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

Area of Science:

• Molecular Biology
• Biochemistry
• Neuroscience
• Vision Research

Background:

• Visual G protein transducin inactivation is crucial for recovery from photoexcitation.
• Regulator of G protein signaling 9-1 (RGS9-1) is a key GTPase-accelerating protein regulating transducin.
• Understanding RGS9-1 regulation is vital for comprehending visual adaptation processes.

Purpose of the Study:

• To identify the kinase responsible for RGS9-1 phosphorylation.
• To determine the specific site of RGS9-1 phosphorylation.
• To investigate the in vivo regulation of RGS9-1 phosphorylation by light and calcium.

Main Methods:

• Phosphorylation assays using [gamma-(32)P]ATP on isolated bovine rod outer segments and recombinant RGS9-1.
• Mass spectrometry to identify the phosphorylation site.
• Characterization of the kinase activity and its sensitivity to various inhibitors and conditions.
• Immunoblotting with a phospho-specific antibody in mouse retinas under different light conditions.

Main Results:

• RGS9-1 is phosphorylated by an endogenous kinase in rod outer segments, with a stoichiometry of 0.2-0.45.
• Mass spectrometry identified Serine 475 (Ser475) as the major phosphorylation site.
• A novel peripheral membrane kinase, distinct from known kinases, phosphorylates RGS9-1 at Ser475.
• RGS9-1 phosphorylation is significantly reduced in light-adapted mouse retinas compared to dark-adapted retinas.
• The kinase activity is sensitive to calcium levels and inhibited by protein kinase C inhibitor bisindolylmaleimide I.

Conclusions:

• RGS9-1 is phosphorylated in vivo on Ser475 by a novel kinase.
• RGS9-1 phosphorylation is dynamically regulated by light and intracellular calcium levels.
• This light- and calcium-dependent phosphorylation of RGS9-1 plays a significant role in visual light adaptation.