G Protein–Coupled Receptor Kinase GRK5 Phosphorylates Moesin and Regulates Metastasis in Prostate Cancer Free

Prostate cancer is the most common noncutaneous neoplasm diagnosed in the Western male population and is the second leading cause of cancer-related mortality in American men (1). Clinically localized prostate cancer can be successfully managed with radiotherapy or surgery, and prostate cancer–related deaths primarily result from cancer metastasis to distant organs (2). Cancer metastasis involves the cell local invasion and migration so that detached cells from the primary tumor mass can colonize at distant organs. Hence, molecular mediators involved in the cancer cell migration and invasion may serve as biomarkers and therapeutic targets.

G protein–coupled receptors (GPCR) are founding members of the superfamily of seven transmembrane-spanning receptors that regulate physiologic and pathophysiologic processes, including initiation and progression of cancer (3–5). Overexpression and activating mutations of GPCRs are linked to tumor growth, angiogenesis, and metastasis (4, 5), and targeting mutated or deregulated GPCRs are promising in experimental cancer therapy (6). GPCR signal transduction is regulated mainly by two groups of proteins, the β-arrestins and the GPCR kinases (GRK). There are seven GRK enzymes that can be divided into three subgroups (7): visual GRKs (GRK1 and GRK7); β-adrenergic receptor kinases (GRK2 and GRK3); and GRK4 subfamily (GRK4, 5, and 6).

Evidence is accumulating that deregulated GRKs, like GPCRs, contribute to human disease, albeit by incompletely understood mechanisms. Specifically, GRK5 was shown to phosphorylate tumor suppressor p53 and to enhance MDM2 binding to and ubiquitination of p53, leading to inhibition of p53-mediated apoptosis in response to DNA damage (8). GRK5 phosphorylates the non-GPCR substrate nucleophosmin, thereby altering sensitivity of breast cancer cells to polo-like kinase inhibitor-induced apoptosis (9). Also, GRK5 phosphorylates receptor tyrosine kinases platelet-derived growth factor receptor (10, 11) and VEGFR (12) that may play a role in cancer. Screening with shRNA library identified GRK5 as a potential regulator of cell-cycle progression (13), and GRK5 expression levels correlate with prostate cell proliferation in vitro and tumor growth in animals (14). Biochemically, depletion of GRK5 expression resulted in G₂–M arrest in prostate (14) and breast (15) cancer cells, reinforcing the idea that GRK5 regulates the cell-cycle progression.

ERM (ezrin–radixin–moesin) proteins link membrane components to actin cytoskeleton and play important roles in the cytoskeleton remodeling and cell adhesions to neighboring cells and to matrix (16). ERMs display similarity in domain organization, where the N-terminal domain (FERM) makes extensive interactions with the C-terminal domain and masks the ligand-binding sites (16). Phosphorylation of ERMs at T567, T564, and T558 of ezrin, radixin and moesin, respectively, by various kinases like PKCα or PKCϵ, weakens the interaction between the N- and C-terminal domains, thereby opening the folded protein and allowing the FERM to interact with membrane components and the C-terminal domain to interact with F-actin. ERMs are required for formation of focal adhesions and stress fibers (17), and altered expression or intracellular distribution of ERMs has been linked to tumor metastasis. In this study, we tested the idea that GRK5 regulates the prostate cancer cell migration in vitro and local invasion and metastasis in animals.

Anti-vinculin, anti-GRK5, anti-moesin, anti-Flag, and anti-GAPDH antibodies were purchased from Sigma; anti-phoshoTyrosine (pTyr) from Millipore; and anti-paxillin from BD Biosciences. Fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc., and rhodamine-conjugated phalloidin from Invitrogen. FBS was from HyClone; cell culture media F12K, RPMI-1640, and DMEM, and penicillin-streptomycin from Mediatech. Fibronectin and puromycin were from Sigma.

Full-length moesin cDNA in pCMV6-XL5 vector was obtained from OriGene Technologies, Inc., and subcloned with a N-terminal Flag-tag into pcDNA3.1(-) vector and sequenced. Mutations of threonine (T) residue to alanine (A) or glutamate (D) residues of the Flag-moesin at amino acid position 66 were performed with QuikChange site-directed mutagenesis kit (Stratagene). Moesin mutation primers were: T66A Forward: 5′-CTC AAT AAG AAG GTG GCT GCC CAG GAT GTG CGG AAG-3′; T66A Reverse: 5′-CTT CCG CAC ATC CTG GGC AGC CAC CTT CTT ATT GAG-3′; T66D Forward: 5′-CTC AAT AAG AAG GTG GAT GCC CAG GAT GTG CGG AAG-3′; T66D Reverse: 5′-CTT CCG CAC ATC CTG GGC ATC CAC CTT CTT ATT GAG-3′. Silencing of expression of moesin was performed by transient transfection with siRNA targeting moesin and scrambled siRNA was used as a negative control (Dharmacon).

The prostate cancer cell lines PC3, DU145, and LNCaP were all obtained from American Type Culture Collection and used within 6 months of purchase. Cells were grown in F12K (PC3), DMEM (DU145), and RPMI-1640 (LNCaP), supplemented with 10% FBS and 100 U penicillin/mL, and 100 μg streptomycin/mL. Cells were grown in a humidified atmosphere with 5% CO₂. For generating stable cell lines, control shRNA and shRNA targeting GRK5 (shGRK5-356: CGA AGG ACC ATA GAC AGA GAT; shGRK5-526: GAA GGA AAT TAT GAC CAA GTA) in lentiviruses were from Sigma (13). Resultant cell lines were selected with 2 μg/mL puromycin for 2 weeks. Rescue of GRK5 expression was performed by stable transfection with shRNA-resistant wobble mutant GRK5r or bovine kinase-inactive GRK5-K215Rr (14).

Cell migration assays were performed using two different approaches: Boyden chamber (8 μm pore size, BD Biosciences) and Xcelligence (Roche). For the Boyden chamber assay, cells were seeded at a density of 4.0 × 10⁴/100 μL in DMEM (DU145) or F12K (PC3) medium containing 0.1% (v/v) FBS in the upper chamber. In the lower chamber, 650 μL of DMEM or F12K media containing 2.5% FBS (i.e., directed movement) were added. After 16 hours of incubation, cells were fixed and stained with Diff-Quik solution. Cells in the upper chamber were removed using a cotton swab, and cells migrating through the membrane were photographed in three randomly selected fields and counted using ImageJ software (NIH). Cell invasion studies were performed using Boyden chamber equipped with membranes precoated with fibronectin (100 μg/mL; Sigma) or Matrigel (BD Biosciences).

For continuous monitoring of cell migration in real time, we used the Xcelligence device according to the manufacturer's protocol. Briefly, 4 × 10⁴ cells were seeded per well of CIM-plate 16 (Roche) in serum-free medium while the equilibrated lower chamber contained 10% FBS as a chemoattractant. The CIM-plate 16 containing the cells was placed in the RTCA DP Analyzer inside a cell culture incubator, and cell migration measurements were taken for 24 hours at 15-minute intervals. The readings were analyzed with RTCA software, replicates were averaged, and the background recording was subtracted to calculate the rate of actual cell migration per unit time.

Exponentially growing cells were washed with PBS and lysed in lysis buffer [25 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 2 μg/mL pepstatin A]. Cleared lysates were used for immunoblot analysis or incubated with antibodies overnight for immunoprecipitation, followed by incubation with protein A/G beads for 1 hour at 4°C. Immune complexes on protein A/G beads were washed with lysis buffer and immunoprecipitated proteins were boiled into SDS-PAGE sample buffer. Western blot analysis was used to determine relative protein expression using an enhanced chemiluminescence substrate detection system.

For detection of focal adhesions or moesin, cells were seeded on fibronectin-coated (25 μg/mL) coverslips for 6 hours in serum-free medium and fixed with 2% formaldehyde. Cells were permeabilized in a buffer containing 10% FBS, 0.2% saponin, and 0.04% NaN3 in PBS and stained using anti-vinculin, anti-paxillin, anti-pTyr, or anti-moesin antibodies. F-actin was visualized by staining cells with rhodamine-conjugated phalloidin. Slides were examined using an epifluorescence microscope (DM 6000B, Leica) equipped with a 63× /1.4–0.6 oil immersion lens, or a Leica confocal microscope (TCS SP5; Leica) equipped with a 63×/1.4–0.6 oil immersion lens. Images were captured and analyzed using the application suite Advanced Fluorescence 2.0.2 software (Leica). For counting focal adhesions, anti-vinculin or anti-paxillin antibody-stained linear structures at the ends of large actin bundles or stress fibers with length of 3 to 10 μm were counted (18).

Isolation of pseudopodia was performed as described by Lafarga and colleagues (19). Briefly, cell culture inserts (Falcon) with 3.0 μm pore PET track-etched membrane were coated with fibronectin (25 μg/mL). Cells were serum starved for 16 hours, detached by HyQtase and seeded onto the top of inserts at 1 × 10⁴ cells in serum-free medium for 2 hours. The serum-free medium in the lower chamber was then replaced with one containing 10% FBS and the cells were incubated for additional 1.5 hour. The membranes were fixed in methanol for 5 minutes, the cell bodies were lysed, and the pseudopodia were scraped off from lower surface of the filter and boiled in Laemmli buffer. The isolated pseudopodia fractions were resolved by SDS-PAGE and subjected to Western blot analysis.

For spreading assay, 2 × 10⁴ cells resuspended in Opti-MEM were seeded on fibronectin-coated (25 μg/mL) coverslips for up to 3 hours. Cells were fixed and processed for immunofluorescence staining and examined by confocal microscopy. For cell spreading, the size of at least 50 cells was measured using the Volocity software (PerkinElmer). For cell adhesion, number of cells attached to the surface was counted from three randomly selected fields under ×40 magnification.

Phosphorylated proteins were isolated from shCon-PC3 and shGRK5-PC3 cells using the phosphoprotein enrichment kit (Pierce) following the manufacturer's protocol. Briefly, cells grown on 15 cm dishes were washed twice with HEPES, pH 7.0, followed by addition of 1.0 mL lysis/binding/wash buffer containing CHAPS and protease and phosphatase inhibitor cocktails (all provided by the manufacturer). Cells were harvested and incubated on ice for 45 minutes with periodic vortexing. Cellular debris was removed by centrifugation and equal amounts of protein (4 mg) were mixed with resin and incubated on a rocker for 30 minutes at 4°C. The eluted enriched phosphoproteins were resolved by SDS-PAGE and the gel was stained with Coomassie blue. Bands showing difference in intensity between control and GRK5 knockdown cells were excised and the proteins were identified by mass spectrometry at the University of Florida Interdisciplinary Center for Biotechnology Research (UF ICBR) Proteomics Core.

In vitro phosphorylation of moesin was performed by coincubating purified moesin (800 nmol/L; Abcam) or tubulin (200 nmol/L; Cytoskeleton) together with GRK5 (200 nmol/L; Sigma) proteins in kinase buffer (20 mmol/L Tris-HCl, pH 7.5, 2 mmol/L EDTA, and 5 mmol/L MgCl2) containing 0.2 mmol/L [γ-³²P]ATP (2.0–7.5 cpm/fmol) for up to 90 minutes at 30°C. SDS-PAGE sample buffer was added to terminate the phosphorylation reaction and proteins were resolved by SDS-PAGE and subjected to autoradiography. To determine site(s) of phosphorylation, the in vitro kinase assay was performed with nonradiolabeled ATP. Following incubation and resolution on SDS-PAGE, the gel was stained with Coomassie blue and the moesin band was excised and subjected to mass spectrometry analysis at the UF ICBR Proteomics Core.

To confirm site of moesin phosphorylation by GRK5, HEK293 cells overexpressing wild-type Flag-moesin or Flag-moesin-T66A–mutated form were subjected to immunoprecipitation using Flag antibody. Immunocomplexes were subjected to in vitro kinase assay using purified GRK5 protein (100 nmol/L), followed by fractionation on SDS-PAGE, gel staining, and autoradiography.

The procedure for renal capsule grafting is described at http://mammary.nih.gov/tools/mousework/Cunha001/index.html. Briefly, male athymic nu/nu homozygous nude mice (6–8 weeks old) were anesthetized, and the kidney was exposed through a dorsal incision. An incision was made in the epithelial layer capsule, and cell pellet (5 × 10⁵) formed in collagen was placed underneath the epithelial cell sheath. After implanting, the kidney was placed back into its position, absorbable sutures were used to close the muscle, and the skin was stapled. Six weeks following tumor cell implantation, mice were sacrificed and the grafts were removed for analysis. All procedures were performed as per Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Florida.

Subrenal tumor grafts or mouse tissues were embedded in paraffin and sectioned (5 μm). These sections were deparaffinized in xylene, rehydrated in graded alcohol, subjected to heat-induced antigen retrieval with Target Retrieval Solution, and blocked with Protein Block. Implanted tumor or mouse tissues were probed with rabbit anti-human LDHA (1:100) or rabbit anti-human Ki-67 (1:100) antibody. Samples were developed with AEC substrate, counter-stained with hematoxylin, and mounted with Faramount. Images were taken using Nikon Eclipse 50i microscope equipped with a DS-Fi1 camera and NIS-Elements BR3.1 software.

Data are presented as the mean ± SEM from at least three independent experiments. Statistical significance was calculated by Student t test or one-way ANOVA with Tukey posttest. Graphs were generated using Prism software (GraphPad) and axis labels were generated using Adobe Illustrator (Adobe).

To provide evidence for GRK5's role in cell migration and invasion, we generated prostate cancer cell lines with stable knockdown of GRK5 (Fig. 1A). The knockdown of GRK5 did not affect expression of family-member GRK2 (data not shown). Control PC3 cells migrated efficiently toward an FBS gradient (Fig. 1B), and the knockdown of endogenous GRK5 expression resulted in a marked decrease in the cell migration (Fig. 1B). Similar results were obtained in DU145 cells (Fig. 1C), excluding the possible cell type-specific response. Quantitation of results indicated that the knockdown of GRK5 attenuated the PC3 cell migration by 65% (Fig. 1B) and DU145 cells by 54% (Fig. 1C).

To gain confidence in the cell migration results using the Boyden chamber assays that were determined at fixed times, we measured the cell migration rate in real-time with the Xcelligence device. GRK5 knockdown in PC3 (Fig. 1D) and DU145 (Fig. 1E) cells evidenced a decreased cell migration rate that was lowered by 3.5-fold in shGRK5-PC3 cells at 24 hours (Fig. 1D) and by 1.7-fold in shGRK5-DU145 cells at 22 hours (Fig. 1E). GRK5 knockdown, however, showed no significant effect on the cell proliferation rate for up to 24-hour incubation (Fig. 1F). These results support the idea that GRK5 is required for the directional prostate cancer cell migration.

Cancer metastasis involves invasion through basement membranes and we examined whether GRK5 affects the cell invasion. Control PC3 and DU145 cells invaded a fibronectin matrix, and knockdown of GRK5 inhibited the cell invasion (Fig. 1G and H). Quantitation of invading cell numbers indicated that decreased expression of GRK5 reduced invasion of shGRK5-PC3 by 67% (Fig. 1G) and shGRK5-DU145 cells by 44% (Fig. 1H). Similar results were obtained using Matrigel as the invaded matrix (Fig. 1I and J); the knockdown of GRK5 (with two distinct shRNA sequences) reduced invasion of shGRK5-PC3 cells by 50% (Fig. 1I) and shGRK5-DU145 cells by 50% (Fig. 1J). Therefore, the expression of GRK5 protein is required for the effective migration and invasion of prostate cancer cells.

Cell migration is a critical step for the dissemination of primary cancer cells to distant organs and we used in vitro assays to estimate the effect of GRK5 expression on several parameters of cell migration. As shown in Fig. 2A, the knockdown of GRK5 expression increased attachment of both DU145 and PC3 cells to a fibronectin-coated surface. Also, control PC3 and DU145 cells exhibited time-dependent increases in spreading on the fibronectin-coated surface, and knockdown of GRK5 enhanced the spreading of both cell types (Fig. 2B). These results establish that GRK5 regulates the cell attachment and spreading.

Cell migration requires constant formation and remodeling of cell–cell and cell–matrix adhesions. Focal adhesions are dynamic supramolecular structures that connect actin cytoskeleton with extracellular matrix. We examined focal adhesions by staining cells with antibodies against vinculin, paxillin, and pTyr, commonly used markers of focal adhesions. Representative images of focal adhesions in DU145 cells observed by visualizing vinculin are shown in Fig. 2C, and Western blot results evidenced a lack of effect for GRK5 knockdown on the total vinculin protein expression (data not shown). Control DU145 cells formed an average of 4 to 5 focal adhesions under our assay conditions (Fig. 2D) and the knockdown of GRK5 increased focal adhesion numbers to 7 to 9 per cell (Fig. 2D). Similar effects of GRK5 knockdown on focal adhesions were observed in PC3 cells (data not shown).

GRK5 may regulate cellular functions in a kinase-dependent or -independent manner (20). We examined whether the effects of GRK5 on prostate cancer cell migration and invasion required its kinase activity. For this purpose, we depleted endogenous GRK5 by stable knockdown, and replenished cells with shRNA-resistant wild-type GRK5 (GRK5r) or kinase-inactive GRK5-K215Rr forms (Fig. 3A). In control PC3 cells, forced overexpression of GRK5r did not impact the cell migration (Fig. 3B), suggesting these cells express sufficiently high levels of endogenous GRK5 to control the cell migration. However, forced overexpression of GRK5-K215Rr decreased the cell migration by 67% (Fig. 3B), suggesting a dominant negative role for the overexpressed kinase-inactive GRK5. In cells with GRK5 knockdown, rescued expression of GRK5 partially restored the cell migration, while replenishing cells with the kinase-inactive GRK5-K215Rr failed to do so (Fig. 3B), implying a requirement for GRK5 kinase activity in cell migration.

We also examined requirement of GRK5 kinase activity on the cell invasion. Similar to the cell migration results, forced overexpression of GRK5r exhibited no effect on the cell invasion, and forced overexpression of the kinase-inactive GRK5-K215Rr reduced invasion of the control cells (Fig. 3C). In GRK5 knockdown cells, rescued expression of GRK5 partially reversed the cell invasion, whereas forced overexpression of GRK5-K215Rr was ineffective (Fig. 3C). Therefore, the kinase activity is needed for GRK5 to regulate the prostate cancer cell migration and invasion.

In addition to phosphorylating GPCRs, GRKs are now recognized to phosphorylate non-GPCR substrates (20). Phosphorylated proteins were isolated from shCon-PC3 and shGRK5-PC3 cells, resolved by gel chromatography, and differentially stained bands (Supplementary Fig. S1) were excised and analyzed by mass spectrometry (Supplementary Table S1). The putative GRK5 substrates may be classified into functional groups, including cytoskeletal remodeling; metabolism for carbohydrates, proteins, fatty acids, and nucleotides; endoplasmic reticulum stress, protein maturation and trafficking; and protease-related proteins. The identified proteins included known GRK5 substrates such as α-actinin (21), thereby increasing confidence in the results. These studies, nonetheless, need to be done in a quantitative manner to validate the identified proteins as bona fide GRK5 substrates. We focused on moesin, which is reported to play a role in cancer cell migration and metastasis. We began by confirming the role of moesin in cell migration and invasions and found that the silencing of moesin expression caused a marked reduction in the prostate cancer cell migration and invasion. (Supplementary Fig. S2).

Similar to the reported expression pattern of GRK5 (14), the level of moesin correlated with aggressiveness of prostate cancer cells, with the highest level in the most tumorigenic and invasive PC3 cells followed by DU145, whereas the noninvasive LNCaP cells showed least amount of moesin (Fig. 4A). Next, we asked whether GRK5 regulated the subcellular distribution of moesin. In control DU145 and PC3 cells, moesin was observed in the cell periphery as filamentous spikes (Fig. 4B). The similar staining of LNCaP cells evidenced a weak signal (data not shown), consistent with the lack of moesin expression in these cells (Fig. 4A). Stable knockdown of GRK5 resulted in the disappearance of moesin-stained filamentous structures and instead, it was distributed around the cell periphery with some cytosolic presence (Fig. 4B). These results indicate that GRK5 regulates the subcellular localization of moesin.

Moesin regulates actin cytoskeleton remodeling that plays active roles in cell migration, and migrating cells form protrusive structures, including pseudopodia (22). We harvested the pseudopodia from migrating PC3 and DU145 cells and could show that they express both GRK5 and moesin (Fig. 4C). Cortactin served as a positive control for the pseudopodia fraction, and PARP was used as a negative control to show absence of total cell body components.

To provide more evidence that GRK5 regulates moesin, we examined whether the two proteins colocalize. Overexpressed Flag-GRK5 was enriched on the plasma membrane and colocalized with endogenous moesin on the plasma membrane of model HEK293 cells (Fig. 4D). Furthermore, endogenous GRK5 coimmunoprecipitated endogenous moesin from both PC3 and DU145 cell lysates (Fig. 4E). The colocalization and complex formation of GRK5 and moesin suggested that moesin serves as a substrate for GRK5, and we tested this possibility by performing in vitro kinase assays using purified proteins. As shown in Fig. 4F, GRK5 catalyzed the phosphorylation of moesin in a time-dependent manner. At 30-minute incubation, the stoichiometry of GRK5 phosphorylation of moesin was calculated to be only 0.02 mol/mol. However, the calculated stoichiometries of GRK5 autophosphorylation as well as phosphorylation of bona fide substrate tubulin were 0.3 and 0.2 mol/mol, respectively, suggesting needed improvements in the assay conditions and/or enzyme quality.

To identify the residue(s) in moesin that are phosphorylated by GRK5, mass spectrometry was performed after the in vitro kinase assay. Results show that GRK5 phosphorylated moesin on T66 (Fig. 5A), a novel phosphorylation site. This site of phosphorylation by GRK5 was also identified with bioinformatics using KinasePhos2.0 software tool with a score of 0.546996 that predicts a modest probability of phosphorylation by GRK5. We also tested for PKC-mediated phosphorylation sites in moesin using this software and T66 residue was not retrieved. Comparative in vitro kinase assay using purified GRK5 and immunoprecipitated Flag-moesin or Flag-moesin-T66A evidenced a markedly reduced phosphorylation content of moesin-T66A (Fig. 5B). To explore the functional consequence of moesin phosphorylation by GRK5, we transfected shCon-PC3 and shGRK5-PC3 cells with cDNAs encoding Flag-moesin, phosphorylation-deficient Flag-moesin-T66A, or phosphomimetic Flag-moesin-T66D, and examined the effect on cell spreading (Fig. 5C). Overexpression of wild-type moesin or moesin-T66D forms inhibited the cell spreading (Fig. 5D and E), whereas forced expression of moesin-T66A increased it (Fig. 5F), consistent with the observations that GRK5 knockdown increased the cell spreading (Fig. 2B).

We examined the role of GRK5 in tumor growth and metastasis. Exemplar PC3 cells with or without GRK5 knockdown were embedded in collagen and implanted underneath the renal capsule of athymic nude mice. Control PC3 cells formed tumors at 6 weeks of implantation (Fig. 6A) and the average tumor weight was 490 ± 58.6 mg (Fig. 6B). Knockdown of GRK5 significantly inhibited the tumor formation (Fig. 6A) and the average weight of these tumors was 59 ± 12.9 mg (Fig. 6B).

We examined the tumor tissues by immunohistochemical staining with anti-human lactate dehydrogenase A (LDHA) antibodies, and measured the invaded parenchyma area of the kidneys (Fig. 6C). Control shCon-PC3 tumors invaded 44% of the total mouse kidney area compared with 14% of the total area invaded by tumor cells harboring GRK5 knockdown (Fig. 6D). We also examined the cancer cell metastasis to regional lymph nodes, which was observed in the renal (RLN) and ureter (ULN) lymph nodes, but not the accessory axillary lymph node (ALN) in mice harboring shCon-PC3 tumors, while in shGRK5-PC3 tumors, the local metastasis to lymph nodes was markedly reduced (Fig. 6E). Specifically, there was about 88% and 89% metastatic probability in RLN and ULN in the control group compared with 22% and 33% probability of metastasis to RLN and ULN in GRK5 knockdown group (Fig. 6F).

Prostate cancer cells metastasize to distant organs, including lungs, liver, and bones. Specific anti-human LDHA and Ki-67 (a proliferation marker; data not shown) staining detected lung and liver metastasis of implanted shCon-PC3 cells (Fig. 6G). The knockdown of GRK5 decreased probability of lung and liver metastases to 44% and 22%, respectively (Fig. 6H), showing that GRK5 regulates the prostate tumor growth and metastasis.

GPCRs are the canonical substrates for GRKs that, in addition, are now recognized to phosphorylate non-GPCR proteins. For example, GRK2 was shown to phosphorylate transcription factor p53 and to regulate its nuclear translocation and transcriptional activities (23). Cytoskeleton proteins, including ezrin and radixin, were also shown to be phosphorylated by GRK2 (24). Uniquely, GRK5 encodes a nuclear localization signal and had been shown to phosphorylate histone deacetylase 6 (25, 26). In this study, we provide evidence that GRK5 phosphorylates cytosolic moesin protein, with functional consequences on actin remodeling, invasion, and metastasis of prostate cancer cells.

ERM proteins contain an N-terminal FERM domain that mediates membrane association, and a C-terminal domain that binds actin. Intramolecular interaction between amino and carboxyl termini negatively regulates the function of ERM proteins (16). For example, the binding of phosphatidylinositol-4, 5-bisphosphate induces conformational changes to expose the membrane-binding site of ERMs (27). Phosphorylation also plays a role in the activation of ERMs that have been reported to be substrates of several kinases, including PKC, NF-κB–inducing kinase, lymphocyte-oriented kinase, and MST4 (28–31). Our results show that ubiquitous GRK5 colocalizes with moesin on the plasma membrane of prostate cancer cells and catalyzes the moesin phosphorylation.

Previously, GRK2 was the only GRK family member reported to phosphorylate ezrin at T567 (32) and radixin at T564 (24) residues. Phosphorylation of moesin by GRK5 suggests that select GRKs may elicit specific regulation of a particular ERM. Also, moesin was demonstrated to be phosphorylated at T558 (33), and we now identified T66 as another phosphorylation site of moesin that may add to the complexity of the regulatory mechanisms governing its function. Remarkably, various GRKs exhibit distinct subcellular distribution patterns and activation mechanisms (20). Hence, extracellular signals that preferentially activate GRK5 (vs. GRK2) may promote the moesin (vs. ezrin/radixin) activation and consequent specific modulation of the actin cytoskeleton.

Moesin has been implicated in cell spreading (34, 35), and our results suggest that phosphorylation of moesin by GRK5 regulates moesin function. Forced overexpression of the phosphomimetic moesin-T66D form attenuated the cell spreading, whereas forced expression of the phosphorylation-deficient moesin-T66A enhanced it. Combined with the observation that knockdown of GRK5 increased the cell spreading, our results suggest that the function of moesin in cell spreading and cell migration is controlled, at least in part, by GRK5.

Cancer metastasis involves the active remodeling of cell–cell and cell–matrix adhesions, both of which are regulated by ERMs. Accumulating evidence suggests a role for ERMs in cancer progression. For example, the expression level of ezrin is increased in metastatic pancreatic and breast cancers (36, 37), and moesin mRNA expression is amplified in head and neck cancers (38). We found that the expression level of moesin, and its regulator GRK5, correlates with aggressiveness of prostate cancer cell lines. Regulation of actin cytoskeleton remodeling through moesin may contribute to the mitogenic effects of GRK5.

In summary, GRK5 regulates prostate cancer cell migration and invasion. GRK5 forms a complex with moesin, phosphorylates moesin principally on T66 residue, and regulates cellular distribution of moesin. In addition, GRK5 is required for the in vivo growth, invasion, and metastasis of prostate cancer. Therefore, GRK5 may serve as a drug target to more effectively treat patients with advanced prostate cancer.

No potential conflicts of interest were disclosed.

This article was prepared while Z. Nie was employed at the University of Florida College of Medicine. The opinions expressed in this article are the author's own and do not reflect the view of the NIH, the Department of Health and Human Services, or the United States government.

Conception and design: P.K. Chakraborty, Z. Nie, Y. Daaka

Development of methodology: P.K. Chakraborty, Y. Zhang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.K. Chakraborty, Y. Zhang, A.S. Coomes, W.-J. Kim, R. Stupay, T. Atkinson, J.I. Kim

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P.K. Chakraborty, Y. Zhang, W.-J. Kim, R. Stupay, T. Atkinson, Z. Nie, Y. Daaka

Writing, review, and/or revision of the manuscript: P.K. Chakraborty, Y. Zhang, T. Atkinson, Z. Nie, Y. Daaka

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P.K. Chakraborty, L.D. Lynch

Study supervision: P.K. Chakraborty, Y. Daaka

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.