VHL: Ligase and Kinase Adaptor

Science's STKE  23 Oct 2007:
Vol. 2007, Issue 409, pp. tw381
DOI: 10.1126/stke.4092007tw381

Elevated activity of the transcription factor nuclear factor κB (NF-κB) contributes to tumorigenesis by decreasing apoptosis caused by cytokines, such as tumor necrosis factor-α (TNF-α). Clear cell renal carcinoma, a common kidney cancer, is frequently associated with mutations in or decreased expression of the gene encoding von Hippel Lindau (VHL), a protein well known for its role as an E3 ubiquitin ligase substrate recognition subunit mediating ubiquitination of the hypoxia-responsive transcription factor HIF-1α. However, several lines of evidence suggest that the role of VHL in these tumors is not related to its regulation of HIF-1α. Cells deficient in VHL activity also often exhibit elevated NF-κB signaling. Yang et al. affinity purified proteins that interacted with VHL and identified the adaptor protein CARD9, which is involved in activation of NF-κB. The interaction between CARD9 and VHL was confirmed using multiple assays--in vitro with recombinant proteins, in transfected cells, and with endogenous proteins. CARD9 consistently appeared as a doublet when assayed by electrophoresis, the top band was absent in CARD9 isolated from VHL-deficient cells, and both molecular forms collapsed into a single faster-migrating form when the samples were treated with phosphatase. VHL interacts with casein kinase 2 (CK2), and CARD9 has two CK2 consensus phosphorylation sites in the C-terminal region. CARD9 was a substrate for CK2 in vitro. Overexpression of VHL increased the interaction between CK2 and CARD9, and purified dephosphorylated CARD9 was phosphorylated by incubation with a VHL-containing cell extract. Inhibition of CK2 activity (by siRNA or with pharmacological agents) decreased the phosphorylation of CARD9. Phosphorylation of CARD9 by CK2 appeared to inhibit the ability of CARD9 to inhibit NF-κB, because cells expressing CARD9 mutants lacking the C-terminal domain or with mutated phosphorylation sites or cells in which CK2 was inhibited pharmacologically exhibited enhanced NF-κB activation. In VHL−/− renal carcinoma cells, knockdown of CARD9 with siRNA attenuated the NF-κB activity, increased apoptosis in response to TNF-α, and decreased tumor growth in a mouse xenograft assay. Thus, VHL appears to have a second important function with relevance for cancer--serving as a kinase adaptor to regulate NF-κB activity.

H. Yang, Y. A. Minamishima, Q. Yan, S. Schlisio, B. L. Ebert, X. Zhang, L. Zhang, W. Y. Kim, A. F. Olumi, W. G. Kaelin Jr., pVHL acts as an adaptor to promote the inhibitor phosphorylation of the NF-κB agonist Card9 by CK2. Mol. Cell 28, 15-27 (2007). [PubMed]