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AIM:To study the detail mechanism of interaction betweenPKC and GRK_2 and the effect of GRK_2 on activity of PKC.METHODS:The cDNA of pleckstrin homology (PH) domainlocated in GRK_2 residue 548 to 660 was amplified by PCRwith the mRNA of human GRK_2 (β1-adrenergic receptorkinase) as template isolated from human fresh placenta,the expression vector pGEX-PH inserted with the abovedcDNA sequence for GRK_2 PH domain protein and theexpression vectors for GST (glutathion-s-transferase)-GRK_2PH domain fusion protein,BTK (Bruton’s tyrosine kinase)PH domain and GST protein were constructed.Theexpression of GRK_2 in culture mammalian cells (6 cell lines:PC-3,MDCK,SGC7901,Jurkat cell etc.) was determined bySDS-PAGE and Co-immunoprecipitation.The binding of GRK_2PH domain,GST-GRK_2 PH domain fusion protein and BTKPH domain to PKC in Vitro were detected by SDS-PAGE andWestern blot,upon prolonged stimulation of epinephrine,the binding of GRK_2 to PKC was also detected by westernblot and Co-immunoprecipitation.RESULTS:The binding of GRK_2 PH domain to PKC in Vitrowas confirmed by western blot,as were the binding uponprolonged stimulation of epinephrine and the binding of 13TKPH domain to PKC.In the present study,GRK_2 PH domainwas associated with PKC and down-regulated PKC activity,but Btk PH domain up-regulated PKC activity as comparedwith GRK_2 PH domain.CONCLUSION:GRK_2 can bind with PKC and down-regulatedPKC activity.
AIM: To study the detail mechanism of interaction between PKC and GRK_2 and the effect of GRK_2 on activity of PKC. METHODS: The cDNA of pleckstrin homology (PH) domainlocated in GRK_2 residue 548 to 660 was amplified by PCR with the mRNA of human GRK_2 (β1 -adrenergic receptorkinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the abovedcDNA sequence for GRK_2 PH domain protein and theexpression vectors for GST (glutathion-s-transferase) -GRK_2PH domain fusion protein, BTK (Bruton’s tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK_2 in culture mammalian cells (6 cell lines: PC-3, MDCK, SGC7901, Jurkat cell etc.) was determined by SDS- PAGE and Co-immunoprecipitation. The binding of GRK_2PH domain , GST-GRK_2 PH domain fusion protein and BTKPH domain to PKC in Vitro were detected by SDS-PAGE and Western blot, upon prolonged stimulation of epinephrine, the binding of GRK_2 to PKC was also detected by westernblot and Co-immunopre cipitation.RESULTS: The binding of GRK_2 PH domain to PKC in Vitrowas confirmed by western blot, as were the binding upon protiant stimulation of epinephrine and the binding of 13TKPH domain to PKC. In the present study, GRK_2 PH domainwas associated with PKC and down- regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK_2 PH domain.CONCLUSION: GRK_2 can bind with PKC and down-regulated PKC activity.