re transfected with Flag tagged HIV 1 Gag and V5 aPKC e pression

re transfected with Flag tagged HIV 1 Gag and V5 aPKC e pression vector. Gag Flag displayed a punc tate e pression pattern in the cytoplasm and a partial co localization with aPKC in cytoplasm and plasma membrane. We performed immunoprecipitation analysis and found that aPKC could bind Gag in cells. We ne t e amined whether aPKC can Regorafenib structure directly phosphorylate HIV 1 Gag protein in vitro. Recombinant GST Gag or GST proteins were e pressed and purified from wheat germ cell free e tract by glutathione sepharose beads and used as substrates for in vitro kinase assays. aPKC was found to phosphorylate GST Gag but not GST, with a prominent auto phosphorylation of aPKC also observed. These data together indicate that aPKC binds and phos phorylates HIV 1 Gag.

aPKC phosphorylates Inhibitors,Modulators,Libraries the Ser487 residue of HIV 1 Gag We ne t sought to determine the sites of aPKC phos phorylation in HIV 1 Gag. GST Gag was incubated with recombinant aPKC for their phosphorylation and this mi ture was then processed for proteomic analysis. Ini tial phosphorylation site analysis was performed using the data dependent of tandem matri assisted laser desorption Ionization time of flight mass spectrometry, followed by in depth analysis with selected peptides through data collection. Fragmen tation of this peptide by MS MS produced a spectrum through which we identified one of the b ions and 10 of the y ions matching the sequence QEPIDKELYPLTpSLR. Tandem mass spectra of the signals at m z 1881. 95, m z 1783. 95 and m z 1801. 97 revealed se quences corresponding to the unmodified, mono phos pho peptide of Gag p6.

Furthermore, a Mascot search result identified the se quence QEPIDKELYPLTpSLR. The Ser487 site was found to Inhibitors,Modulators,Libraries be located at Ser40 of Gag p6 domain in close pro imity to both LYP nL and L LF motif. Based on our MS analysis, we constructed a GST tagged p6 and its site directed mutant Inhibitors,Modulators,Libraries GST p6 Ser487Ala and GST p6 Ser461Ala as a negative control. Subsequent in vitro kinase assay results demonstrated that GST p6 is phosphorylated by aPKC, but not GST p6 S487A. These results suggested that aPKC indeed phosphorylates the Ser487 residue of HIV 1 Gag in vitro. To further assess the phosphorylation of Gag at Inhibitors,Modulators,Libraries Ser487, we generated a polyclonal antibody against phosphoryated Ser487. We initially confirmed the specificity and sensitivity of the antibody using the AlphaScreen Carfilzomib system.

We found that our antibody recognized only Ser487 phos phorylated peptides but neither a non phosphorylated peptide nor a peptide harboring a Ser487 to Ala sub stitution. We then used this antibody for in depth cell culture study. 293T Oligomycin A mw cells were transfected with V5 tagged wild type aPKC or a kinase negative mutant, together with wild type Gag Pol. A marked increase in the level of Gag phosphorylation at Ser487 was observed in cells e pressing the wild type aPKC, whereas there was no obvious increase in the amounts of phos phorylation in either aPKC Kn or mock transfected cells. These observations clearly indicate that the

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