Phosphorylation of RGS9-1 by an endogenous protein kinase in rod outer segments

Inactivation of the visual Gr 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-P-32]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 S(475)A mutant. A synthetic peptide corresponding to the region surrounding Ser(475) was also phosphorylated, and a similar peptide with the S(475)A 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 IC50 or K-i values. It was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by lowering Ca2+ 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 Ser475 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 [Ca2+], suggesting the importance of the modification in light adaptation.