Phosphorylation of the regulator of G protein signaling RGS9-1 by protein kinase a is a potential mechanism of light- and Ca2+-mediated regulation of G protein function in photoreceptors

N. Balasubramanian, K. Levay, T. Keren-Raifman, E. Faurobert, Vladlen Z Slepak

Research output: Contribution to journalArticle

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Abstract

In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein β subunit Gβ5. Phosphorylated RGS9-1 remains associated with Gβ5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser427 and Ser428. Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca2+ ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.

Original languageEnglish
Pages (from-to)12619-12627
Number of pages9
JournalBiochemistry
Volume40
Issue number42
DOIs
StatePublished - Oct 23 2001

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GTP-Binding Protein Regulators
Phosphorylation
GTP-Binding Proteins
Protein Kinases
Light
Transducin
GTPase-Activating Proteins
Labeling
Vertebrate Photoreceptor Cells
Rhodopsin
Mitogen-Activated Protein Kinase Kinases
Phosphoric Diester Hydrolases
Protein Subunits
Protein Kinase Inhibitors
Cyclic AMP-Dependent Protein Kinases
Guanosine Triphosphate
Catalytic Domain
Substitution reactions
Phosphotransferases
Ions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation of the regulator of G protein signaling RGS9-1 by protein kinase a is a potential mechanism of light- and Ca2+-mediated regulation of G protein function in photoreceptors. / Balasubramanian, N.; Levay, K.; Keren-Raifman, T.; Faurobert, E.; Slepak, Vladlen Z.

In: Biochemistry, Vol. 40, No. 42, 23.10.2001, p. 12619-12627.

Research output: Contribution to journalArticle

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abstract = "In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30{\%}, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90{\%}, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein β subunit Gβ5. Phosphorylated RGS9-1 remains associated with Gβ5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser427 and Ser428. Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca2+ ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.",
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AU - Slepak, Vladlen Z

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N2 - In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein β subunit Gβ5. Phosphorylated RGS9-1 remains associated with Gβ5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser427 and Ser428. Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca2+ ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.

AB - In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein β subunit Gβ5. Phosphorylated RGS9-1 remains associated with Gβ5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser427 and Ser428. Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca2+ ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.

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