Mutations of von Hippel–Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B–elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.

The suppressor of cytokine signaling (SOCS) box consists of the BC box and the cullin 5 (Cul5) box, which interact with Elongin BC and Cul5, respectively. SOCS box-containing proteins have ubiquitin ligase activity mediated by the formation of a complex with the scaffold protein Cul5 and the RING domain protein Rbx2, and are thereby members of the cullin RING ligase superfamily. Cul5-type ubiquitin ligases have a variety of substrates that are targeted for polyubiquitination and proteasomal degradation. Here, we review the current knowledge on the identification of Cul5 and the regulation of its expression, as well as the signaling pathways regulated by Cul5 and how viruses highjack the Cul5 system to overcome antiviral responses.

The ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly induced by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. However, how ISGylation contributes to innate immune responses is not clear. The dsRNA-dependent protein kinase (PKR) inhibits translation by phosphorylating eIF2α to exert its anti-viral effect. ISG15 and PKR are induced by interferon, suggesting that a relationship exists between ISGylation and translational regulation. Here, we report that PKR is ISGylated at lysines 69 and 159. ISG15-modified PKR is active in the absence of virus infection and phosphorylates eIF2α to down-regulate protein translation. The present study describes a novel pathway for the activation of PKR and the regulation of protein translation.

The Elongin complex was originally identified as a positive regulator of RNA polymerase II and is composed of a transcriptionally active subunit (A) and two regulatory subunits (B and C). The Elongin BC complex enhances the transcriptional activity of Elongin A. “Classical” SOCS box-containing proteins interact with the Elongin BC complex and have ubiquitin ligase activity. They also interact with the scaffold protein Cullin (Cul) and the RING domain protein Rbx and thereby are members of the Cullin RING ligase (CRL) superfamily. The Elongin BC complex acts as an adaptor connecting Cul and SOCS box proteins. Recently, it was demonstrated that classical SOCS box proteins can be further divided into two groups, Cul2- and Cul5-type proteins. The classical SOCS box-containing protein pVHL is now classified as a Cul2-type protein. The Elongin BC complex containing CRL family is now considered two distinct protein assemblies, which play an important role in regulating a variety of cellular processes such as tumorigenesis, signal transduction, cell motility, and differentiation.

Dia2 is an F‐box protein, which is involved in the regulation of DNA replication in the budding yeast Saccharomyces cerevisiae . The function of Dia2, however, remains largely unknown. In this study, we report that Dia2 is associated with the replication fork and regulates replication fork progression. Using modified yeast two‐hybrid screening, we have identified components of the replisome (Mrc1, Ctf4 and Mcm2), as Dia2‐binding proteins. Mrc1 and Ctf4 were ubiquitinated by SCFDia2 both in vivo and in vitro . Domain analysis of Dia2 revealed that the leucine‐rich repeat motif was indispensable for the regulation of replisome progression, whereas the tetratricopeptide repeat (TPR) motif was involved in the interaction with replisome components. In addition, the TPR motif was shown to be involved in Dia2 stability; deleting the TPR stabilized Dia2, mimicking the effect of DNA damage. ChIP‐on‐chip analysis illustrated that Dia2 localizes to the replication fork and regulates fork progression on hydroxyurea treatment. These results demonstrate that Dia2 is involved in the regulation of replisome activity through a direct interaction with replisome components.