Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family and has been shown to play a key role in determining the desensitization and resensitization patterns of a variety of G protein-coupled receptors. In this report, we show that GRK2 is actively degraded by the proteasome proteolytic pathway, unveiling a new mechanism for the rapid regulation of its expression levels. Interestingly, activation of beta2-adrenergic receptors (beta2AR) markedly increases GRK2 ubiquitination and degradation through the proteasome pathway. In addition, blocking GRK2 degradation notably alters beta2AR signaling and internalization, consistent with a relevant physiological role for GRK2 proteasomal degradation. Activity-dependent modulation of GRK2 cellular levels emerges as an important mechanism for modulating the cellular response to agonists acting through G protein-coupled receptors.
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PMID:Degradation of the G protein-coupled receptor kinase 2 by the proteasome pathway. 985 63

G-protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of G-protein-coupled receptors (GPCRs). GRK2 expression is altered in several pathological conditions, but the molecular mechanisms that modulate GRK2 cellular levels are largely unknown. We recently have described that GRK2 is degraded rapidly by the proteasome pathway. This process is enhanced by GPCR stimulation and is severely impaired in a GRK2 mutant that lacks kinase activity (GRK2-K220R). In this report, we find that beta-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of beta-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with beta-arrestin mutants that display an impaired c-Src interaction. The presence of an inactive c-Src mutant or of tyrosine kinase inhibitors strongly inhibits co-transfected or endogenous GRK2 turnover, respectively, and a GRK2 mutant with impaired phosphorylation by c-Src shows a markedly retarded degradation. This pathway for the modulation of GRK2 protein stability puts forward a new feedback mechanism for regulating GRK2 levels and GPCR signaling.
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PMID:Beta-arrestin- and c-Src-dependent degradation of G-protein-coupled receptor kinase 2. 1156 77

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.
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PMID:MAPK-dependent degradation of G protein-coupled receptor kinase 2. 1273 76

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation.
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PMID:G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation. 1455 68

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195-199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenomenon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3.
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PMID:MAP kinase protects G protein-coupled receptor kinase 2 from proteasomal degradation. 1580 51

Arrhythmia-prone subepicardial border zone (EBZ) tissue demonstrates decreased G protein receptor kinase 2 (GRK2) activity and increased sensitivity to isoproterenol 6-24 h after coronary artery ligation (CAL) in the dog. With the use of a semiquantitative immunofluorescence technique, the relative fluorescence intensity (RF) of GRK2 in EBZ decreased to 24% of that in a remote site (RS) (P < 0.01, n = 30 cells from 3 dogs), whereas GRK5 RF did not change. Confocal studies of cardiac tissue from transgenic mice overexpressing GRK2 validated the use of a semilogarithmic relationship between RF and GRK2 activity. As shown with the use of quantitative real-time RT-PCR, both GRK2 and GRK5 mRNA were not decreased at 24 h in EBZ (n = 6 dogs) relative to RS control, indicating that the decrease of GRK2 in the EBZ is likely due to posttranscriptional degradation following CAL. Pretreatment of six dogs with the selective proteasome inhibitor bortezomib provided 100% (EBZ) and 50% (infarct) protection against loss of GRK2 at 24 h. There was an absence of rapid (>300 beats/min) and very rapid (>360 beats/min) ventricular triplets that are highly predictive of sudden cardiac death during ECG monitoring in the bortezomib-pretreated animals in contrast to nonpretreated infarcted animals. We have demonstrated that the dramatic decrease in GRK2 in cardiac ischemic tissue can be largely blocked by prior proteasome blockade and that this is associated with significant cardioprotection against malignant ventricular tachyarrhythmias.
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PMID:Proteasome degradation of GRK2 during ischemia and ventricular tachyarrhythmias in a canine model of myocardial infarction. 1599 60

The effect of exercise on beta-adrenergic receptor (beta-AR) trafficking was investigated in rat adipocytes. The binding sites of a hydrophilic ligand, [(3)H]CGP12177, increased immediately (0 h) and at 3 h after exercise (3 h) but decreased at 24 h after exercise (24 h). The data of immunoblotting revealed that the alterations in the binding sites mainly paralleled the alterations in the beta2-AR proteins in membrane fractions. The protein expressions of both G-protein-coupled receptor kinase (GRK)-2 and beta-arrestin-2 were reduced, with a decline in beta2-AR ubiquitination at 0 h and 3 h. The protein expressions of beta2-AR, GRK-2, beta-arrestin-2, the beta2-AR/beta-arrestin-2 complex, and beta2-AR ubiquitination returned to their respective control levels at 24 h, whereas the beta2-AR mRNA level was reduced. Administration of either lactacystin or propranolol did not alter GRK-2 and beta2-AR protein expressions after exercise. Thus, the mechanism underlying the increased density of beta2-AR up to at least 3 h may involve alterations in a multistep event involving the coordinate interaction among proteins mediating beta2-AR trafficking, in which both the receptor-agonist interactions and ubiquitin-proteasome pathway have a key role. However, the decreased protein expression of beta2-AR at 24 h might be due to some change occurring at the translational levels.
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PMID:Beta-adrenergic receptor trafficking by exercise in rat adipocytes: roles of G-protein-coupled receptor kinase-2, beta-arrestin-2, and the ubiquitin-proteasome pathway. 1636 19

Cellular levels of G protein-coupled receptor kinase (GRK)3 determine the sensitivity of the alpha(2A/B)-adrenoceptor (alpha(2)-AR) to agonist-induced down-regulation. Using human neuroblastoma BE(2)-C cells, this study examines how cellular GRK3 levels are affected by several mechanisms reported to influence stability and degradation of other GRKs. We first examined the interaction between the 90-kDa heat shock protein (Hsp90) and GRK3; Hsp90 reportedly affects the maturation and stability of GRK2. In unstimulated cells, GRK3 coimmunoprecipitates with Hsp90, suggesting a physical interaction. Moreover, when GRK3 protein expression was increased by 24-h epinephrine (EPI) treatment, Hsp90 protein expression increased with a similar but slightly delayed time course. To investigate the influence of Hsp90 on GRK3 protein stability, we determined the effect of the Hsp90 inhibitor geldanamycin (GA) on cellular GRK3 levels. GA eliminated the interaction between Hsp90 with GRK3 and produced a rapid, proteasome-mediated, 70% decrease in GRK3 levels within 24 h. To investigate the influence of Hsp90 on up-regulation of GRK3 expression, we examined the effect of GA on EPI-induced up-regulation. GA reduced the absolute increase in GRK3; however, the percentage of increase in GRK3 by EPI was not significantly different in the absence versus presence of GA (141 +/- 41 versus 94 +/- 12%). Finally, we examined the influence of Ca(2+)-activated proteases on cellular GRK3. Treatment with the calcium ionophore ionomycin produced a rapid decrease in GRK3 levels that was inhibited by the calpain inhibitor calpeptin. In conclusion, several mechanisms influence the degradation of GRK3 and therefore have the potential to affect GPCR signaling by regulating GRK3 levels in neurons.
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PMID:Role of 90-kDa heat shock protein (Hsp 90) and protein degradation in regulating neuronal levels of G protein-coupled receptor kinase 3. 1717 67

G protein-coupled receptor kinase 2 (GRK2) modulates G protein-coupled receptor desensitization and signaling. We previously described down-regulation of GRK2 expression in vivo in rat neonatal brain following hypoxia-ischemia. In this study, we investigated the molecular mechanisms involved in GRK2 down-regulation, using organotypic cultures of neonatal rat hippocampal slices exposed to oxygen and glucose deprivation (OGD). We observed a 40% decrease in GRK2 expression 4 h post-OGD. No changes in GRK2 protein occurred after exposure of hippocampal slices to glucose deprivation only. No significant alterations in GRK2 mRNA expression were detected, suggesting a post-transcriptional effect of OGD on GRK2 expression. Blockade of the proteasome pathway by MG132 prevented OGD-induced decrease of GRK2. It has been shown that extracellular signal-regulated kinase-dependent phosphorylation of GRK2 at Ser670 triggers its turnover via the proteasome pathway. However, despite a significant increase of pSer670-GRK2 after OGD, inhibition of the extracellular signal-regulated kinase pathway by PD98059 did neither prevent the hypoxia-ischemia-induced increase in pSer670-GRK2 nor the down-regulation of GRK2 protein. Interestingly, inhibition of phosphoinositide-3-kinase with wortmannin inhibits both OGD-induced phosphorylation of GRK2 on Ser670 and the GRK2 decrease. In conclusion, OGD-induced phosphoinositide-3-kinase-dependent phosphorylation of GRK2 on Ser670 is a novel mechanism leading to down-regulation of GRK2 protein via a proteasome-dependent pathway.
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PMID:Down-regulation of GRK2 after oxygen and glucose deprivation in rat hippocampal slices: role of the PI3-kinase pathway. 1743 35

Arrhythmia-prone epicardial border zone (EBZ) tissues demonstrate decreased G protein-coupled receptor kinase-2 (GRK2) activity and increased sensitivity to isoproterenol 6-24 h after coronary artery ligation in the dog. We previously demonstrated that the ischemia-mediated decrease in GRK2 in cardiac ischemic tissue was largely blocked by proteasome blockade initiated 1 h before the onset of ischemia, and this was associated with significant cardioprotection against malignant ventricular tachyarrhythmias. For application to clinical circumstances, it is desirable to determine whether a clinical window exists following the onset of ischemia for such a protective effect. The treatment of six dogs with the selective proteasome inhibitor bortezomib 1 h after the surgical induction of left coronary artery ischemia provided 80% (EBZ) and 42% (infarct) protection (by immunoblot) against the loss of GRK2 at 24 h. There was no significant increase of heat shock protein 70(72) in the EBZ of bortezomib-treated animals compared with control. There was a striking absence of rapid (>300 beats/min) and very rapid (>360 beats/min) ventricular triplets that is highly predictive of sudden cardiac deaths (SCDs) during electrocardiogram monitoring of the first 24 h in the bortezomib-treated animals in contrast with nontreated infarcted animals. There were no SCDs in the 6 treated animals (0%) and five SCDs in the 14 control animals (36%). Assay of whole blood proteasome activity demonstrated the expected decrease over the 24-h observation period. These data support the concept that proteasome inhibition within a window of time following myocardial infarction may be of use in suppressing malignant tachyarrhythmias and SCD.
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PMID:Proteasome inhibition 1 h following ischemia protects GRK2 and prevents malignant ventricular tachyarrhythmias and SCD in a model of myocardial infarction. 1819 26


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