β₂-Adrenergic Receptor Stimulated, G Protein-Coupled Receptor Kinase 2 Mediated, Phosphorylation of Ribosomal Protein P2

Abstract

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.

Arrestin-2 and G Protein-coupled Receptor Kinase 5 Interact with NFκB1 p105 and Negatively Regulate Lipopolysaccharide-stimulated ERK1/2 Activation in Macrophages

“…Recent studies have suggested that GRKs and arrestins could play much broader roles in cell signaling (16,40). Although non-GPCR targets for GRKs have been identified (41)(42)(43)(44)(45)(46), arrestins have recently been shown to be involved in receptor signaling pathways that are not considered within the typical GPCR paradigm (27,29,37,47). These include receptor signaling from a variety of ligands ranging from insulin to cytokines.…”

G Protein-coupled Receptor Kinase 2-mediated Phosphorylation of Downstream Regulatory Element Antagonist Modulator Regulates Membrane Trafficking of Kv4.2 Potassium Channel

“…Detection of DREAM after immunoprecipitation was performed with an affinity-purified rabbit polyclonal antibody (Ab1014) raised against recombinant fulllength DREAM (30). For pulldown studies, GRK2 or GRK6 and its truncated forms were 35 S-labeled in vitro using the transcription/translation T7-TNT system (Promega). Equimolar amounts, confirmed by PAGE and silver staining (data not shown), of recombinant GST and GST-DREAM proteins (20 pmol) bound to glutathione-Sepharose (Amersham Biosciences) were incubated with the labeled proteins in interaction buffer (20 mM K-Hepes, pH 7.5, 10% glycerol, 150 mM KCl, 2 mM MgCl 2 , 0.5 mM EGTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 0.5% Blotto (Bio-Rad)) containing protease inhibitor mixture (Calbiochem) and with the addition of 2 mM CaCl 2 or 4 mM EDTA for calcium-dependence experiments.…”

Functional specialization of β-arrestin interactions revealed by proteomic analysis

“…Although ribosomal proteins are often observed as contaminants of coimmunoprecipitation, it is tempting to speculate that ␤-arrestins may associate with ribosomal proteins in the cytoplasm, nucleolus, or ribosomal assembly machinery and regulate their activities. Previously a good correlation between GPCR activation and the translational control of gene expression mediated by G protein-coupled receptor kinase 2 activation and ribosomal protein P2 phosphorylation was demonstrated (17). Thus, it is plausible that ␤-arrestins maybe involved in such processes.…”