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Originally published In Press as doi:10.1074/jbc.M307637200 on October 13, 2003

J. Biol. Chem., Vol. 278, Issue 51, 50908-50914, December 19, 2003
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G Protein-coupled Receptor Kinase Interaction with Hsp90 Mediates Kinase Maturation*

Jiansong Luo and Jeffrey L. Benovic{ddagger}

From the Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received for publication, July 15, 2003 , and in revised form, September 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
G protein-coupled receptor kinases (GRKs)1 are a family of serine/threonine protein kinases that specifically phosphorylate the agonist-activated form of G protein-coupled receptors (GPCRs). Receptor phosphorylation functions to promote arrestin binding resulting in receptor desensitization and trafficking (1-3). Recent studies have revealed that GRKs themselves are also subject to various regulatory processes including phosphorylation and ubiquitination (4, 5). For example, GRK2 phosphorylation by cAMP-dependent protein kinase (6), protein kinase C (7), and c-Src (8) results in activation, whereas phosphorylation by ERK1/2 effectively inhibits GRK2 activity (9, 10). Interestingly, c-Src phosphorylation also promotes the degradation of GRK2 via a process that is regulated by receptor stimulation and may involve GRK2 ubiquitination and targeting to the proteasome pathway (11). GRKs also interact with numerous additional proteins including G protein {alpha} (12-14) and {beta}{gamma} (15, 16) subunits, clathrin (17), the GRK-interacting protein GIT1 (18), caveolin-1 (19), phosphoinositide 3-kinase-{alpha} and -{gamma} (20), cytoskeletal proteins such as tubulin and actin (21-23), and various calcium-binding proteins (reviewed in Ref. 24). Many of these interactions are thought to be important for regulating the localization and enzymatic activity of GRKs. In an effort to identify proteins that interact with endogenous GRKs, here we used immunoprecipitation and mass spectrometry analysis and demonstrated that GRK2 interacts with heat shock protein 90 (Hsp90).

Hsp90 is a highly conserved protein chaperone that interacts with a diverse group of regulatory and signaling proteins including various protein kinases (25-27), heterotrimeric G proteins (28, 29) and G protein-coupled receptors (30). The functional role of Hsp90 interaction is protein-dependent. For example, Hsp90 can maintain the active state of the serine/threonine kinase Akt by preventing its dephosphorylation (27), whereas Hsp90 interaction with the tyrosine kinases c-Src (31) and Lck (32) functions in protein maturation. Hsp90 contains three functional domains: an N-terminal region that binds ATP, a middle segment that has been implicated in binding client proteins, and a conserved C-terminal region (33, 34). The interaction of Hsp90 with client proteins is dependent on its ability to bind and hydrolyze ATP and can be effectively disrupted by ATP-mimetic drugs including the antibiotics geldanamycin (GA) and herbimycin-A (25, 33). These antibiotics bind tightly to the Hsp90 ATP/ADP binding pocket often resulting in proteasome-dependent degradation of proteins that require Hsp90 for conformational maturation (35, 36).

In our search for proteins that regulate the function of endogenous GRK2, we found that GRK2 interacts with Hsp90 in HL60 cells. Disruption of GRK2/Hsp90 interaction with geldanamycin resulted in increased degradation of GRK2 mainly through the proteasome pathway. Moreover, additional GRK family members are also regulated by interaction with Hsp90. These studies suggest that heat shock proteins likely play a general role in regulating GRK maturation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials—HL60 and COS-1 cells were from the American Type Culture Collection (Manassas, VA). Anti-GRK2/3 and anti-GRK4-6 mouse monoclonal antibodies were from Upstate Biotechnology (Lake Placid, NY) and an anti-GRK2 mouse monoclonal antibody (3A10) was purified from a hybridoma cell culture supernatant. Anti-Hsp90 monoclonal antibodies were from Stressgen Biotechnologies (Victoria, Canada) and Sigma, whereas purified Hsp90 was from Stressgen Biotechnologies. Geldanamycin was from Sigma, and [35S]methionine/cysteine labeling mixture was from Amersham Biosciences. SYPRO ruby protein gel stain was from Molecular Probes (Eugene, OR), lactacystin and calpeptin were from Calbiochem (La Jolla, CA), and FuGENE 6TM was from Roche Applied Science.

Cell Culture and Transfection—HL60 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate at 37 °C in a humidified atmosphere containing 5% CO2. COS-1 cells were maintained in Dulbecco's modified Eagle's medium and were transfected at a confluence of ~70% with 1 µg of total DNA/6-cm plate using FuGENE 6TM following the manufacturer's instructions.

Immunoprecipitation of GRK2—HL60 cells (~108) grown in 15-cm plates to a density of ~2 x 106 cells/ml were harvested by centrifugation, washed two times with cold phosphate-buffered saline, and homogenized with 2 ml of buffer A (20 mM HEPES, pH 7.5, 10 mM EDTA, 150 mM NaCl, 10 µg/ml leupeptin, 2 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml benzamidine, and 0.1% Triton) using a Brinkman Polytron (14,000 rpm, 2 x 30 s). The homogenate was centrifuged at 100,000 x g for 30 min, and the supernatant was pre-cleared by incubation with protein G-agarose for 1 h at 4 °C. The supernatant was then incubated on a rocker with 20 µl of anti-GRK2/3 or anti-GRK4-6 mAb for 1 h at 4 °C followed by the addition of 100 µl of 50% protein G-agarose pre-equilibrated in lysis buffer and a 1-h incubation at 4 °C. Samples were then centrifuged, and the pellets were washed three times with 1 ml of buffer A and one time with 20 mM Tris-HCl, pH 7.5, 2 mM EDTA on a rocker for 15 min at 4 °C. Bound proteins were eluted by addition of 50 µl of SDS sample buffer followed by boiling for 10 min. Samples were electrophoresed on a 7.5% polyacrylamide gel and stained with Coomassie Blue or SYPRO ruby stain following the manufacturer's instructions. Stained protein bands were excised from the polyacrylamide gel and stored at -20 °C until further analysis.

Mass Spectrometry Analysis—Proteins in polyacrylamide gel slices were digested with 20 ng/µl trypsin (Promega, Madison, WI) in 25 mM NH4HCO3 buffer for 16 h at 37 °C. Mass spectra of tryptic peptides were acquired using surface-enhanced laser desorption/ionization on a hydrophobic H4 chip using a Ciphergen PBS II Instrument (Fremont, CA). Proteins were identified by comparing observed peptide mass fingerprints with those theoretically derived from the NCBInr data base using the Profound data base searching algorithm (Rockefeller University).

Immunoblotting—To analyze co-immunoprecipitation of GRK2 and Hsp90, lysates from HL60 or transfected COS-1 cells were immunoprecipitated with anti-GRK2/3 mAb as described above, electrophoresed on a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted using anti-Hsp90 mAb, horseradish peroxidase-labeled goat anti-mouse secondary antibodies, and chemiluminescence (Pierce). To analyze GRK levels in transfected COS-1 cells, cells were homogenized in buffer A and centrifuged, and the supernatants were electrophoresed on a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted using anti-GRK2/3 or anti-GRK4-6 mAbs, horseradish peroxidase-labeled goat anti-mouse secondary antibodies, and chemiluminescence. Some blots were stripped and reprobed using an {alpha}-tubulin mAb (Sigma).

Metabolic Labeling—HL60 cells (4 x 107) were harvested and washed two times with 10 ml of Dulbecco's modified Eagle's medium without methionine and cysteine and then incubated in 10 ml of the same medium with or without 1 µM geldanamycin for 1 h. The cells were then incubated in 4 ml of medium containing 200 µCi/ml [35S]methionine/cysteine labeling mixture with or without 1 µM geldanamycin for 30 min (pulse), washed two times with RPMI 1640 containing 10% fetal bovine serum, and chased with the same medium in the presence of geldanamycin or vehicle for 2-18 h. In an additional series of experiments, geldanamycin was not added until the beginning of the chase period. The cells were then lysed, and GRK2 was immunoprecipitated, electrophoresed on a 10% polyacrylamide gel, and detected by fluorography. GRK2 levels were quantified using liquid scintillation counting (PerkinElmer Life Sciences).

Substrate Phosphorylation—Wild-type GRK2 was overexpressed and purified from Sf9 cells as described in Ref. 37. GRK2 activity was assayed by incubating 22 nM kinase and purified Hsp90 (0, 15, 150, or 1500 nM) with either rod outer segment membranes (4 µM rhodopsin) or tubulin (1 µM) in 20 mM Tris-HCl, pH 8.0, 4 mM MgCl2, and 0.1 mM [{gamma}-32P]ATP (~1,000 cpm/pmol). Samples were incubated for 5 min (rhodopsin) or 10 min (tubulin) at 30 °C in room light, quenched with SDS buffer, and electrophoresed on a 10% polyacrylamide gel. Gels were dried and autoradiographed, and and 32P-labeled proteins were excised and counted to determine pmol of phosphate transferred.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Identification of Proteins Associated with Endogenous GRK2 in HL60 Cells—GRKs were initially identified via their ability to phosphorylate and regulate the activity of agonist-occupied GPCRs (1, 2). Although GRKs themselves are also subject to numerous regulatory interactions and post-translational modifications (4, 5), the majority of studies have utilized over-expression or in vitro analysis to analyze these processes. Here we attempted to identify proteins that interact with and potentially regulate the function of endogenous GRKs. The strategy used involved immunoprecipitation of endogenous GRKs from cell lysates followed by SDS-PAGE and mass spectrometry. In the present study, we used HL60 cells because they have high endogenous levels of GRK2 (~170,000 molecules/cell) and GRK6 (~27,000 molecules/cell) (38). An HL60 cell lysate (from ~108 cells) was incubated with either anti-GRK2/3 monoclonal antibodies that selectively immunoprecipitate GRK2 and -3, or anti-GRK4-6 monoclonal antibodies that immunoprecipitate GRK4, -5, and -6 (39). The resulting protein complexes were electrophoresed on a 7.5% polyacrylamide gel and then stained with Coomassie Blue or SYPRO ruby stain. Protein bands of ~90, ~80, and ~52 kDa were clearly observed in the GRK2/3 immunoprecipitation that were not evident in the GRK4-6 lane (Fig. 1). In contrast, there were no protein bands selectively immunoprecipitated in the GRK4-6 lane. This may reflect the 7-fold lower level of GRK6 compared with GRK2 in HL60 cells (38) and the lower efficiency of the GRK4-6 antibodies in immunoprecipitation.2 The 80-kDa band in the GRK2/3 lane was identified as GRK2 by immunoblotting using an anti-GRK2-specific monoclonal antibody. The 90- and 52-kDa bands were excised from the gel, digested with trypsin, and analyzed by mass spectrometry. The 90-kDa protein was identified as Hsp90-{beta} (19 matching peptides and 31% overall sequence coverage), whereas the 52-kDa protein was a proteolytic fragment of GRK2.



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  		FIG. 1.
Identification of GRK2-interacting proteins in HL60 cells. GRK2-interacting proteins were isolated by immunoprecipitation of GRK2 from HL60 cell lysates using an anti-GRK2/3 mAb and an anti-GRK4-6 mAb as a control as described under "Experimental Procedures." The samples were electrophoresed on a 7.5% SDS-polyacrylamide gel and then stained with SYPRO ruby protein stain. The arrows depict the proteins immunoprecipitated with anti-GRK2/3 mAb that were distinct from the bands immunoprecipitated with anti-GRK4-6 mAb. The strongest band at ~80 kDa was identified as GRK2 using an anti-GRK2 antibody, whereas the ~90- and ~52-kDa bands were excised, digested with trypsin, and analyzed by mass spectrometry. Western blotting with same source secondary anti-mouse IgG antibodies identified the heavy (HC) and light chain (LC) IgG bands.

 

To confirm the interaction of Hsp90 and GRK2, we used co-immunoprecipitation and immunoblotting. GRK2 from HL60 cells was immunoprecipitated using the anti-GRK2/3 mAb, electrophoresed on a 10% polyacrylamide gel, and analyzed for Hsp90 using an anti-Hsp90 monoclonal antibody (Fig. 2A). Interestingly, a 90-kDa band that was slightly larger than the Hsp90 band in the HL60 lysate was specifically detected in the anti-GRK2/3 mAb immunoprecipitation. This band was not detected using Hsp90 antibodies specific for either the {alpha} or {beta} isoforms (not shown), suggesting that the ~90-kDa Hsp90 that co-immunoprecipitates with GRK2 may be a different variant. Indeed, a number of Hsp90 variants have been identified including various polymorphisms of Hsp90-{alpha} and -{beta} (40) and Hsp90N, a 75-kDa Hsp90 that lacks the ansamycin-binding domain (41). In addition, analysis of the NCBI protein data base reveals a 737-amino acid Hsp90 variant (NCBI accession number T46243 [GenBank] ) that is 99.6% identical to Hsp90-{beta} over the first 709 residues but contains 13 additional amino acids within a divergent C-terminal tail. It is also possible that the Hsp90 that co-immunoprecipitates with GRK2 is post-translationally modified. However, treatment of the GRK2 immunoprecipitate with alkaline phosphatase did not change the mobility of the Hsp90 band, suggesting that it is not phosphorylated (not shown).



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  		FIG. 2.
Characterization of GRK2/Hsp90 interaction by immunoprecipitation. A, the interaction of GRK2 with Hsp90 was also demonstrated by immunoprecipitation of GRK2 from HL60 cell lysates using anti-GRK2/3 mAb and detection of Hsp90 using an anti-Hsp90 antibody as described under "Experimental Procedures." No Hsp90 co-immunoprecipitated with the anti-GRK4-6 mAb (right lane). B, to further verify that Hsp90 co-immunoprecipitated with GRK2, HL60 cells were treated with the Hsp90-specific inhibitor geldanamycin for 2 h at 37 °C. The cells were then lysed, immunoprecipitated with anti-GRK2/3 mAb, and analyzed by SDS-PAGE and Western blotting (WB) using an anti-Hsp90 mAb. The blot was also stripped and probed for GRK2 using an anti-GRK2 mAb, whereas the lysate was blotted for total Hsp90 and GRK2. IP, immunoprecipitate.

 
Geldanamycin specifically binds to the ATP/ADP pocket of Hsp90 and effectively disrupts Hsp90 interaction with a number of proteins (42). As further evidence that Hsp90 interacts with GRK2, we incubated HL60 cells with 1 µM geldanamycin for 2 h at 37 °C. Geldanamycin treatment was found to effectively block Hsp90 association with GRK2 as assessed by GRK2 immunoprecipitation (Fig. 2B). Thus, endogenous GRK2 appears to specifically associate with Hsp90 in HL60 cells.

Effect of Geldanamycin on the Expression, Maturation, and Degradation of Endogenous GRK2—An in vivo requirement for Hsp90 has been established for steroid hormone receptors (43), various protein kinases such as v-Src, Wee-1, Cdk4, Raf, and Akt (27, 44-46), and additional proteins such as nitric-oxide synthase (47) and calcineurin (48). Geldanamycin destabilizes the interaction between Hsp90 and its associated client proteins leading to accelerated degradation and loss of function. To assess the effect of Hsp90 interaction on GRK2 expression, HL60 cells were treated with various concentrations of geldanamycin for 24 h, and GRK2 levels were analyzed by immunoblotting. Geldanamycin induced down-regulation of GRK2 expression in a dose-dependent manner with 50% inhibition observed at ~0.3 µM and ~65% lower GRK2 levels at 10 µM geldanamycin (Fig. 3, A and B). This effect was time-dependent with 50% of the maximal reduction in GRK2 levels observed after an ~8-h treatment with 5 µM geldanamycin (Fig. 3C). The reduction in GRK2 levels suggests that association of GRK2 with Hsp90 is required for GRK2 stability.



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  		FIG. 3.
Inhibition of GRK2 expression in HL60 cells by geldanamycin. HL60 cells were treated with 0, 0.08, 0.4, 2, or 10 µM geldanamycin for 24 h at 37 °C, and total GRK2 levels were then analyzed by Western blotting (WB) using an anti-GRK2 mAb. A, shows a representative immunoblot of GRK2 and {alpha}-tubulin expression after geldanamycin treatment. B, shows the relative levels of GRK2 expression from three independent experiments quantified by densitometry. C, shows the time course of geldanamycin treatment. HL60 cells were incubated with 5 µM geldanamycin for 0, 1, 3, 9, or 27 h, harvested, and immunoblotted for total GRK2 and reprobed for {alpha}-tubulin as described under "Experimental Procedures."

 
One important function for Hsp90 is to catalyze the proper folding of newly synthesized client proteins. In the absence of Hsp90 action, the protein may be subject to rapid degradation. Thus, a decline in the steady state level of GRK2 following geldanamycin treatment could result from changes in the rate of GRK2 synthesis and/or degradation. To characterize these processes, HL60 cells were metabolically labeled with [35S]methionine/cysteine for 30 min to label newly synthesized proteins, chased for 0, 2, 6, or 18 h with complete medium, and then analyzed for GRK2 levels by immunoprecipitation, SDS-PAGE, and autoradiography.

To assess whether disrupting Hsp90/GRK2 interaction affects de novo GRK2 synthesis, geldanamycin was present during the entire pulse and chase periods (GA-1), whereas to assess whether Hsp90 affects GRK2 degradation, geldanamycin was present only during the chase period (GA-2). The half-life of GRK2 in HL60 cells in the absence of geldanamycin treatment was ~20 h (Fig. 4), similar to our previous studies that reported a half-life of 24 h in HL60 cells (38). When geldanamycin was present only during the chase period, the half-life of GRK2 was decreased to ~12 h, suggesting that Hsp90 interaction with GRK2 has a relatively modest stabilizing effect on the rate of GRK2 degradation (Fig. 4). In contrast, when geldanamycin was present during the pulse and chase periods, there was a dramatic increase in the rate of GRK2 degradation, with the half-life of GRK2 decreasing to <2 h (Fig. 4). In addition, although a single protein band of ~80 kDa was observed in the untreated and GA-2 samples, multiple protein bands were observed in the GA-1 samples. Our data also showed that degradation of GRK2 was mainly accelerated at the early chase times (Fig. 4B), suggesting that disruption of Hsp90 interaction during the synthesis of GRK2 results in proteolytic degradation of GRK2. Thus, the interaction of GRK2 and Hsp90 appears to inhibit proteolytic degradation of newly synthesized GRK2 and likely aids in GRK2 folding and maturation. It is interesting that the half-life for newly synthesized GRK2 in geldanamycin-treated HL60 cells (<2 h) is similar to the ~1-h half-life reported by Penela et al. (11, 49) in untreated C6 glioma, Jurkat, and transfected HEK-293 cells. This may reflect differences in co- and/or post-translational processes between these cell types, or it might suggest that Hsp90 interaction with GRK2 is lacking in some cells.



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  		FIG. 4.
Pulse-chase analysis of GRK2 expression in HL60 cells. A, HL60 cells were incubated for 30 min at 37 °C with medium containing [35S]methionine and [35S]cysteine and then chased with nonradioactive medium for the indicated time. Geldanamycin (1 µM) was either not present (Untreated) or was present during the entire pulse-chase period (GA-1) or just the chase period (GA-2). Cells were then washed and lysed, and total GRK2 was immunoprecipitated as described under "Experimental Procedures." A representative fluorogram is shown. B, the level of GRK2 expression from two to three independent experiments was quantified by densitometry and plotted.

 
Because Hsp90 has been shown to regulate the activity of some proteins (27, 30), we also assessed whether Hsp90 regulates GRK2 activity. GRK2 phosphorylation of the GPCR rhodopsin was found to be unchanged (94 + 10%) in the presence of 1.5 µM purified Hsp90 compared with a control performed in the absence of Hsp90. Similarly, 1.5 µM Hsp90 had no significant effect on GRK2 phosphorylation of the nonreceptor substrate tubulin (120 + 9% of control). Taken together, these results suggest that Hsp90 plays a primary role in GRK2 folding and maturation and does not appear to regulate the activity of the mature kinase.

Hsp90 binds numerous protein kinases primarily when they are in relatively inactive conformations. Hsp90 association with inactive kinases reflects its essential positive role in facilitating kinase folding, maturation, and activation rather than recognition of repressed kinase molecules themselves (46). However, it has been controversial whether Hsp90 is vital for normal maturation of cellular kinases. Our results suggest that the interaction of GRK2 with Hsp90 mainly stabilizes newly synthesized GRK2 including protein folding and maturation. This observation parallels the report of Xu et al. (31) that suggested that Hsp90 is necessary for the maturation of c-src and v-src. The discovery of Hsp90 regulating the folding and maturation of GRK2 has potential physiological significance, because increased GRK2 expression has been reported in several diseases. For example, increased levels of GRK2 have been reported in congestive heart failure (50) and hypertension (51, 52) patients. Thus, the use of ansamycins holds promise for the treatment of human diseases that overexpress GRK2.

Effect of Protease Inhibitors on Geldanamycin-promoted Degradation of GRK2—Most intracellular protein degradation is catalyzed by calpains, lysosomal proteases, and the ubiquitin-proteasome system (53). In an effort to determine whether any of these pathways are involved in the geldanamycin-promoted degradation of GRK2, various protease inhibitors were incubated with HL60 cells during a 3-h treatment with geldanamycin. Although geldanamycin treatment resulted in a modest decrease (~15%) in steady state GRK2 levels under these conditions, this decrease was effectively attenuated by the proteasome inhibitors lactacystin and MG132 and was partially blocked by the calpain inhibitor calpeptin (Fig. 5A). In contrast, the lysosomal protease inhibitor leupeptin had no effect on geldanamycin-promoted degradation of GRK2. Interestingly, a small amount of GRK2 was also found in a Triton-insoluble fraction, and this significantly increased in the geldanamycin-treated cells (Fig. 5B, compare first two lanes). Moreover, the accumulation of Triton-insoluble GRK2 was dramatically increased when cells were treated with both geldanamycin and the proteasome inhibitors (middle two lanes), whereas there was no significant increase observed with either the calpain or lysosomal protease inhibitors (last two lanes).



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  		FIG. 5.
Effects of protease inhibitors on geldanamycin-induced degradation of GRK2 in HL60 cells. A, HL60 cells were pretreated with or without lactacystin (10 µM), MG132 (10 µM), calpeptin (50 µM), or leupeptin (50 µg/ml) for 1 h followed by addition of geldanamycin for 3 h in the presence or absence of protease inhibitors. Cells were lysed, and GRK2 levels in Triton-soluble and -insoluble fractions were analyzed by Western blotting. The relative levels of GRK2 from four experiments were quantified by densitometry and plotted. *, p < 0.001 compared with control; **, p < 0.05 compared with geldanamycin alone. B, a representative immunoblot of Triton-insoluble GRK2 in the presence or absence of geldanamycin and various protease inhibitors.

 
In an effort to confirm these findings, we also looked at the effect of geldanamycin on GRK2 transiently expressed in COS-1 cells. Geldanamycin induced down-regulation of GRK2 expression in a dose-dependent manner with 50% inhibition observed at ~0.1 µM and ~90% lower GRK2 levels at 10 µM geldanamycin (Fig. 6A). Cells incubated with geldanamycin for 15 h in the presence or absence of various protease inhibitors suggested a role for the proteasome in GRK2 degradation (Fig. 6B), similar to our results in HL60 cells. Moreover, there was also a dramatic increase in the accumulation of Triton-insoluble GRK2 in the cells treated with geldanamycin and the proteasome inhibitors (Fig. 6C). This insoluble GRK2 showed a faint laddering pattern typically observed for ubiquitinated proteins, suggesting that ubiquitination of GRK2 might be involved in the degradation process. Interestingly, a similar laddering pattern is observed for Raf and Akt in the presence of Hsp90 inhibitors (36, 54). Taken together, our results suggest that Hsp90 interaction with GRK2 facilitates proper folding of the kinase and that disruption of this interaction leads to a misfolded insoluble form of GRK2 that is targeted for degradation by the proteasome pathway.



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  		FIG. 6.
Effect of geldanamycin and protease inhibitors on GRK2 expression in transfected COS-1 cells. A, COS-1 cells were transiently transfected with GRK2, and after 48 h the cells were treated with 0, 0.01, 0.1, 1, or 10 µM geldanamycin for 24 h at 37 °C. GRK2 expression was analyzed in cell lysates by Western blotting (WB) using an anti-GRK2 mAb. B, COS-1 cells were incubated in the presence or absence of geldanamycin and various protease inhibitors (lactacystin (10 µM), MG132 (10 µM), calpeptin (50 µM), or leupeptin (50 µg/ml)) for 15 h at 37 °C. Cells were lysed, and GRK2 levels in Triton-soluble (upper panel) and -insoluble (lower panel) fractions were analyzed by Western blotting. The lane numbers in panel B represent the addition of compounds in the experiments: 1, no addition, 2, GA; 3, GA + lactacystin; 4, GA + MG132; 5, GA + calpeptin; and 6, GA + leupeptin. C, the relative levels of soluble and insoluble GRK2 from two to three experiments were quantified by densitometry and plotted.

 
Previous studies have suggested that GRK2 degradation occurs through the ubiquitin-dependent proteasome pathway (49). Our results demonstrate that the degradation of GRK2 induced by Hsp90 inhibitors also mainly goes through this pathway. Although the mechanism by which GRK2 is targeted to the proteasome after Hsp90 inhibitor treatment is unknown, the ability of the purified 20 S proteasome to degrade GRK2 (49) suggests that disruption of the Hsp90-GRK2 complex may facilitate the direct interaction of GRK2 with the proteasome. Our data also suggest that GRK2 degradation induced by geldanamycin was accompanied by ubiquitination. Indeed, ubiquitination has been shown to be necessary for ansamycin-induced degradation of several protein kinases (54, 55).

Potential Role of Hsp90 on Expression of Other GRKs—The seven known mammalian GRKs can be divided into three subfamilies based on their overall structural organization and homology: GRK1 and -7; GRK2 and -3; and GRK4, -5, and -6. Common features shared by the GRKs include a central ~330-amino acid catalytic domain most related to those of other AGC kinases, such as cAMP-dependent protein kinase, akt, protein kinase C, and PDK1, an ~180-residue N-terminal region that contains a regulator of G protein signaling homology domain, and an ~60-160-amino acid C-terminal lipid-binding domain that varies in structure (1-3). In an effort to assess whether Hsp90 might have a role in stabilizing additional GRKs, we compared the effects of geldanamycin treatment on GRK2, GRK3, GRK5, and GRK6 transiently expressed in COS-1 cells. A 24-h treatment with 1 µM geldanamycin resulted in >80% down-regulation of all four GRKs, suggesting that Hsp90 may play a general role in promoting GRK folding and maturation during synthesis (Fig. 7).



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  		FIG. 7.
Effect of geldanamycin on GRK2, -3, -5, and -6 expression in transfected COS-1 cells. COS-1 cells were transiently transfected with GRK2, GRK3, GRK5, or GRK6, and after 48 h the cells were treated with or without 1 µM geldanamycin for 24 h at 37 °C. The levels of the indicated GRK were determined by Western blot analysis. The immunoblot is representative of two independent experiments.

 
An increasing number of signal transduction proteins appear to be dependent on Hsp90 interaction for folding and maturation (56). In terms of AGC protein kinases, only PDK1 and Akt have previously been demonstrated to be regulated by Hsp90. PDK1 interacts with Hsp90 via its kinase domain, and Hsp90 inhibitors result in proteasome-dependent degradation of PDK1 in HEK293T cells (57). It is interesting that the catalytic domain of PDK1 is most related to the catalytic domains of the GRKs (58). This suggests that the GRK catalytic domain is the region most likely to be involved in Hsp90 interaction. Treatment of cells with Hsp90 inhibitors also results in ubiquitination and proteasome-dependent degradation of Akt (54). However, Hsp90 has also been suggested to stabilize the active form of Akt (54), and Fontana et al. (59) have shown that Hsp90 enhances phosphorylation of endothelial nitric-oxide synthase by Akt, suggesting that Hsp90 may act as a scaffold facilitating Akt/endothelial nitric-oxide synthase interaction. Although our results suggest that Hsp90 plays an important role in GRK folding and maturation, we cannot rule out additional roles for Hsp90 in GRK function. Indeed, the demonstration that Hsp90 interacts with GPCRs (30), G proteins (28, 29), and GRKs may suggest a role for Hsp90 in formation of GPCR signaling complexes.


   FOOTNOTES
 
The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number T46243 [GenBank] .

* This work was supported by Grant GM44944 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 215-503-4607; Fax: 215-923-1098; E-mail: jeff.benovic{at}mail.tju.edu.

1 The abbreviations used are: GRK, G protein-coupled receptor kinase; GA, geldanamycin; Hsp90, heat shock protein 90; GPCR, G protein-coupled receptor; mAb, monoclonal antibody. Back

2 A. Pronin and J. L. Benovic, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. T. Som for valuable suggestions and discussions, Dr. A. Marchese for critical reading of the manuscript, and R. Wassell for mass spectrometry analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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