Phosphorylation of Ralb Is Important for Bladder Cancer Cell Growth and Metastasis

Published OnlineFirst October 12, 2010; DOI: 10.1158/0008-5472.CAN-10-0952 Published OnlineFirst on October 12, 2010 as 10.1158/0008-5472.CAN-10-0952

Therapeutics, Targets, and Chemical Biology Cancer Research Phosphorylation of RalB Is Important for Bladder Cancer Cell Growth and Metastasis

Hong Wang1, Charles Owens1, Nidhi Chandra1, Mark R. Conaway2, David L. Brautigan3,4, and Dan Theodorescu5

Abstract RalA and RalB are monomeric G proteins that are 83% identical in amino acid sequence but have paralogue- specific effects on cell proliferation, metastasis, and apoptosis. Using in vitro kinase assays and phosphosite- specific antibodies, here we show phosphorylation of RalB by protein kinase C (PKC) and RalA by protein kinase A. We used mass spectrometry and site-directed mutagenesis to identify S198 as the primary PKC phosphorylation site in RalB. Phorbol ester [phorbol 12-myristate 13-acetate (PMA)] treatment of human bladder carcinoma cells induced S198 phosphorylation of stably expressed FLAG-RalB as well as endogenous RalB. PMA treatment caused RalB translocation from the plasma membrane to perinuclear regions in a S198 phosphorylation–dependent manner. Using RNA interference depletion of RalB followed by rescue with wild-type RalB or RalB(S198A) as well as overexpression of wild-type RalB or RalB(S198A) with and without PMA stimulation, we show that phosphorylation of RalB at S198 is necessary for actin cytoskeletal organization, anchorage-independent growth, cell migration, and experimental lung metastasis of T24 or UMUC3 human bladder cancer cells. In addition, UMUC3 cells transfected with a constitutively active RalB(G23V) exhibited enhanced subcutaneous tumor growth, whereas those transfected with phospho-deficient RalB(G23V-S198A) were indistinguishable from control cells. Our data show that RalA and RalB are phosphorylated by different kinases, and RalB phosphorylation is necessary for in vitro cellular functions and in vivo tumor growth and metastasis. Cancer Res; 70(21); OF1–10. ©2010 AACR.

Introduction whereas RalB was found to be essential for survival of a variety of tumor cell lines (15, 16). Studies using geranylger- The Ral (Ras-like) proteins in the superfamily of small anyltransferase I inhibitors (GGTI-2417) showed expression GTPases are the closest relatives of Ras, with 55% amino acid of GGTI-resistant farnesylated RalB, but not farnesylated sequence identity (1). The two Ral genes, RalA and RalB, are RalA, conferred resistance to the proapoptotic and anti– expressed in human tissues and produce proteins that are anchorage-dependent growth effect of GGTI-2417. Conversely, >80% identical in sequence. Ral proteins are downstream ef- farnesylated RalA, but not farnesylated RalB, rescued anchor- fectors of Ras and contribute to Ras-induced transformation age-independent cell proliferation in MiaPaCa2 cells (17). (2–6). Ral proteins have also been implicated in the regulation Knockdown of RalA in pancreatic cancer cells impaired tu- of endocytosis, exocytosis, actin cytoskeletal dynamics, and mor formation (12), whereas knockdown of RalA in prostate gene transcription (5, 7, 8). In addition, RalA and RalB contrib- cancer cells inhibited bone metastasis (11). RalB depletion re- ute to cancer cell migration, invasion, and metastasis (9–14). duced bone metastasis in DU145 but not PC3 cells (11), where- Ral GTPases have both overlapping and distinctive roles in as it inhibited metastasis by pancreatic cells, suggesting the these cellular processes. Studies using RNA interference cellular context may be important in determining the role of (RNAi) revealed that depletion of RalA severely impaired Ral paralogues in cancer. In bladder cancer, RalB but not RalA the anchorage-independent proliferation of cancer cell lines, depletion by RNAi inhibited migration, whereas combined knockdown of both RalA and RalB inhibited growth (10). In

Authors' Affiliations: Departments of 1Molecular Physiology and vesicle transport, active RalA but not RalB was found to pro- Biological Physics, 2Public Health Sciences, and 3Microbiology and mote the assembly of the exocyst and enhance polarized de- 4Center for Cell Signaling, University of Virginia School of Medicine, livery of membrane proteins (18). Together, these findings Charlottesville, Virginia and 5University of Colorado Comprehensive Cancer Center, Denver, Colorado show that, despite extensive sequence identity, these Ral proteins have different cellular functions. Hence, we anticipate Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). that Ral paralogues respond to different signals and/or Corresponding Author: Dan Theodorescu, University of Colorado Com- engage different downstream effectors, but this has not yet prehensive Cancer Center, Denver, CO 80045. Phone: 303-724-7135; been shown. Fax: 303-724-3162; E-mail: [email protected]. Pathways that activate Ral GTPases include the GDP/GTP doi: 10.1158/0008-5472.CAN-10-0952 cycle regulated by guanine nucleotide exchange factors (GEF) 2+ ©2010 American Association for Cancer Research. and elevation of intracellular Ca levels leading to interaction

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of Ral GTPases with calmodulin (7). Recently, phosphorylation Cells, transfection, immunoprecipitation, and of RalA was found to regulate its activity in cell transformation Western blot (19–21). RalA was found as a phosphoprotein by searching for UMUC3, RT112, and T24 human bladder cancer cells and Aurora-A kinase substrates using small pool expression HEK293T cells were obtained from American Type Culture screening. Aurora-A kinase phosphorylates RalA at S194, in- Collection and cultured as described (http://www.atcc.org/). creasing RalA activity and translocating RalA (19, 21). Proteo- The cells were transfected using FuGene 6 (Roche) according mic analyses found RalA in complex with the Aβ subunit of to the manufacturer's instructions. T24 cells were transfected protein phosphatase 2A (PP2A). PP2A dephosphorylates RalA using Lipofectin (Invitrogen). UMUC3 cells or T24 cells stably at S183 and S194 and inactivates the protein (20). Currently, expressing wild-type FLAG-Ral or mutant Ral or GFP-Ral or nothing is known regarding RalB phosphorylation, but the S183 mutant GFP-Ral were selected in medium containing G418. and S194 sites in RalA are not conserved in RalB. These data led Surviving cells were pooled, and exogenous Ral protein expres- us to hypothesize that phosphorylation may be paralogue spe- sion was analyzed by Western blot. Surviving GFP-transfected cific and that different kinases may regulate the Ral paralogues. cells were pooled and sorted using fluorescence-activated cell Here, we test this hypothesis and show that RalB is phosphor- sorting (FACS; Flow Cytometry Core Facility, University of ylated at S198 by protein kinase C (PKC). RalA and RalB are Virginia) to get stable cells with equal expression. For immu- phosphorylated differentially by protein kinase A (PKA) and noprecipitation, the extracts were incubated with anti-FLAG PKC. The S198 phosphorylation site is necessary for the effects M2 agarose or specific antibodies with beads for 2 hours at of RalB on anchorage-independent growth and cell motility 4°C. The beads were washed and then either used as substrates in vitro and the development of lung metastasis in vivo.Further- for kinase assay or subjected to SDS-PAGE for Western blot. more, S198 is necessary for manifestation of the growth promot- Lentiviral short hairpin RNA (shRNA) expression vectors tar- ing effect of GTP loaded RalB on in vivo tumor growth. Together, geting RalB were purchased from Sigma, and UMUC3 stable RalB these data suggest RalB phosphorylation is an independent knockdown cells were established as manufacturer's instruc- mechanism of regulating the biological activity of RalB. tions. RalB depleted cells were transfected with shRNA-resistant FLAG-RalB or mutant and selected in the medium containing Materials and Methods G418. Surviving cells were pooled and analyzed by Western blot.

Biochemical reagents and DNA constructs In vitro kinase assay Anti-RalB antibodies were from R&D Systems and Milli- GST-RalA or GST-RalB fusion proteins or immunoprecipi- pore. Monoclonal anti-FLAG M2 antibody, EZview Red Anti- tates of FLAG-RalA, FLAG-RalB, or the mutants were incu- FLAG M2 Affinity Gel, and phorbol 12-myristate 13-acetate bated with purified kinases in kinase reaction buffer with (PMA) were from Sigma-Aldrich. PKC kinase inhibitor [γ-32P]ATP for 30 minutes at 30°C. The products were ana- Go-6983, calyculin-A, and anti-α-tubulin monoclonal antibody lyzed by SDS-PAGE and autoradiography. Phosphorylation were from Calbiochem. Anti-green fluorescent protein (GFP) was quantified by excising the bands corresponding to the antibody was from Santa Cruz. [γ-32P]ATP was purchased proteins and measuring radioactivity with a Beckman Model from Perkin-Elmer Life and Analytical Sciences. NH2 terminal LS6500 scintillation counter. tagged glutathione S-transferase (GST)–RalA or GST-RalB fusion proteins were expressed in Escherichia coli strain Cell fractionation BL21 (DE3) and purified by GST-Bind kits (Novagen) accord- RT112 cells were treated with or without 100 ng/mL PMA ing to the manufacturer's instructions. PKA was purified to ho- and 20 nmol/L calyculin-A for 10 minutes after overnight mogeneity from bovine heart (22). PKC was purified by serum starvation. Cell membranes were prepared with sequential chromatography as multiple PKC isoforms from ProteoExtract Native Protein Extraction kit (Calbiochem) fol- human RBC (23). Recombinant human Aurora-A kinase was lowing the manufacturer's instructions. Protein concentra- expressed in bacteria and purified on Ni-NTA agarose (24). tions were determined by BCA protein assay (PIERCE). Plasmids encoding full-length human RalA and RalB were constructed in pCMV4-FLAG vector (Sigma-Aldrich) provid- Immunofluorescence ing an NH2 terminal FLAG tag. GFP-RalB was constructed in For immunofluorescence, the cells were plated on the cover pEGFP-C1 vector (Clontech). G23V mutants for active RalB or glasses. The cells were fixed with 4% paraformaldehyde and phosphosite mutants, RalB(S198A) and RalB(G23V-S198A), permeabilized with 0.25% Triton X-100. The cells were stained phosphomimetic RalB(S198D), were generated by QuickChange with phalloidin-Alexa Fluor 594 (Molecular Probes) to visual- site-directed mutagenesis (Stratagene). ize actin filaments and 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Images were acquired using Zeiss LSM 510 Generation of phospho-specific RalB antibody Meta confocal microscope. Phosphospecific RalB antibody was generated by AbboMax, Inc. Briefly, two rabbits were immunized with KHL-conjugated Wound assay and Transwell migration assay RalB peptide centered on phospho-S198. The antisera Wound assay was performed as previously described (25). were adsorbed against immobilized nonphosphopeptide, T24 stable cells (2 × 105) were plated in a six-well plate in and the phospho-specific antibody affinity was purified with triplicate and incubated for 2 days. The cells were serum phosphopeptide. starved in HyQ-CCM1 medium for 24 hours, scraped, and

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assayed with or without PMA. The assay was terminated Results when the wound of PMA-stimulated FLAG vector cells was almost closed. The wound was quantified using ImageJ. Cell RalB is phosphorylated at S198 by PKC migration was performed as described (10). UMUC3 stable The phosphorylation of RalA by Aurora-A has been cells (2 × 104) were seeded to the upper chamber of Transwell reported, but the corresponding region of RalB has a differ- filters (8.0-μm pores, Becton Dickinson) in serum-free media ent sequence, computationally predicted to be a PKC sub- in triplicate in a 24-well tissue culture plate. The lower cham- strate. Thus, we used in vitro kinase assays to compare the bers were filled with media supplemented with 2% fetal bo- phosphorylation of RalA and RalB by PKC, Aurora-A kinase, vine serum (FBS). Cells that migrated through the chambers andPKA.PurifiedrecombinantGST-RalBandGST-RalA were stained with crystal violet after 6 hours of migration fusion proteins were incubated with purified kinases plus and counted. [γ-32P]ATP, and incorporated radioactivity was visualized by autoradiography and quantified by scintillation counting. Anchorage-independent growth GST-RalB was phosphorylated more effectively by PKC com- Cells (2 × 104) were suspended in 3-mL MEM medium pared with either PKA or Aurora-A (Supplementary Fig. S1A, containing 2% FBS and 0.4% SeaPlaque low melting temper- left). In contrast, under the same conditions, GST-RalA ature agarose (Cambrex) with or without PMA. The suspension was phosphorylated much more extensively by PKA than was added onto a layer of MEM containing 1% SeaPlaque by either PKC or Aurora-A kinase (Supplementary Fig. S1A, agarose in triplicate in a six-well plate. Plates were incubated right). Furthermore, FLAG-RalB and FLAG-RalA immuno- at 37°C, and medium with or without stimulators replaced precipitated from 293T cells were also preferentially phos- every 3 to 4 days. Colony numbers were quantified after phorylated in vitro by different kinases. FLAG-RalB was 3 weeks using ImagePro 4.5 software. phosphorylated by PKC, but not by either PKA or Aurora-A, whereas FLAG-RalA was phosphorylated to the same extent In vivo xenograft experiments as FLAG-RalB, but by PKA and not by either PKC or Aurora-A Four-week-old athymic nude female mice were obtained (Supplementary Fig. S1B). from the National Cancer Institute breeding facility. For To identify phosphorylation sites in FLAG-RalB, we used subcutaneous tumorigenicity, 1 × 106 of UMUC3 stable mass spectrometry and mutagenesis. Mass spectrometry cells were injected s.c. into right and left flanks of mice. did not reveal any phosphorylated tryptic peptides, but the Five mice were injected for each cell line. Subcutaneous analysis did not cover the COOH terminal region from resi- tumors were measured by calipers weekly, and tumor due 175 to 206 (Supplementary Fig. S2). In the COOH termi- was volume calculated. To evaluate the ability of develop- nal region of RalB residue S198 corresponds to the position ment of experimental lung metastases of UMUC3 cells, 1 × of S194 in RalA, which has been reported as a site of phos- 106 of UMUC3 stable cells were injected into mice by tail phorylation (19–21). We introduced a S198A mutation that vein. Ten mice were injected for each cell line. The detect- reduced in vitro FLAG-RalB phosphorylation by PKC ∼70%, able lung lesions were evaluated by Xenogen biolumines- relative to wild-type RalB as a control (Fig. 1A). These results cent imaging (Caliper Life Sciences, Hopkinton; ref. 26). indicated S198 is the primary but not the only PKC phos- For the overall survival calculation, a mouse was counted phorylation site(s) in RalB. Other sites in the COOH terminal as “deceased” if it died spontaneously or if any triggers to region of FLAG-RalB reactive with PKC were not identified; euthanasia were encountered, such as weight loss >20%, skin however, we at least excluded S182 and S192 as potential ulceration, signs of chronic pain, or distress. Euthanasia was sites of phosphorylation, which were predicted by algorithms carried out according to AVMA Guidelines on Euthanasia in NetPhospho 2.0 server (http://www.cbs.dtu.dk/services/ (Rev. June 2007). NetPhos/). Double mutants S198A-S182A and S198A-S192A and a triple mutant S198A-S182A-S192A were [32P]phosphor- Statistical analysis ylated by PKC to the same extent as single mutant RalB Student's t tests were used for comparisons in cell mi- (S198A) (data not shown). gration and anchorage-independent cell growth assays. Re- peated measures models were used to compare tumor RalB is phosphorylated at S198 by endogenous PKC growth among groups. The models allowed the variability in living cells in tumor volumes to increase with time and F tests were We transiently expressed FLAG-RalB in 293T cells (Fig. 1B) used for pair-wise comparisons of groups and for compari- or UMUC3 human bladder cancer cells (Fig. 1C) and treated sons over specific time points. For pooled analyses, tumor the cells with PMA to activate endogenous PKC with or with- growth across multiple separate and independent experi- out pretreatment with PKC kinase inhibitor Go6983. RalB ments was analyzed using repeated measures models that al- phosphorylation was assayed by immunoblotting with a lowed rates of tumor growth to differ across experiments. phosphosite-specific RalB(pS198) antibody we developed. Significance was considered at P < 0.05. Analyses were car- There was phosphorylation of S198 in FLAG-RalB in 293T ried out in SAS version 9.1, PROC MIXED. Kaplan-Meier cells that was increased by PMA addition to the cells. Pretreat- curves were used to estimate overall survival in the metasta- ment of the cells with Go6983 effectively blocked PMA- sis experiment in vivo. The log-rank test was used to com- induced phosphorylation of S198 in RalB (Fig. 1B). Mutation pare groups. of S198 in FLAG-RalB eliminated phosphorylation of this site,

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as expected, and no immunoblotting with the phosphosite antibody was seen with S198A RalB or the G23V-S198A activated RalB even in cells treated with PMA (Fig. 1B). Equivalent expression of the RalB proteins is shown by immunoblotting for FLAG. To generalize these observations, we evaluated UMUC3 human bladder cancer cells. In these cells, phosphorylation of S198 in FLAG-RalB was observed with PMA stimulation, and this response was effectively blocked by pretreatment with Go6983 (Fig. 1C). To show phosphorylation of endogenous RalB, we used RT112 human bladder cancer cells that have abundant RalB. Cells were treated with or without PMA to activate PKC and RalB was immunoprecipitated from a membrane fraction with anti-RalB antibody. Endogenous RalB phosphorylation in response to PMA was observed by immunoblotting with RalB(pS198) antibody (Fig. 1D). Taken together, these results show PKC phosphorylation of RalB at S198 in different cell lines.

Phosphorylation of S198 in RalB affects RalB intracellular localization Given that RalA phosphorylation affects its localization (21), we used fluorescent microscopy to evaluate whether phosphorylation of S198 in RalB may also regulate cellular

Figure 1. Phosphorylation of RalB by PKC. A, FLAG-RalB wild-type or S198A mutant were overexpressed in 293T cells and immunoprecipitated by anti-FLAG agarose. In vitro kinase assay was carried out as described in Materials and Methods. Phosphorylation was analyzed by SDS-PAGE followed by scintillation counting of excised bands and presented as the average pmol of incorporated 32P for two independent experiments. The bottom blot panel is Coomassie staining of immunoprecipitated FLAG- RalB and S198A mutant used in the kinase reaction. B, FLAG-RalB or their mutants were expressed in HEK293T, and the cells were treated with 100 ng/mL PMA for 15 min with or without 1 h pretreatment of 1 μmol/L Go-6983. The FLAG-RalB was immunoprecipitated, and phosphorylation of RalB was analyzed by immunoblotting with anti-phospho-RalB(pS198) antibody (top). Anti-FLAG blots were used as the loading control (bottom). C, FLAG-RalB or their mutants were expressed in UMUC3 human bladder carcinoma cells, the cells were treated the same as in B following overnight serum starvation, and RalB phosphorylation was analyzed as in B. Figure 2. PKC-mediated internalization of RalB depends on S198. A, T24 D, endogenous RalB phosphorylation. RT112 human bladder carcinoma cells cells stably expressing GFP-RalB or GFP-RalB(S198A) were serum starved were treated with or without 100 ng/mL PMA and 20 nmol/L calyculin-A for overnight following -PMA/+PMA treatment. The cells were fixed, and 10 min following overnight serum starvation. Membrane fractions were images were acquired using Zeiss LSM 510 Meta confocal microscope. prepared. RalB was immunoprecipitated with anti-RalB antibody. The samples Green,GFP-RalBorGFP-RalB(S198A);blue,DAPI.B,immunoblot were subjected to SDS-PAGE and analyzed with anti-phospho-RalB(pS198) analysis of protein expressing of GFP-RalB wild-type or mutant RalB(S198A) antibody for phosphorylation or with anti-RalB for loading control. by anti-RalB and anti-GFP antibodies. GFP was used as a control.

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Figure 3. Effects of RalB phosphorylation on anchorage-independent growth of UMUC3 cells. A, immunoblot analysis of UMUC3 stable cell lines expressing FLAG-RalB wild-type or mutant RalB(S198A) by anti-RalB for Ral protein expression or anti-α-tubulin immunoblot for loading control. The cells were treated with 100 ng/mL PMA for 15 min. The FLAG-RalB and FLAG-RalB(S198A) were immunoprecipitated, and the phosphorylation of RalB was analyzed with anti-phospho-RalB(pS198) antibody. Immunoblotting with anti-FLAG was used as the control for recovery of RalB in immunoprecipitation. B, anchorage-independent growth of UMUC3 stable cells with and without PMA (0.05 nmol/L). Colony numbers relative to control cells with empty vector are expressed as mean ± SEM from triplicate plates of two experiments. P value is from Student's t test. C, immunoblot analysis of RalB in UMUC3 stable cell lines, which were lentivirally infected with a scramble control (Con) or shRNA against RalB, complemented by an empty vector (vector), shRNA-resistant RalB or RalB(S198A) mutant. Immunoblot with anti-RalB indicated the deletion of endogenous RalB and ectopic expressed RalB, with anti-α-tubulin used as loading control. D, effect on anchorage-independent growth of the indicated UMUC3 stable cell lines from C. Colony numbers relative to scramble control (Con) cells are expressed as mean ± SEM from triplicate plates of two experiments. P value is from Student's t test. localization of this paralogue in T24 cells that we have stud- these PMA-treated cells (Fig. 3A). We compared these UMUC3 ied before with regard to Ral function (25). We found that cell lines for their ability to form colonies in an anchorage- GFP-RalB was retained in the plasma membrane of T24 cells, independentgrowthassayandobservedadoublingofthe but PMA treatment translocated wild-type RalB from the number of colonies in response to PMA stimulation in cells plasma membrane to a perinuclear region, whereas phosphosite- expressing wild-type RalB, a significant increase relative to mutated RalB(S198A) protein was not translocated in re- controls treated with vehicle. In contrast, UMUC3 cell lines sponse to PMA (Fig. 2A). The protein expression levels of stably expressing phosphosite mutant RalB(S198A) yielded GFP-RalB wild-type or mutant RalB(S198A) were shown by the same number of colonies as controls, irrespective of PMA anti-RalB and anti-GFP antibodies, and GFP was used as a treatment (Fig. 3B). Thus, responsiveness to PMA required control (Fig. 2B). The results suggest that PKC activation by S198 in RalB, consistent with PKC-dependent phosphorylation PMA mediates relocalization of RalB, and this response de- of this site contributing to anchorage-independent growth. pends on the phosphorylation of S198. We also established UMUC3 cell lines stably knocked down for RalB by infection with lentivirus encoding RalB shRNA Phosphorylation of RalB at S198 and anchorage- (Fig. 3C). As a test of RalB function, these knocked down cells independent growth of bladder cancer cells were transfected with plasmids encoding shRNA-resistant We established UMUC3 cell lines that stably express wild- forms of wild-type RalB or RalB(S198A). Immunoblotting with type FLAG-RalB or the phosphosite mutant RalB(S198A) (Fig. 3A). anti-RalB showed the effective shRNA knockdown of endogenous The phosphorylation of wild-type RalB but not the S198A RalB, relative to a scrambled sequence as control, and subse- mutant was detected with RalB(pS198) phosphosite antibody quent expression of shRNA-resistant FLAG-RalB at levels sim- by immunoblotting anti-FLAG immunoprecipitates from ilar to the endogenous RalB in the control cells (Fig. 3C). We

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found that RalB depletion significantly impaired anchorage- either of these phenotypes (Fig. 4A and B). In addition to independent growth, reducing the number of colonies by a third effects on the cytoskeleton, RalB knockdown with lentivirus (Fig. 3D). This phenotype was reversed by ectopic expression encoding RalB shRNA impaired Transwell cell migration in of wild-type RalB protein; however, the phosphosite mutant UMUC3 cells (Fig. 4B), and this paralleled the effects on RalB(S198A) did not restore anchorage-independent growth the actin cytoskeleton (Fig. 4A and B). This phenotype was (Fig. 3D). These data show that S198 in RalB is required for partially rescued by reexpression of wild-type RalB but not anchorage-independent growth of human bladder cancer by RalB(S198A). There was a statistically significant differ- cells. We concluded that phosphorylation of S198 modulates ence in the rescue by wild-type versus S198A RalB. RalB function. We previously showed that PMA treatment stimulates cell motility in T24 human bladder tumor cells (25). We RalB S198 is required for cell motility and actin established T24 stable cells expressing either (a) FLAG-RalB cytoskeletal organization wild-type, (b) nonphosphorylatable FLAG-RalB S198A, or (c) We have previously shown that the knockdown of RalB a phosphomimetic mutant FLAG-RalB S198D. In the wound produced a reduction in actin stress fibers and a marked migration assay, chemokinesis of these cells was stimulated increase in filopodia extensions (10) and have recapitulated by PMA, resulting in wound closure of the control T24 these findings here (Fig. 4A). Interestingly, expression of the cells and T24 cells expressing wild-type FLAG-RalB protein shRNA-resistant wild-type RalB in knockdown cells restored (Fig. 4C). However, in the same time period, T24 cells expres- the actin stress fibers and reduced the prominent filopodia, sing RalB(S198A) migrated much less, leaving most of but expression of the RalB(S198A) mutant did not affect the wound space open. Conversely, wounds were almost

Figure 4. Effects of RalB phosphorylation on the actin cytoskeleton and cell migration. A, effects on actin cytoskeleton. The indicated UMUC3 stable cells as described in Fig. 3C were fixed and stained with phalloidin-AlexaFluor 594 (red) to visualize actin filaments and DAPI (blue) to visualize nuclei. Arrows indicate filopodial extensions. B, Transwell migration and cell actin. Transwell migration was carried out with the indicated UMUC3 stable cell lines as described in Fig. 3C. Light gray columns, numbers of cells that migrated on the opposite side of the filter (“Migrated Cells,” ^) relative to scramble control cells (Con) set at 100 are expressed as mean ± SEM from triplicate plates. *, P < 0.001. P value is from Student's t test relative to Con. Dark gray columns, the percentage of cells with actin stress fiber relative to untransfected cells (&) as a function of transfected construct. Most cells with shRNA RalB had actin reduction/absence and were used as “actin negative” in the scoring of other mutants. Typical images are shown in Fig. 4A. *, P < 0.001. P value is from Student's t test relative to Con. C, wound assay. The assay was carried out with T24 human bladder carcinoma cells stably expressing FLAG-RalB or mutants RalB(S198A) and RalB(S198D). FLAG vector was used as control as described in Materials and Methods. Representative of the fields used to quantitate motility using ImageJ (top) and generate the graph (bottom). The average distances (microns) of wounds were shown as mean ± SEM from multifields of two experiments. *, P < 0.001.

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Figure 5. RalB phosphorylation is required for subcutaneous growth and experimental lung colonization by UMUC3 cells. A, UMUC3 cell lines stably expressing activated GFP-RalB(G23V), phosphosite mutant GFP-RalB(G23V-S198A), or GFP vector were injected s.c. into mice. The average tumor volumes (mm3)ofeachgroup(n = 5) as indicated were shown as mean ± SEM. Figure shown is one representative of three experiments. Data were analyzed using repeated measures models. (See text for pooled data analysis of all three experiments and Supplementary Table 1 for averages of all three experiments.) B, proportion of tumors of each group present at different time points out of total possible sites (n =10).C,protein expression in subcutaneous tumors. The lysates of tumor tissues were subjected to SDS-PAGE and analyzed by anti-GFP or anti-RalB antibodies. D, UMUC3 stable cell lines as described in Fig. 3C, knockdown RalB complemented by an empty vector, and shRNA-resistant RalB or RalB(S198A) mutant were injected into mice by tail vein. The detectable metastasis was determined by Xenogen (inset shows typical example of mice with lung metastasis at 6 wk). Kaplan-Meier curves were used to estimate disease-specific overall survival (“death” surrogates in animals with Xenogen detectable metastasis). The log-rank test with adjustment for multiple comparisons was used to compare groups (n = 10). completely closed by T24 cells expressing RalB(S198D), even activated GFP-RalB(G23V), phosphosite-mutated GFP-RalB without PMA treatment (Fig. 4C). Data from multiple inde- (G23V-S198A), or GFP only (Fig. 5A–C). GFP fusion proteins pendent experiments were quantified to show the lack of were used to select for equal protein expression by FACS response to PMA by cells expressing RalB(S198A) and the sorting. Tumor growth was compared in three independent enhanced baseline and PMA-independent response of cells experiments, consisting of 158 measurements of tumor vo- expressing RalB(S198D). Taken together, these data indicate lumes from 45 animals, 15 in each of the three groups (Sup- S198 in RalB is involved in actin cytoskeletal organization plementary Table S1), with measurement times ranging and cell motility. from 1 to 4 weeks postinjection. The combined experiments were analyzed using repeated measures models that al- RalB phosphorylation at S198 is required for lowed rates of tumor growth to differ across experiments. promotion of human bladder cancer tumor The combined analyses indicated significant overall dif- growth by activated RalB ferences in tumor growth between all groups (P <0.001). We evaluated tumors resulting from s.c. inoculation of Tumor growth in animals injected with GFP-RalB(G23V) UMUC3 human bladder cancer cells stably expressing either cells had significantly greater tumor growth (P < 0.0001)

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than animals injected with either GFP or GFP-RalB(G23V- occurring at another site. Mutation of S182 and S192, in addi- S198A). Tumor growth in animals injected with GFP and tion to S198, did not eliminate phosphorylation of RalB, and GFP-RalB(G23V-S198A) were similar (Fig. 5A). Comparison mass spectrometry did not locate other sites of phosphoryla- of overall tumor burden at each time point revealed that tion up to residue 175 of RalB. Whereas the identity of second- control GFP cells and those expressing GFP-RalB(G23V- ary PKC phosphorylation site(s) in RalB remains unknown, S198A) had lower tumor formation at earlier time points S198 clearly is the primary PKC phosphorylation site with than those expressing GFP-RalB(G23V) (P < 0.041; Fig. 5B). functional consequences. Similarly, RalA was shown to be Importantly, at the end of the experiment, we isolated tumors phosphorylated at multiple sites; besides major S194 sites, and found similar levels of GFP proteins in all tumors by im- S183 was phosphorylated in response to suppression of phos- munoblotting with anti-GFP or anti-RalB antibody (Fig. 5C). phatase PP2A Aβ subunit (20). Together, these observations indicate that enhancement The phosphorylation of S198 is important for anchorage- of in vivo tumor growth by activated RalB requires S198 independent cell growth, cell motility, actin cytoskeletal phosphorylation. organization, and in vivo bladder cancer tumorigenicity driven by active RalB. We also evaluated the role of RalB RalB S198 is required for the development of experimental phosphorylation in regulating experimental lung metastasis lung metastasis by UMUC3 cells in bladder cancer by using loss of function experiments. Sim- We evaluated the development of experimental lung me- ilar to what was noted in prostate cancer for bone metastasis tastasis resulting from UMUC3 cell lines stably knocked for the DU145 cell line (11), RalB depletion by RNAi reduced down for RalB, knockdown RalB complemented by an empty lung colonization and increased survival in UMUC3 cells. Im- vector, shRNA-resistant RalB, or RalB(S198A) as described in portantly, when we restored Ral expression to endogenous Fig. 3C. Following injection of cells into the tail vein of mice, levels by expressing wild-type RalB and phosphosite mutant the metastases were detected and monitored weekly by bio- RalB(S198A), only the former restored the metastatic com- luminescence using a Xenogen instrument (26). Kaplan-Meier petence of the cells. This indicates that phosphorylation of curves were used to estimate disease-specific and overall RalB is important for the development of lung metastasis survival, and the log-rank test with Bonferroni correction for in human bladder cancer cells. multiple comparisons was used to compare groups (10 mice in Our finding that RalA and RalB are phosphorylated by dif- each; Fig. 5D; Supplementary Table S2). Once a bioluminescent ferent kinases may explain in part the different cellular ac- signal was detected and shown to increase over two consecutive tions of these paralogues, despite nearly identical protein weeks, mice were considered to have a lung metastasis and sequence. The situation with RalA and RalB may be similar were thus no longer disease free. Mice that exhibited surrogates to the Ras family member Rap1, which is phosphorylated by of death as defined by our animal care and use committee were PKA. Phosphorylation alters the ability of Rap1 to associate considered as dead of disease. Mice injected with UMUC3 cells and regulate its downstream targets (27–30). Previous re- that had RalB knockdown survived with no metastasis, whereas ports claimed that phosphorylation of RalA at S194 by Auro- most mice injected with UMUC3 cells expressing control shRNA ra-A kinase regulates RalA activity as measured by effector died with obvious metastases up to 9 weeks. Mice with UMUC3 RalBP1 binding and function in cells (19, 21). We compared cells knocked down for RalB but expressing a shRNA-resistant the binding of wild-type and S198A RalB with downstream wild-type RalB protein developed metastases at a similar rate effectors RalBP1, Sec5, and Exo84, but coprecipitation with to mice expressing control shRNA (P = 0.1278 in Supplemen- these effectors was not different. The phosphorylation of tary Table S2). In contrast, mice expressing the phosphosite RalB may affect binding to another yet undetermined protein mutant RalB(S198A) did not restore RalB-dependent metasta- in bladder cancer cells. Phosphorylation of RhoA by PKA sis (Fig. 5D; P = 1.00 in Supplementary Table S2). Together, induces relocalization of RhoA (31, 32). Recently, it was this indicates that phosphorylation at S198 of wild-type RalB reported that RalA phosphorylation at S194 by Aurora-A is necessary for the development of experimental lung kinase translocated RalA from the plasma membrane (21). metastasis of human bladder cancer cells. Not surprisingly, Here, in human bladder cells, we observed phosphorylation disease-specific survival paralleled overall survival (Supple- of RalB by PKC induced RalB translocation from plasma mentary Table S2). membrane to perinuclear regions. This might be the functional consequence of RalB phosphorylation. Discussion RalB phosphorylation by PKC has interesting implications for human bladder cancer. RalB has been implicated in blad- This study reports phosphorylation of RalA and RalB by der cancer migration and metastasis (10), and PKC is known different protein kinases. RalB is phosphorylated at S198 by to play an important role in cellular differentiation and in PKC, whereas RalA is phosphorylated at S194 by PKA. The malignancy and metastasis (33–35). PKC activity is elevated S198 site is phosphorylated in stably expressed RalB and, in invasive human bladder carcinoma cell line EJ and inhibi- more importantly, in endogenous RalB in living cells acti- tion of kinases by staurosporine inhibited invasion of this cell vated by phorbol ester. The S198 site (KKS198FK) in RalB line (36). PMA also upregulated vascular endothelial growth conforms to a motif recognized by PKC [(R/K)X(S)(Hyd)(R/K)], factor (VEGF) expression via PKC signaling in human blad- and mutation of this Ser to Ala reduced PKC phosphorylation der transitional carcinoma cell line RT4, and this was inhib- by ∼70%, with the other 30% of phosphorylation presumably ited by staurosporine. VEGF is known as a key factor in

OF8 Cancer Res; 70(21) November 1, 2010 Cancer Research

RalB Phosphorylation by PKC

human tumorigenesis and metastatic potential (37) and has RalGEFs and PKCs are coincidently activated as conditions been shown to be regulated by Ral GTPases (38). In colon for RalB action. Because PKCs are implicated in bladder can- carcinoma cells, PKCα contributes to high migratory activity cer progression and react with S198, inhibition of PKC, activa- (39), and a PKC inhibitor effectively inhibits migration and tion of the S198 phosphatase, or mimicking the phosphosite to invasion of bladder carcinoma cells (40). Taken together, interfere with RalB targeting could prove useful for treating there seems to be substantial evidence for involvement of metastatic bladder cancer. RalB and PKC in human bladder progression. Importantly, by linking PKC and RalB, our results may expose one of Disclosure of Potential Conflicts of Interest the underlying mechanisms whereby PMA and other PKC ac- tivators act as tumor promoters in cancer because RalB is No potential conflicts of interest were disclosed. involved in multiple cancer types (9–14). Based on these data, work to determine which PKC isoforms affect S198 in RalB Acknowledgments seems warranted. In summary, RalB phosphorylation translocates RalB from We thank Dr. M.A. Schwartz for helpful suggestions. plasma membrane to perinuclear regions in an S198-dependent fashion and is necessary for RalB effects on cell migration and Grant Support in vitro anchorage-independent growth and tumor growth NIH grants GM56362 (D.L. Brautigan) and CA104106 (D. Theodorescu). and metastasis in vivo, irrespective of RalB activation by The costs of publication of this article were defrayed in part by the payment GTP loading. Importantly, these results suggest that RalB of page charges. This article must therefore be hereby marked advertisement in functionsasan“AND” gate in signaling, wherein both GTP accordance with 18 U.S.C. Section 1734 solely to indicate this fact. loading and S198 phosphorylation are required for full bio- Received 03/17/2010; revised 08/27/2010; accepted 09/01/2010; published logical activity. We should look for circumstances where OnlineFirst 10/12/2010.

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OF10 Cancer Res; 70(21) November 1, 2010 Cancer Research

Phosphorylation of RalB Is Important for Bladder Cancer Cell Growth and Metastasis

Hong Wang, Charles Owens, Nidhi Chandra, et al.

Cancer Res Published OnlineFirst October 12, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-10-0952

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