Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase.
OBJECTIVE
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) domain located in GRK(2) residue 548 to 660 was amplified by PCR with the mRNA of human GRK(2) (beta1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK(2) PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK(2) PH 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(2) PH domain, GST-GRK(2) PH domain fusion protein and BTK PH 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 western blot and Co-immunoprecipitation.


RESULTS
The binding of GRK(2) PH domain to PKC in Vitro was confirmed by western blot, as were the binding upon prolonged stimulation of epinephrine and the binding of BTK PH domain to PKC. In the present study, GRK(2) PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK(2) PH domain.


CONCLUSIONS
GRK(2) can bind with PKC and down-regulated PKC activity.
Yang
Xing-Long
XL
Institute of Gastroenterology, NanFang Hospital, First Military Medical University 510515, Guangzhou, Guangdong Province, China. foru3000@hotmail.com
Zhang
Ya-Li
YL
Lai
Zhuo-Sheng
ZS
Xing
Fei-Yue
FY
Liu
Yu-Hu
YH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004837:Epinephrine / Q000494:pharmacology; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D010920:Placenta / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D064546:Protein Kinase C beta; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D001798:Blood Proteins; D017931:DNA Primers; D010446:Peptide Fragments; D010750:Phosphoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C039077:platelet protein P47; D011494:Protein Kinases; C475887:protein kinase C kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051552:beta-Adrenergic Receptor Kinases; D004837:Epinephrine