Pietrek M, Brinkmann MM, Glowacka I, Enlund A, Havemeier A, Dittrich-Breiholz O, Kracht M, Lewitzky M, Saksela K, Feller SM, Schulz TF

Pietrek M, Brinkmann MM, Glowacka I, Enlund A, Havemeier A, Dittrich-Breiholz O, Kracht M, Lewitzky M, Saksela K, Feller SM, Schulz TF. from KSHV-infected endothelial cells and recognized associated proteins by label-free quantitative mass spectrometry. Cellular proteins interacting with pK15 point to previously unappreciated cellular processes, such as the endocytic pathway, that could be involved in the function of pK15. We found that the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2, which is usually involved in the endocytosis of activated receptor tyrosine kinases and their signaling from intracellular organelles, interacts and colocalizes with pK15 in vesicular Rabbit Polyclonal to SCTR structures abundant in the perinuclear area. Further functional analysis revealed that PI3K-C2 contributes to the pK15-dependent phosphorylation of PLC1 and Erk1/2. PI3K-C2 also plays a role in SU11274 KSHV lytic replication, as evidenced by the reduced expression of the viral lytic genes K-bZIP and ORF45 as well as the reduced release of infectious computer virus in PI3K-C2-depleted KSHV-infected endothelial cells. Taken together, our results suggest a role of the cellular PI3K-C2 protein in the functional properties of the KSHV pK15 protein. IMPORTANCE The nonstructural membrane protein encoded by open reading frame K15 of Kaposi’s sarcoma-associated herpesvirus (KSHV) (HHV8) activates several intracellular signaling pathways that contribute to the angiogenic properties of KSHV in endothelial cells and SU11274 to its reactivation from latency. A detailed understanding of how pK15 activates these intracellular signaling pathways is usually a prerequisite for targeting these processes specifically in KSHV-infected cells. By identifying pK15-associated cellular proteins using a combination of immunoprecipitation and mass spectrometry, we provide evidence that pK15-dependent signaling may occur from intracellular vesicles and rely on the endocytotic machinery. Specifically, a class II PI3K, PI3K-C2, is usually recruited by pK15 and involved in pK15-dependent intracellular signaling and viral reactivation from latency. These findings are of importance for future intervention strategies that aim to disrupt the activation of intracellular signaling by pK15 in order to antagonize KSHV productive replication and tumorigenesis. (25, 35, 42). Taken together, the above-described observations show that KSHV pK15 can recruit several cellular proteins that play an important role in the activation of angiogenic and inflammatory pathways and that might provide a survival advantage to the infected cell (19, 22, 24,C26, 40, 41). In this study, we have used immunoprecipitation followed by mass spectrometry (MS) SU11274 in order to identify additional pK15-interacting partners and elucidate their functions. We found that the -isoform of the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2, which is usually involved in receptor endocytosis and signaling activation following the binding of cognate ligands, is usually a novel pK15-interacting partner. We show that PI3K-C2 colocalizes with pK15 in intracellular vesicles. Furthermore, much like its role in receptor-mediated signaling, PI3K-C2 plays a role in the activation of pK15-mediated signaling as well as KSHV lytic reactivation in infected endothelial cells. Taken together, our results here demonstrate the importance of PI3K-C2 in pK15-mediated signaling and point to a role for this nonstructural viral membrane protein in mediating signaling from intracellular membrane vesicles as a key step in computer virus reactivation. RESULTS Identification of K15-interacting proteins by label-free quantitative proteomics. Endothelial cells are among the targets of KSHV contamination (4, 7,C9) that play an important role in the pathogenesis of KS. The KSHV K15 protein, which we recently showed to be expressed in KS lesions (19), contributes to the invasiveness and angiogenic properties of infected endothelial cells in culture and to the formation of endothelial spindle cells as well as KSHV lytic replication (19, 24, 26). In order to identify new interacting partners of the pK15 protein, a conditionally immortalized endothelial cell collection (HuARLT2), stably infected with a recombinant KSHV (HuARLT2-rKSHV), was used (see Materials and Methods). A rat monoclonal antibody to pK15, 6E7, directed against the PPLP SH3-binding motif in the cytoplasmic domain name of pK15, was covalently cross-linked to protein A/G beads by using the pierce cross-link immunoprecipitation (IP) kit, which enables efficient antigen immunoprecipitation with less IgG contamination from your antibody. 6E7-coated beads were used to immunoprecipitate endogenous pK15 from HuARLT2-rKSHV cells, which were either left untreated or treated with a cocktail of a baculovirus encoding KSHV RTA and sodium butyrate (SB) to induce the lytic replication cycle (Fig. 1A). Cellular lysates were also prepared from uninfected parental HuARLT2 cells that had been treated similarly and used in parallel as a control. The efficiency of the IP was assessed by Western blot analysis of a small aliquot from each preparation (Fig. 1B). After scaling up the experiment to 5 108 KSHV-infected and uninfected HuARLT2 cells for each immunoprecipitation, the immunoprecipitated proteins were resolved on SDS-PAGE gels SU11274 and visualized by staining with Coomassie amazing blue dye. Open in a separate windows FIG 1.