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. 2003 Apr;9(4):800-3.
doi: 10.3748/wjg.v9.i4.800.

Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase

Xing-Long Yang  1 , Ya-Li Zhang, Zhuo-Sheng Lai, Fei-Yue Xing, Yu-Hu Liu
Affiliations

Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase

Xing-Long Yang et al. World J Gastroenterol. 2003 Apr.

Abstract

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) 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.

Conclusion: GRK(2) can bind with PKC and down-regulated PKC activity.

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Figures

Figure 1
Figure 1
The binding of PH domain to PKC by Western blot with the antibody to PKC. Lane 1, 2, 3, 4 and 5 were the GST-PH domains of IRS-1, GRF/c, β-ARK1, PLCγ/n and Btk, respectively; lane 6 was the GST; lane 7 was lysate of Jurkat cells.
Figure 2
Figure 2
The expressing of GRK2 by Western blot with the antibody to GRK2. Lane 1, 2, 3, 4 were the results of Western blot in PC-3, MDCK, 7901 and Jurkat cell line respectively. Lane 5,6 was GST.
Figure 3
Figure 3
After stimulation of epinephrine, Co-immunopre-cipitation was performed with anti-GRK2 and anti-PKC in co-precipitation and detection of PKC and GRK2. Lane 1 was PKC detected in precipitate of GRK antibody, lane 2 was GRK2 de-tected in precipitate of PKC antibody. Lane 3 indicated the PKC did not bind to GRK2 in the intact cells without stimulation of epinephrine , lane 4 was the lysate of Jurkat cell.
Figure 4
Figure 4
Effects of the PH domain on PKC activity measured by protein kinase C Assay System. The volume was percent-age of PKC activity. The data were representation of two experiments. Lanes 1, 2, 3, 4 and 5 were GST-PH domain of IRS-1, GRF/c, β-ARK1, PLC γ/n and Btk, respectively; lane 6 wasthe GST; lane 7 was lysate of Jurkat cells.
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