**5. Function of pVHL**

Following identification and cloning of the VHL tumor suppressor gene, its sequence did not immediately suggest a function for the protein. However, early immunoprecipitation experiments indicated that pVHL forms a complex with elongin B and elongin C [34]; cullin 2, a member of the Cdc53 family of proteins [35]; and the RING-box protein Rbx1 [36, 37]. Importantly, the binding of pVHL to elongins B and C could be blocked by specific ccRCC-associated mutations in the VHL gene, strongly suggesting that these two proteins contribute to the tumor suppressor activity of VHL [34]. The subsequent identification of mutations in the TCEB1 gene, which encodes elongin C, in ccRCC tumors that have wild-type VHL further emphasizes the importance of this complex in ccRCC formation [25, 32, 38].

Elongins B and C, cullin 2 and Rbx1 are all components of an E3-ligase complex that adds polyubiquitin chains to specific proteins and thus targets them for degradation by the proteasome [39, 40]. This suggested that pVHL might act as the recognition component of a pVHL ligase complex. In a separate line of work, dysregulation of the hypoxia-inducible factor (HIF) transcription factors had been identified in VHL-defective ccRCC cells [41]. It was subsequently shown that pVHL directly interacted with HIF, leading to polyubiquitination and subsequent proteasomal degradation of its alpha-subunits [42, 43]. Again, the pVHL-HIF interaction could be blocked by specific ccRCC-associated mutations in VHL, leading to overexpression of HIF and underlining the importance of HIF in the development of ccRCC [42]. Importantly, this interaction was not only altered by pathogenic VHL mutations but was also regulated in an oxygen-dependent manner [44, 45]. This indicated that the pVHL-HIF interaction was integral to the mechanism of cellular oxygen-sensing.

The central role of HIF in ccRCC biology has been further underscored in numerous studies. In particular, in xenograft and transgenic mouse models of

#### *HIF Pathways in Clear Cell Renal Cancer DOI: http://dx.doi.org/10.5772/intechopen.96539*

VHL-defective ccRCC, tumor growth is dependent upon the presence of HIF [46–51]. Specifically, tumor growth is dependent on the DNA binding activity of HIF, which is required for it to transactivate its target genes [48]. Thus, HIF and its associated transcriptional response are key mediators of tumorigenesis in ccRCC.

In addition to HIF, pVHL can interact with a number of other proteins, although the biological significance of these interactions is incompletely understood [52]. Some of these interactions can lead to ubiquitination of other proteins aside from HIF. For example, pVHL has been reported to interact with and ubiquitinate two de-ubiquitinase enzymes (VDU1 and VDU2) leading to their degradation [53, 54]. In turn, VDU2 but not VDU1 may de-ubiquitinate HIF-1α, potentially providing another level of control to the HIF pathway [55]. In addition, pVHL can bind to and ubiquitinate two subunits of the RNA polymerase 2 complex, POL2RA (RPB1) and POL2RG (RPB7) [56–58]. Importantly, the pVHL-RPB1 interaction was shown to be oxygen-dependent, involving a mechanism similar to that regulating pVHL interaction with HIF [58]. Similarly, the erythropoietin receptor (EPOR), which lies downstream of the canonical HIF-target gene, erythropoietin (EPO), may also be bound and ubiquinated by pVHL in response to oxygen [59]. pVHL can also interact with and ubiquitinate the regulatory domain of atypical protein kinase C (PKC), a serine–threonine kinase that has roles in cell polarity and cell growth, leading to its degradation [60–62]. Again, this interaction may be regulated by oxygen [62]. Similarly, an oxygen-dependent interaction between pVHL and sprouty homolog 2 (SPRY2), which modulates the action of receptor tyrosine kinases, has been reported [63]. Taken together, these findings indicate that pVHL may contribute to oxygen signaling more extensively than simply through regulation of HIF.

pVHL may also play a non-canonical role in extra-cellular matrix assembly, independently of HIF. Specifically, pVHL can interact directly with the alpha-chain of collagen 4 and is important in maintaining the collagen 4 network [64, 65]. This molecule is heavily hydroxylated, and as will be explained below, hydroxylation is important in the recognition of HIF-alpha (as well as collagen 4) by pVHL. Importantly, this interaction can be dissociated by ccRCC-associated VHL mutations. Similarly, fibronectin co-immunoprecipitates with pVHL, and consistently the extracellular fibronectin matrix produced by VHL-defective ccRCC cells is also disrupted [66]. However, the contribution of this phenomenon to cellular oxygen sensing and ccRCC tumorigenesis is still unclear.
