The Kaposi’s sarcoma (KS)-associated herpesvirus is a lymphotropic virus strongly implicated

The Kaposi’s sarcoma (KS)-associated herpesvirus is a lymphotropic virus strongly implicated in the pathogenesis Rabbit Polyclonal to TPH2. of KS and several lymphoproliferative disorders. WT or mutant K1 expression vectors cells were lysed and extracts were prepared for immune precipitation. We found that HA-tagged K1 protein although efficiently recognized by anti-HA on Western blots cannot be efficiently precipitated by the same antibody (not shown). Therefore we generated a plasmid made up of the WT K1 sequence or the Y>F double mutant with the signal sequences replaced by the CD8 signal sequence and a Flag tag in-frame with K1. The Flag-tagged K1 activated the NFAT-luciferase reporter similarly to WT and HA-tagged K1 and by flow cytometry was shown to be expressed around the cell surface (data not shown). The Flag antibody (IBI Kodak New York) was used to immunoprecipitate K1 and the immunoprecipitates were separated by SDS/PAGE blotted to membranes and probed with Sal003 antiphosphotyrosine antibody (4G10). As shown in Fig. ?Fig.11lane 6). This experiment also shows that neither Sal003 of the two tyrosines present in the K1 cytoplasmic tail outside of the ITAM are stably phosphorylated. The single tyrosine mutants were assayed for phosphorylation in the HA-tagged K1 constructs by using antiphosphotyrosine antibody (4G10) for immunoprecipitation and antibody to the HA tag for immunoblotting. In this experiment the HA-tagged K1 is usually immunoprecipitated but neither of the single Y>F mutant K1 proteins is usually immunoprecipitated (data not shown) a result that is also in accord with findings in other ITAM-bearing proteins. This finding suggests that either the phosphorylations at this site are highly and reciprocally cooperative or more likely that lesions that impair SH2 recognition of the ITAM (e.g. by syk family members) leave the remaining phosphotyrosines unshielded from the action of cellular phosphatases (36). K1 Forms Homo-Multimers. To examine K1-K1 interactions we took advantage of the two tagged forms of K1. Raji cells were cotransfected with HA-K1 or HA-K1DC and either empty vector or Flag-K1 and the anti-Flag antibody was used to immunoprecipitate Sal003 the transfected cell lysates. The anti-Flag antibody was capable of immunoprecipitating HA-K1 or HA-K1DC only in presence of Flag-K1 indicating that Flag-K1 and HA-K1 multimerize (Fig. ?(Fig.11gene have been inactivated Sal003 by homologous recombination (31). As shown in Fig. ?Fig.3320-fold). No activation is seen when the vector alone was cotransfected with the human syk construct (Fig. ?(Fig.33and data not shown). Western blots of the supernatants show the presence of comparable amounts of Syk in all extracts (Fig. ?(Fig.33C lanes 4-6). This same blot subsequently was probed with the anti-HA antibody to verify that Sal003 as in Fig. ?Fig.11C only WT K1 is immunoprecipitated by 4G10 (Fig. ?(Fig.33C). Syk Interacts with the Doubly Phosphorylated K1 ITAM Peptide. In other well-studied cases syk phosphorylation and activation is the result of direct conversation with the ITAM (21 38 To examine the conversation of K1 and syk we first attempted coimmunoprecipitation. WT or mutant HA-K1 genes were transfected into Raji cells and lysates were prepared. After precipitation with anti-syk precipitates were examined by immunoblotting with anti-HA mAb. We were unable to consistently observe an conversation between syk and K1 in this fashion (not shown); perhaps the mutation in the K1 ITAM lowers the affinity of syk binding to K1. In another approach we used biotinylated synthetic peptides made up of the K1 ITAM and flanking sequences either unphosphorylated doubly or singly phosphorylated at the ITAM tyrosine residues. Raji cell lysates were incubated with these peptides and peptide-bound proteins were collected on avidin-agarose beads. After elution and separation by SDS/PAGE the eluates were examined by immunoblotting with anti-syk antibody. The doubly phosphorylated peptide is able to precipitate high levels of syk (Fig. ?(Fig.33D) whereas the unphosphorylated (Fig. ?(Fig.3D)3D) or singly phosphorylated peptides displayed substantially reduced binding. Thus the K1 ITAM can enter into complexes.