G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation.
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.
Luo
Jiansong
J
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Benovic
Jeffrey L
JL
D016227:Benzoquinones; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D054770:G-Protein-Coupled Receptor Kinase 3; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D018922:HL-60 Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes; D011809:Quinones; D043562:Receptors, G-Protein-Coupled; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517898:GRK5 protein, human; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C001277:geldanamycin