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J. Biol. Chem., Vol. 276, Issue 25, 22287-22295, June 22, 2001
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From the 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 [
Phosphorylation of RGS9-1 by an Endogenous Protein Kinase in Rod
Outer Segments*
,,¶, and
**
Verna and Marrs McLean Department of
Biochemistry and Molecular Biology, Baylor College of Medicine,
Houston, Texas 77030 and the Departments of § Ophthalmology,
Chemistry, and ** Pharmacology, University of Washington,
Seattle, Washington 98195
-32P]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,
Ser475. The kinase responsible catalyzed robust
phosphorylation of recombinant RGS9-1 and not of an S475A
mutant. A synthetic peptide corresponding to the region surrounding
Ser475 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 IC50 or
Ki 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 Ser475 in vivo, and the
phosphorylation level is regulated by light and by
[Ca2+], suggesting the importance of the modification in
light adaptation.
*
This work was supported by United States Public Health
Service Research Grants EY08061 and EY11900 and Training Grant EY07001, the Welch Foundation, a grant from Research to Prevent Blindness (RPB)
(to the University of Washington, Department of Ophthalmology), and a
grant from the E. K. Bishop Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
An RPB Senior Investigator.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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