In order to dissect, whether this effect of PknG is a direct interaction or pathway mediated, we performed kinase activity of PknG. PknG undergoes autophosphorylation (Fig. 6D, lane 1, 92 kDa band) and phosphorylates CYT387 order it’s self cleavage product (Fig. 6D, lane
1, 32 kDa band) but does not phosphorylate PKC-α (Fig. 6D, lane 2) or histone (Fig. 6D, lane 4). PKC-α phosphorylates histones (Fig. 6D, lane 3, 25 kDa band) which confirms that PKC-α used in assay was active. To test if there is any possibility that PknG dephosphorylates PKC-α, the immunoprecipitated PKC-α (contain adequate amount of phosphorylated form PKC-α too) was treated with purified PknG. To our surprise, levels of PKC-α and phosphorylated PKC-α were reduced upon treatment with PknG suggesting degradation of PKC-α (Fig. 6E). This also
suggests that the observed reduced level of phosphorylated form in earlier experiments was due to decrease in total PKC-α protein. However, PknG treatment did not affected PKC-δ (which is used as control in the experiment) confirming the specifiCity of PknG for PKC-α rather than general protease activity (Fig. 6E). For better understanding of the direct effect of PknG on PKCα, we incubated click here macrophage lysate with purified PknG and observed degradation of PKC-α (Fig. 6F). To further look for the degradation of PKC-α in a time dependent manner, we treated purified PKC-α with PknG. The immunoblotting with PKC-α antibody showed that PknG cleaves PKC-α proteolytically and the resulting product was detectable with anti-PKC-α antibody (Fig. 6G). Figure 6 Mechanism of downregulation of PKC-α by PknG. (A) Tideglusib Cloning of pknG in pIRES2-EGFP vector; M, DNA ladder; 1, pIRES2-EGFP-pknG undigested; 2, pIRES2-EGFP undigested; 3, pIRES2-EGFP digested with BamHI; 4, pIRES2-EGFP-pknG digested with HindIII; 5, pIRES2-EGFP-pknG digested with BamHI, right oriented recombinants will produce 1.6 kb fragment; (B) and (C) pIRES2-EGFP-pknG was transfected in THP-1 cells and after 48 h cells were lysed and immunoblotted with
anti-serum against PknG and with PKC-α antibodies, lane 1 macrophages transfected with vector alone and lane 2 transfected with pIRES2-EGFP-pknG. (D) 5 μg PknG was incubated with immunoprecipitated PKC-α in kinase buffer for 30 min in presence of [γ32P]-ATP then resolved by 8% SDS-PAGE and exposed to X-Ray film., lane 1 PknG alone; lane 2 PKC-α and PknG, lane 3 PKC-α and Histone-4 and lane 4 PknG and Histone-4. (E) THP-1 cell lysate was immunoprecipitated with either antibodies against PKC-α or PKC-δ using protein G Sepharose. The immunoprecipitated proteins were incubated with 5 μg purified PknG for 1 h and immunoblotted with PKC-α and PKC-δ antibodies. (F) Macrophage cell lysate (50 μg) was incubated with 5 μg purified PknG or buffer alone for indicated times and immunoblotted with PKC-α antibodies.
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