Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2.
G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol of protein, respectively). The phosphorylation reactions exhibit apparent K(m) values in the range of 40-100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G beta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.
Ruiz-Gómez
A
A
Departamento de Biología Molecular and Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, E-28049 Madrid, Spain.
Humrich
J
J
Murga
C
C
Quitterer
U
U
Lohse
M J
MJ
Mayor
F
F
D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D054769:G-Protein-Coupled Receptor Kinase 2; D020555:GTP-Binding Protein Regulators; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D013379:Substrate Specificity; D051552:beta-Adrenergic Receptor Kinases
D002352:Carrier Proteins; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D009419:Nerve Tissue Proteins; C098789:PDCL protein, human; D010750:Phosphoproteins; C065631:phosducin; D017868:Cyclic AMP-Dependent Protein Kinases; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2