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Viruses depend upon the host cell for manufacturing components of progeny virions. To mitigate the inextricable dependence on host cell protein synthesis, viruses can modulate protein synthesis through a variety of mechanisms. We report that the viral protein kinase (vPK) encoded by open reading frame 36 (ORF36) of Kaposi's sarcoma-associated herpesvirus (KSHV) enhances protein synthesis by mimicking the function of the cellular protein S6 kinase (S6KB1). Similar to S6KB1, vPK phosphorylates the ribosomal S6 protein and up-regulates global protein synthesis. vPK also augments cellular proliferation and anchorage-independent growth. Furthermore, we report that both vPK and S6KB1 phosphorylate the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) and that both kinases promote endothelial capillary tubule formation.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.04.045 Byline: Janna E. Slessareva (1), Sheri M. Routt (2), Brenda Temple (1), Vytas A. Bankaitis (2), Henrik G. Dohlman (1) Abstract: In the yeast Saccharomyces cerevisiae, the G protein [beta][gamma] subunits are essential for pheromone signaling. The G[alpha] subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1.sup.Q323L mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein [beta] subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-G[beta] subunit assembly and function at the endosome rather than at the plasma membrane. Author Affiliation: (1) Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA (2) Department of Cell Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Article History: Received 4 October 2005; Revised 6 February 2006; Accepted 28 April 2006 Article Note: (miscellaneous) Published: July 13, 2006

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)‐conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N‐terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix–turn–helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase‐Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3‐dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2‐E3‐mediated polyubiquitination. Synopsis: Ubiquitin (Ub)‐conjugating E2 enzymes control substrate ubiquitination by dictating the linkage specificity of polyubiquitin chains and interacting with E3 ligases via specific surfaces, but the contribution of additional surface regions is unclear. Here, variable ubiquitin interactions of the E2 helix‐turn‐helix (HTH) motif are found to differentially modulate polyubiquitination in diverse E2s. Phylogenetic analysis reveals the HTH motif as a variable region, yet harboring functionally important and conserved charged residues within the distinct E2 family members.Ubiquitin binding to the E2HTH can be repurposed by different E2s, such as cell cycle regulators UBE2R/CDC34 and UBE2S, and tune intrinsic E2 activity.The positively charged UBE2R‐HTH facilitates acceptor ubiquitin recruitment and efficient chain elongation by dictating the dynamics of the gating loop near the enzyme active site.The negatively charged UBE2S‐HTH interacts with the donor Ub, influences the "open" and "closed" states of the UBE2S~Ub conjugate, and enhances ubiquitination by the E3 APC/C by reducing the conformation space of the "open" states. [ABSTRACT FROM AUTHOR]

Abstract Background The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization. Results By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIVMAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface. Conclusions We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.

An x-ray crystal structure and function of the Vps15 WD domain that revealed a seven-bladed propeller resembling that of typical G[beta] subunits are reported. The results revealed that the Vps15 G[beta]-like domain served as a scaffold to assemble Gpa1 and Atg14, while the kinase and intermediate domains are required for proper signaling at the endosome.

The study describes the solution structures of the DNA dodecamer duplexes with and without a cisplatin 1,2-d(CG) intrastrand cross-links, comparing them with the oxaliplatin-CG structure with the same DNA duplex in the same order. The results show that the various conformational differences found in the structures are attributed to the ability of the DNA repair proteins, DNA binding proteins and DNA polymerases to discriminate between the cisplatin and oxaliplatin adducts.