We found that pVHL associates with the NF-κB agonist Card9, which is reported to act in concert with Bcl10 upstream of NF-κB (Bertin et al., 2000
; Gross et al., 2006
). pVHL does not direct the destruction of Card9. Instead pVHL facilitates the phosphorylation of Card9 by CK2 on C-terminal residues that regulate Card9's ability to activate NF-κB. Card9 mutants lacking these phosphorylation sites are NF-κB superagonists while elimination of Card9 in VHL
−/− renal carcinoma cells normalizes NF-κB activity and promotes apoptosis and suppresses tumorigenesis. In contrast, deregulation of HIF does not, based on our genetic experiments, appear to play a prominent role in promoting NF-κB activity when pVHL function is compromised.
Our biochemical studies suggest that pVHL, bound to CK2, brings CK2 into proximity with Card9 and thereby increases the local concentration of the substrate. Therefore pVHL can serve as an adaptor for a kinase, CK2, as well as for the ubiquitin conjugating machinery that directs the polyubiquitination of HIFα subunits (). Phosphorylation of the Card9 C-terminus by CK2 might promote an inhibitory intra or intermolecular interaction or prevent the binding of Card9 to an accessory protein required for its activity. In this regard, we have obtained preliminary data that Card9 can interact with NEMO (also called IKKγ)(data not shown), which might conceivably play a role here.
We observed decreased Card9 phosphorylation, and increased NF-κB activity, in mouse livers after VHL
inactivation, indicating that regulation of Card9 and NF-κB by pVHL occurs under physiological conditions. To date the role of Card9 in NF-κB activation has been most clearly documented during the myeloid immune response to particular infections (Colonna, 2007
). Clearly it will be important to determine the physiological and pathological signals that govern the interaction of pVHL and Card9.
mutations play a causal role in renal carcinomas, which are notoriously resistant to chemotherapy, radiotherapy, and proapoptotic cytokines. Increased NF-κB activity, which has been documented in renal carcinomas before (Oya et al., 2001
; Oya et al., 2003
), is likely to play a role here (Karin et al., 2002
; Nakanishi and Toi, 2005
). Indeed, there is already preclinical evidence that suppression of NF-κB in renal carcinomas with interferon alpha, which is widely used in the treatment of this disease, can increase their sensitivity to chemotherapeutic agents or other cytokines (Bukowski, 2000
; Steiner et al., 2001
). Further elucidation of Card9 regulation and function might reveal additional opportunities to manipulate NF-κB activity in tumor cells.
All renal carcinoma-associated missense VHL
mutants are defective for the regulation of HIF but some preserve the ability to regulate Card9 (data not shown). This is consistent with HIF playing a dominant role in VHL
−/− renal tumors. Nonetheless, many of the VHL
mutations found in renal carcinomas are deletion, truncation, or nonsense mutations that would predictably eliminate pVHL function (Gnarra et al., 1996
). Our data suggests that, in this setting, deregulation of Card9 would confer a more aggressive phenotype and resistance to therapy.
A number of HIF-responsive genes implicated in renal carcinogenesis, including VEGF, cyclin D1, and MMP2/9, are also NF-κB targets, suggesting that HIF and NF-κB might act cooperatively (Karin et al., 2002
; Nakanishi and Toi, 2005
). Agents that inhibit HIF-responsive growth factors such as VEGF have demonstrated activity in kidney cancer clinical trials (Kim and Kaelin, 2004
) and might now be rationally combined with agents that modulate NF-κB function.