several additional hours. This regulation is clearly different from previously reported responses to higher doses (0.25 to 10 mM) of hydrogen peroxide (15
; M. L. Ignacak and M. F. Czyzyk-Krzeska, unpublished results), where fast (within minutes of exposure) and pVHL-independent phosphorylation of Rpb1 Ser5 phosphorylation occurs and where such doses of H2
result in significant cell death (15
). We chose low doses of H2
, in the range between 10 and 50 μM, to mimic the subtle changes in intracellular H2
concentrations that may occur during physiological or pathophysiological variations in the endogenous metabolism but which do not lead to cell death.
Unlike the effects of pVHL on HIF-αs, we did not detect any apparent degradation of Rpb1 in response to hydroxylation/ubiquitylation. These conclusions differ from the previously suggested role of pVHL-mediated polyubiquitylation in the degradation of Rpb1 as an adaptation to UV-induced DNA damage in PC12 cells (25
). Thus, pVHL-mediated ubiquitylation of Rpb1 may have different regulatory activities toward Rpb1 that function in different contexts and can either lead to or prevent its degradation. pVHL not only directly targets proteins for ubiquitylation but also ubiquitylates and targets for degradation the deubiquitylating enzymes (28
), a process which, in a secondary manner, may affect ubiquitylation of some currently unidentified substrates. Recent evidence has demonstrated that pVHL can induce assembly of polyubiquitin chains on the HIF-1α substrate, not only through ubiquitin K48 but also through other lysines (37
). This strongly supports the possibility that the role of pVHL in protein ubiquitylation may extend beyond targeting for proteasomal degradation. The differences in pVHL-mediated effects on constitutive levels of Ser5-phosphorylated Rpb1 in PC12 and RCC cells may also result from tissue-specific characteristics. There is support for this idea in the literature. For example, a lack of correlation between pVHL-dependent tumor formation and HIF accumulation has been described in the case of type 2C VHL disease, where certain mutations encoded within the VHL gene, such as V84L and L188V, cause pheochromocytoma tumors without promoting RCC (6
). However, the effects of oxidative stress on P1465 hydroxylation, Ser5 phosphorylation, and ubiquitylation of Rpb1 were very similar in PC12 (data not shown) and RCC cells, an indication that this is a more general mechanism of regulation.
In conclusion, our data demonstrate that pVHL and PHDs regulate Rpb1 in an HIF-independent manner. By stimulating hydroxylation of P1465, phosphorylation of CTD Ser5, and nondegradative ubiquitylation, pVHL and PHDs could fine-tune RNAPII activities and gene expression. These effects on RNAPII could then participate in the tumor-suppressing or growth-promoting activities of pVHL in renal cancer.