Phosphorylation of Ser166 in RGS5 by protein kinase C causes loss of RGS function.
RGS5 is a member of regulators of G protein signaling (RGS) proteins that attenuate heterotrimeric G protein signaling by functioning as GTPase-activating proteins (GAPs). We investigated phosphorylation of RGS5 and the resulting change of its function. In 293T cells, transiently expressed RGS5 was phosphorylated by endogenous protein kinases in the basal state. The phosphorylation was enhanced by phorbol 12-myristate 13-acetate (PMA) and endothelin-1 (ET-1), and suppressed by protein kinase C (PKC) inhibitors, H7, calphostin C and staurosporine. These results suggest involvement of PKC in phosphorylation of RGS5. In in vitro experiments, PKC phosphorylated recombinant RGS5 protein at serine residues. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Galpha subunits than did the unphosphorylated RGS5. In cells expressing RGS5, the inhibitory effect of RGS5 on ET-1-induced Ca(2+) responses was enhanced by staurosporine. Mass spectrometric analysis of the phosphorylated RGS5 revealed that Ser166 was one of the predominant phosphorylation sites. Substitution of Ser166 by aspartic acid abolished the binding capacity to Galpha subunits and the GAP activity, and markedly reduced the inhibitory effect on ET-1-induced Ca(2+) responses. These results indicate that phosphorylation at Ser166 of RGS5 by PKC causes loss of the function of RGS5 in G protein signaling. Since this serine residue is conserved in RGS domains of many RGS proteins, the phosphorylation at Ser166 by PKC might act as a molecular switch and have functional significance.
Moroi
Kayoko
K
Department of Biochemistry and Molecular Pharmacology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.
Nishiyama
Mariko
M
Kawabata
Shin-ichirou
S
Ichiba
Hideaki
H
Yajima
Takehiko
T
Kimura
Sadao
S
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D019332:Endothelin-1 / Q000494:pharmacology; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020710:RGS Proteins / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology
D019332:Endothelin-1; D044385:GTP-Binding Protein alpha Subunits; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D020710:RGS Proteins; C491349:RGS5 protein, human; D011994:Recombinant Proteins; D012694:Serine; C016658:phorbolol myristate acetate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium