Phosphorylation of RGS9-1 by an endogenous protein kinase in rod outer segments.
Inactivation of the visual G protein transducin, during recovery from photoexcitation, is regulated by RGS9-1, a GTPase-accelerating protein of the ubiquitous RGS protein family. Incubation of dark-adapted bovine rod outer segments with [gamma-(32)P]ATP led to RGS9-1 phosphorylation by an endogenous kinase in rod outer segment membranes, with an average stoichiometry of 0.2-0.45 mol of phosphates/mol of RGS9-1. Mass spectrometry revealed a single major site of phosphorylation, Ser(475). The kinase responsible catalyzed robust phosphorylation of recombinant RGS9-1 and not of an S475A mutant. A synthetic peptide corresponding to the region surrounding Ser(475) was also phosphorylated, and a similar peptide with the S475A substitution inhibited RGS9-1 phosphorylation. The RGS9-1 kinase is a peripheral membrane protein that co-purifies with rhodopsin in sucrose gradients and can be extracted in buffers of high ionic strength. It is not inhibited or activated significantly by a panel of inhibitors or activators of protein kinase A, protein kinase G, rhodopsin kinase, CaM kinase II, casein kinase II, or cyclin-dependent kinase 5, at concentrations 50 or more times higher than their reported IC(50) or K(i) values. It was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by lowering Ca(2+) to nanomolar levels with EGTA; however, it was not stimulated by the addition of phorbol ester, under conditions that significantly enhanced rhodopsin phosphorylation. A monoclonal antibody specific for the Ser(475)-phosphorylated form of RGS9-1 recognized RGS9-1 in immunoblots of dark-adapted mouse retina. Retinas from light-adapted mice had much lower levels of RGS9-1 phosphorylation. Thus, RGS9-1 is phosphorylated on Ser(475) in vivo, and the phosphorylation level is regulated by light and by [Ca(2+)], suggesting the importance of the modification in light adaptation.
Hu
G
G
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Jang
G F
GF
Cowan
C W
CW
Wensel
T G
TG
Palczewski
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D020710:RGS Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012243:Rhodopsin / Q000302:isolation & purification; D012374:Rod Cell Outer Segment / Q000201:enzymology / Q000378:metabolism*
D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D020710:RGS Proteins; D011994:Recombinant Proteins; C116925:regulator of g-protein signaling 9; D012243:Rhodopsin; D011494:Protein Kinases