Proteome Analysis of NIH3T3 Cells Transformed by Activated Gα12

Proteome Analysis of NIH3T3 Cells Transformed by Activated Gr12: Regulation of Leukemia-Associated Protein SET Rashmi N. Kumar, Rangasudhakar Radhakrishnan, Ji Hee Ha, and N. Dhanasekaran* Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Received June 28, 2004; Revised Manuscript Received September 2, 2004

GR12, the R-subunit of the G12 family of heterotrimeric G proteins is involved in the regulation of cell proliferation and neoplastic transformation. GTPase-deficient, constitutively activated mutant of GR12 (GR12Q229L or GR12QL) has been previously shown to induce oncogenic transformation of NIH3T3 cells promoting serum- and anchorage-independent growth. Reduced growth-factor dependent, autonomous cell growth forms a critical defining point at which a normal cell turns into an oncogenic one. To identify the underlying mechanism involved in such growth-factor/serum independent growth of GR12QLtransformed NIH3T3, we carried out a two-dimensional differential proteome analysis of GR12QLtransformed NIH3T3 cells and cells expressing vector control. This analysis revealed a total of 22 proteinspots whose expression was altered by more than 3-folds. Two of these spots were identified by MALDIMS analysis as proliferating cell nuclear antigen (PCNA) and myeloid-leukemia-associated SET protein. The increased expressions of these proteins in GR12QL cells were validated by immunoblot analysis. Furthermore, transient transfection studies with NIH3T3 cells indicated that the expression of activated GR12 readily increased the expression of SET protein by 24 h. As SET has been previously reported to be an inhibitor of phosphatase PP2A, the nuclear phosphatase activity was monitored in cells expressing activated GR12. Our results indicate that the nuclear phosphatase activity is inhibited by greater than 50% in GR12QL cells compared to vector control cells. Thus, our results from differential proteome analysis presented here report for the first time a role for SET in GR12-mediated signaling pathways and a role for GR12 in the regulation of the leukemia-associated SET-protein expression. Keywords: G protein • GR12 • SET • PP2A • PCNA • oncogene • cell transformation • cell proliferation

Introduction Oncogenesis is the result of accumulation of genetic and molecular mutations in a cell and is predominantly characterized by increased cell growth and proliferation, decreased apoptotic potential, and reduced dependency of cells on growth factors.1 GR12, the R-subunit of the heterotrimeric G-Protein G12, has been shown to be a potent activator of oncogenic transformation by us and several other groups.2-6 NIH3T3 cells that are transfected with the activated mutant of GR12 (GR12Q229L or GR12QL) undergo neoplastic transformation and show an oncogenic phenotype.2-6 These cells show enhanced cell proliferation along with serum- and anchorage-independent growth. The underlying mechanism(s) by which activated GR12 transforms NIH3T3 cells is still not fully understood.7 To gain a comprehensive picture of one aspect of the oncogenic pathways activated by GR12, namely the reduced growth-factor dependency for cell growth, we adopted two strategies. The first was differential DNA microarray analysis to monitor global changes in mRNA levels (transcript profile) of GR12QL expressing NIH3T3 cells compared to vector control NIH3T3 cells. Our * To whom correspondence should be addressed. Phone: (215) 707 1941. Fax: (215) 707 5963. E-mail: [email protected]. 10.1021/pr049896n CCC: $27.50

 2004 American Chemical Society

results and validation using this strategy has been previously published.8 While microarrays are informative, they have certain limitations. The mRNA levels of a given cell may not always correlate to its corresponding protein. In addition, the transcript profiling method is limited by its inability to detect protein stability and post-translational changes in the protein expression. With this reasoning, we carried out differential proteome analysis in order to monitor changes in expression of all the proteins (proteome profile) of GR12QL expressing NIH3T3 cells compared to vector control cells. Our results from such proteome analysis, presented here, indicate that the levels of the nuclear protein proliferating cell nuclear antigen9,10 and the leukemia-associated nuclear protein SET11,12 are greatly enhanced in GR12QL-transformants. We also show that the transient expression of GR12QL increases the levels of SET by 2- to 3-fold in NIH3T3 cells. Consistent with the observation that SET is an inhibitor of protein phosphatase 2A,13-15 GR12QL-transfectants showed a decrease in nuclear phosphatase activity. Since expression of SET has been shown to be closely associated with cell proliferation and tumorigenesis,11-19 our results presented here point to the possible novel mechanism through which GR12 can activate oncogenic pathway by stimulating the increase in the levels of SET. Thus, our studies Journal of Proteome Research 2004, 3, 1177-1183

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research articles reported here, identify novel signaling locus in GR12-mediated oncogenic signaling.

Experimental Section Cell Lines Cell Culture. NIH3T3 cells were maintained by serial passage in Dulbecco’s modified Eagle’s medium (DMEM, Cellgro, VA) containing 5% calf serum (Life Technologies, Inc. MA), 50 U/mL penicillin, and 50µg/mL streptomycin at 37 °C in a 5% CO2 incubator. Serum-deprivation of NIH3T3 cells was done by incubation for 24 h in DMEM supplemented with 10 mM HEPES (pH 7.4) and 0.2% BSA. The pcDNA3-NIH3T3 and GR12Q229L-NIH3T3 cell-lines have already been described.20 For serum starvation, of vector-control and GR12QL-NIH3T3 cells either 4 × 105 cells (60 mm tissue culture plate) or 1 × 106 cells (100 mm tissue culture plate) were plated and allowed to grow for 24 h, at this point, the cells were serum starved for 24 h and then subjected to lysis. Transient Transfection of NIH3T3 Cells. Transient transfection of NIH3T3 cells was carried out using TransIT-3T3 Transfection Kit according to manufacturer’s (Mirus Corporation, WI) protocol. Briefly, NIH3T3 cells (2 × 105 cells/35 mm dish) grown to 50-80% confluence for 24 h and were transfected with 2 µg of pcDNA3-vector or vector containing the cDNA insert encoding GR12QL. Cells were collected and lysed 24 h post-transfection for further analysis. Cell Cycle Analysis. NIH 3T3 Cells (5 × 105) were plated and grown for 16 h in DMEM containing 5% calf serum following which they were synchronized by serum-starvation for 24 h. Cells were trypsinized, washed, and resuspended in 1 mL of phosphate-buffered saline containing 1% FBS. These cells were fixed by the dropwise addition of 100% ethanol at 4 °C while vortexing and stored at -20 °C. Fixed and permeabilized cells were washed twice in phosphate buffered saline and treated with 100 µg/mL ribonuclease for 5 min at room temperature. Cells were stained with propidium iodide (50 µg/mL) and analyzed in a Becton Dickinson FAC Scan analyzer according to the manufacturer’s procedure. Two-Dimensional Polyacrylamide Gel Electrophoresis. Twodimensional (2-D) electrophoresis was carried out according to the methods of O′Farrell21 by Kendrick Laboratories Inc., WI as follows: Protein lysates from pcDNA3- or GR12QL-NIH3T3 cells (275 µg each) were separated by isoelectricfocusing. Isoelectricfocusing was carried out in a glass tube of inner diameter 2.0 mm using 2% pH 3.5-10 ampholines (Amersham Biosciences Corp., NJ) for 9600 V-hours. One µg of an IEF internal standard, tropomyosin, was added to the sample. The isoelectrofocused tube-gel was equilibrated for 10 min in a buffer containing 10% glycerol, 50 mM DTT, 2.3% SDS, and 0.0625 M Tris, pH 6.8, after which the tube-gel was sealed to the top of a stacking gel overlaying a 10% ployacrylamide slab gel. SDS electrophoresis in the second dimension was carried out for 4 h at 12.5 mA/gel. The following proteins were added as molecular weight markers: myosin (MW 220 000), phosphorylase A (MW 94 000), catalase (MW 60 000), actin (MW 43 000), carbonic anhydrase (MW 29 000), and lysozyme (MW 14 000). These standards appear along the basic end of the silver-stained 10% acrylamide slab gels. The gels were dried between sheets of cellophane with the acidic edge to the left. Computerized Comparisons. Triplicate gels were obtained as described above. Each gel was scanned with a laser densitometer (Model PDSI, Molecular Dynamics, CA). The scanner was checked for linearity prior to scanning with a calibrated neutral density filter set (Melles Griot, CA). The images were 1178

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analyzed using Progenesis software (version 2002.01, Nonlinear Technology) such that all major spots and all changing spots were outlined, quantified, and matched on all the gels. The comparison of pcDNA3- or GR12QL-NIH3T3 gels included computerized automatic spot finding, spot quantification, automatic background subtraction (mode of nonspot), and automatic spot matching in conjunction with detailed manual spot finding, checking, and matching functions. Averaged gel spot % and fold-differences in spot % were calculated for each spot by Progenesis software. Spot % is equal to spot integrated density (volume) expressed as a percentage of total density of all spots measured. Difference is defined by what fold-change the percentage of a particular spot differs from that of the spot’s matched counterpart in the comparison gel. Protein Identification by Mass Spectroscopy. Identifications of differentially expressed proteins were carried out by the Protein Chemistry Core Facility, Howard Hughes Medical Institute, Columbia University, New York, NY. Spots showing higher expression in GR12QL-NIH3T3 gel compared to pcDNA3NIH3T3 gel were excised from the respective gels and subjected to in-gel digestion with trypsin. The tryptic digests of each of these spots analyzed by matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS). Immunoblot Analysis. Control pcDNA3- and GR12QLNIH3T3 cells were washed three times with cold PBS (pH 7.4) and lysed in 100-200 µL modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride, 1 mM PMSF, 1 mM sodium vanadate, 2 µg/mL leupeptin, 4 µg/mL aprotinin. Nuclear protein extraction was carried out using previously published procedures.22 Total cell lysate protein (50 µg) or nuclear protein (25 µg) was separated by 10% SDS-PAGE and electroblotted onto poly(vinylidene difluoride) (PVDF) membranes (Millipore, Bedford, MA) in 10 mM CAPS (pH 11) buffer containing 20% methanol. The membranes were probed with antibodies specific for PCNA (sc56) and GR12 (sc-409, Santa Cruz, CA), SET antibody (kindly provided to us by Dr. T. D Copeland, NIH), anti-β-actin (Sigma, MO) or monoclonal anti-GAPDH (Ambion, Inc., TX). Horseradish peroxidase (HRP) conjugated anti-rabbit (Promega, Madison, WI) or anti-mouse antibodies (Amersham Biosciences Corp., NJ) were used as secondary antibodies and the immunoblots were developed with using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences Inc., MA). Chemiluminescence was detected by exposing the blots to Kodak Scientific Imaging film for different lengths of times ranging from 5 s to 5 min. Quantification of the bands was carried out using Kodak 1-D Image Analysis Software. Phosphatase Assay. Nuclear proteins were extracted from GR12QL- and pcDNA3-NIH3T3 cells according to the established procedure.22 Phosphatase activity was measured using disodium hexahydrate salt of para-nitrophenyl phosphate (pNPP; Sigma-Aldrich, MO) as a substrate.23 2.5-5 µg of GR12QL-NIH3T3 or pcDNA3-NIH3T3 nuclear extract was taken and added to 100 µL of reaction mixture containing 20 mM TrisHCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 20 mM pNPP. Reactions were allowed to proceed for 1 h to 16 h at 37 °C and were terminated by addition of 250 mM NaOH. The absorbance was measured at 405 nm using Perkin-Elmer Bio-Assay Reader HT5 7000 Plus.

Proteome Analysis of Gr12-Transformants

Figure 1. Activated GR12 stimulates G1-S phase progression in NIH3T3 cells (A) Lysates (50 µg) from pcDNA3- and GR12QLNIH3T3 cells (prepared as described under materials and methods) were separated on a 10% SDS-PAGE and subjected to immunoblot analysis with GR12 antibody to monitor the expression levels of GR12QL. The blot was stripped and re-probed with GAPDH antibody to monitor equal loading. (B) NIH3T3-GR12QL and NIH3T3-pcDNA3 (5 × 105) were plated on 100-mm plates and distribution of cells in different phases of cell cycle grown in medium containing 0% serum was monitored by FACS analysis. Four independent FACS analyses were carried out and the results from a typical analysis are presented.

Results Activated Gr12 Stimulates G1-S Phase Progression in NIH3T3 Cells. Prior to the proteome analysis, the basic characteristics of the proteomes were established in terms of GR12 expression and GR12-mediated proliferation. Previous studies have shown that GR12QL-transformed NIH3T3 cells show increased cell proliferation even in the absence of serum in the growth medium, presumably through the activation of yet to be identified autocrine mechanisms.18 To monitor the stimulatory effect of GR12QL on cell growth and its ability to progress the cells from G1- to S-phase of cell cycle in the absence of any growth factors, the distribution of GR12QLtransformants in S phase was compared to that of control cells. Equal numbers of pcDNA3- and GR12QL-NIH3T3 cells were deprived of serum in growth medium for 24 h and analyzed in a fluorescence-activated cell sorter. As shown in Figure 1, the distribution of cells in S-phase was greater in the case of GR12QL-transformants compared to pcDNA3-transfectants (Figure 1A, B). Even under serum-starved conditions the distribution of GR12QL-NIH3T3 cells in S-phase was 2.5-fold higher than that of the control cells. Together with the observation that these cells continued to grow in serum deprived medium for at least 72 h,20 these findings point to the presence of altered signaling components in GR12QL-NIH3T3 proteome compared to the control proteome. With the reasoning that the compara-

research articles tive analysis of pcDNA3- and GR12QL-proteome can identify the factors that are involved in the genesis and/or maintenance of GR12QL-mediated oncogenic phenotype, we carried out twodimensional gel analyses of pcDNA3-NIH3T3 and GR12QLNIH3T3 cell lysates. Two-Dimensional Gel Analysis. Changes in protein profiles of GR12QL cells and vector control cells were monitored by high-resolution two-dimensional (2-D) polyacryamide gel electrophoresis. Cell lysates prepared from 24-h serum-starved GR12QL- and vector control pcDNA3-NIH3T3 cells were fractionated on 2-D gel to monitor differences in the respective proteomes. Silver-stained gels were scanned with laser densitometer and differential protein spots were identified using limited computerized comparison as described under experimental procedures. Of the 528 discernible protein spots identified (Figure 2), analysis of protein spots showing 3-fold changes over pcDNA3-NIH3T3 levels indicated that a total of 22 proteins that were differentially regulated in GR12QL-proteome (Figure 2C-E). Sixteen polypeptide spots showed more than 3-fold increase and 4 spots showed 3-fold decrease in GR12QL-proteome (Figure 2C) compared to that of the vector control (Figure 2D). These polypeptides were ranging from 15 kDa to 76 kDa in their estimated molecular weights and 4.6 to 7.9 in their estimated pI values (Table 1). A polypeptide with an estimated molecular weight of 43 kDa was present only in pcDNA3-NIH3T3 proteome (Figure 2E). Determination of the spot volume (spot percentage, Table 1) as a percentage of total density of all the measured spots indicated that these proteins are present in GR12QL-proteome in varying levels ranging from extremely low (Spot Nos. 5, 49, 503: 0.01%) to moderately higher levels (Spot No. 325: 1.38%). Protein Identification by Mass Spectrometry. Since the polypeptide-spots that show differential expression in GR12QLNIH3T3 proteome are likely to represent the critical proteins involved in the stimulation and/or maintenance of the transformed phenotype, we sought to identify these proteins. Although some of the polypeptides showed higher folds of increase, their relative expression levels were too low for further analysis (Table 1). Excluding them from the present analysis, we focused on characterizing two polypeptide spots whose average expression levelssas indicated by the spot intensitiess were relatively high with a fold difference of g3 compared to pcDNA3-NIH3T3 proteome (Figure 2A,B). To characterize these protein spots by mass spectrometry, the lysates from GR12QLNIH3T3 cells were refractionated on a 2-D gel and the silverstained spot nos. 256 and 325 were excised out. The excised spots were digested with trypsin and analyzed by MALDI-mass spectrometry. The results from such analyses identified Spot No. 256 as SET protein and Spot No. 325 as proliferating cell nuclear antigen commonly referred as PCNA (Figure 3). Validation of Proteome Analysis. To validate the upregulation of PCNA and SET as indicated by 2-D proteome analysis, immunoblot analyses were carried out using the lysates from pcDNA3-NIH3T3 and GR12QL-NIH3T3 cells. Expression of PCNA was monitored by immunoblot analysis of the nuclear extracts from pcDNA3- and GR12QL-NIH3T3 cells with antibodies raised against PCNA. Expression of β-actin was used to monitor equal loading of nuclear proteins.24,25 The results indicate that GR12QL-NIH3T3 cells show an increase in the expression of PCNA levels compared to the vector control cells (Figure 4). Quantification of these results using Kodak 1-D analysis software indicated that the PCNA-levels were increased Journal of Proteome Research • Vol. 3, No. 6, 2004 1179

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Figure 2. Two-dimensional gel analysis. Representative (n ) 3) silver stained 2-D gel images obtained by fractionating 275 µg of the total cell lysate of 24 h serum starved pcDNA3-NIH3T3 (A) and GR12QL-NIH3T3 cells (B) are presented. These gels were scanned and analyzed to identify the polypeptides spots that showed 3-folds or above increase (C) decrease (D) or absence (E) in GR12QL-NIH3T3 cell lysate compared to pcDNA3-NIH3T3 cell lysate (see text for details). Table 1. Differentially Regulated Polypeptides in GR12QL-NIH3T3 Cellsa

spot no.

reference pI

reference MW

spot no. pcDNA3NIH3T3

spot no. GR12QLNIH3T3

GR12QL-NIH3T3 vs pcDNA3-NIH3T3 difference

82 90 157 201 214 256 284 313 325 343 365 367 396 413 456 468 226 5 49 68 147 503

5.26 4.86 5.18 4.82 5.19 4.84 5.14 5.02 5.03 7.35 5.59 6.00 4.68 6.97 6.77 7.87 6.82 7.70 5.74 7.22 6.91 5.74

76229 73762 57081 50640 48729 41484 38897 36018 34852 33315 30314 29946 27808 26576 21068 18914 43378 158207 89769 80331 59888 15671

0.005 0.02 0.02 0.03 0.02 0.26 0.02 0.06 0.41 0.03 0.03 0.02 0.01 0.03 0.02 0.01 0.09 0.03 0.04 0.16 0.15 0.02

0.02 0.08 0.06 0.08 0.06 0.79 0.06 0.32 1.38 0.11 0.10 0.07 0.04 0.13 0.06 0.15 N.D 0.01 0.01 0.05 0.05 0.01

+ 3.91 + 3.22 + 3.23 + 3.20 + 3.56 + 3.09 + 4.03 + 5.20 + 3.39 + 3.81 + 3.33 + 4.43 + 3.35 + 4.33 + 3.64 + 17.64 * - 5.06 - 5.97 - 3.13 - 3.06 - 3.27

a

Polypeptides spots showing differential expression of 3-folds and above are tabulated along with Reference spot numbers, pI- and MW-values. Spot percentages indicate relative abundance of the polypeptide. The differences were calculated from spot percentage (individual spot density divided by total density of all the measured spots). An asterisk indicates polypeptide not detected (N.D) in GR12QL-NIH3T3 lysate.

by 10-folds in GR12QL-NIH3T3 cells (Figure 4). The findings that PCNA levels are increased in GR12QL-NIH3T3 cells further substantiate and confirm the view that the activated GR12 promotes oncogenic signaling in NIH3T3 cells.7 Similar im1180

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Figure 3. Identification of the upregulated proteins in GR12QLNIH3T3 cells. Polypeptide spot nos. 325 (Panel A) and 256 (Panel B) that showed 3-fold increase in GR12QL-NIH3T3 were excised out and identified by MALDI-MS analysis following tryptic digestion as PCNA and SET, respectively. The respective spots are circled with dotted line and are pointed with the white arrows. The black arrow in panel B points to the tropomyosin internal standard in the 2-D analysis.

munoblot analysis carried out with total lysates using antibodies raised against SET protein indicated that GR12QL-

Proteome Analysis of Gr12-Transformants

Figure 4. Increased expression of PCNA in GR12QL-NIH3T3 cells. Nuclear proteins (25 µg) extracted from 24 h serum starved pcDNA3- and GR12QL-NIH3T3 were separated on 10% SDS-PAGE and immunoblot analysis was carried out using monoclonal antibodies raised against PCNA (Upper Panel). The blot was stripped and re-probed with β-actin antibodies to monitor equal loading. The immuno-reactive bands were quantified as described in experimental procedures and plotted to illustrate the relative expression levels of PCNA in pcDNA3- versus GR12QLNIH3T3 cells. The experiment was repeated at least three times and the results from a typical experiment are presented.

NIH3T3 cells show 3-folds increase in the expression of SET protein (Figure 5A), thereby validating the 2-D proteome analysis. The slow migrating immuno-reactive band above the major SET-protein band (Figure 5A) appears to be the 41-kDa splice variant, SET/TAF-1R. Activated Gr12 Stimulates the Expression of SET. At this juncture, it should be noted that the increased levels of SET protein in GR12QL-NIH3T3 cells is a completely novel and significant finding that can have a far reaching implications in GR12 -signaling pathway. SET protein, which was initially identified as SET-CAN fusion protein in a patient with acute undifferentiated leukemia,11,12 is a phosphoprotein with a predominant localization in the nucleus,11-18 Although, increased expression of SET has been observed in different tumors,16-19 the mechanism by which its levels are regulated is not known. To test whether GR12QL is involved in regulating the expression of SET proteinsas opposed to the view that the increased expression is a consequence of the transformed phenotype of the GR12QL-NIH3T3 transformantsswe monitored the expression of SET in NIH3T3 cells in response to transiently expressed GR12QL. NIH3T3 cells were transfected with GR12QL or the vector pcDNA3 for 24 h, and the lysates from these cells were subjected to immunoblot analysis using SET-antibodies. Our results indicate that the expression of SET is increased in cells transiently expressing GR12QL suggesting that the expression of SET is stimulated by GR12QL-mediated signaling pathway (Figure 5B). Functional Role of SET in Gr12QL-NIH3T3 Cell Proteome. Of the different functions attributed to SET, its inhibitory effect on PP2A is well characterized.13-15 Since the inhibition of nuclear PP2A has been shown to promote potent activation of CDKs26 and SET can interact with both PP2A13-15 and cyclin/ CDK complexes,27-30 it can be speculated that SET accelerates cell cycle progression, thereby promoting cell growth in tumor

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Figure 5. Enhanced expression of SET in GR12QL expressing NIH3T3 cells. (A) Lysates (50 µg) from 24 h serum starved pcDNA3- and GR12QL-NIH3T3 were separated on 10% SDS-PAGE and immunoblot analysis was carried out using polyclonal antibodies raised against SET protein (upper panel). The stripped blot was re-probed with GAPDH antibodies to monitor equal protein loading. The experiment was repeated at least 3 times and the results are from a typical experiment. The immunoreactive bands were quantified as described in experimental procedures and plotted (n ) 3; mean ( SEM) to illustrate the relative expression levels SET protein in pcDNA3- versus GR12QL-NIH3T3 cells (lower panel). (B) Lysates (50 µg) from NIH3T3 cells transiently transfected with the pcDNA3 vector or vector encoding GR12QL (as described under Experimental Procedures) were separated on a 10% SDS-PAGE and subjected to immunoblot analysis with SET-antibodies. The results are from a typical experiment (Upper Panel). The immuno-reactive bands were quantified (n ) 3; mean ( SEM) and plotted to demonstrate the relative expression-levels SET protein in pcDNA3- versus GR12QL-NIH3T3 cells (Lower Panel)

cells by inhibiting PP2A. Considering the observation that the kinetics of CDK-activation is accelerated in GR12QL-cells (Radhakrishnan & Dhanasekaran, unpublished observation), it is possible that GR12-stimulated SET plays a contributory role in cell cycle progression by downregulating the activity of nuclear PP2A. It is significant to note here that an increase in the levels of SET can also be seen in the nuclear compartment (data not shown). Therefore, we investigated whether GR12mediated increase in SET levels accompanies a corollary inhibition of nuclear phosphatase activity. Phosphatase activity was monitored in the nuclear extracts prepared from pcDNA3and GR12QL-NIH3T3 cells. Results from these studies indicated that the phosphatase activity was reduced by 55% in GR12QLcells compared to the control cells (Figure 6). The reduction in PP2A activity does not appear to be due to a reduction in the PP2A levels in GR12QL-NIH3T3 cells (Kumar and Dhanasekaran, unpublished observation). Although it is possible that the observed reduction in phosphatase activity is due to factors other than SET and/or PP2A, the increase in the levels of SET along with a reduction in the nuclear phosphatase activity, points to a strong correlation between the increased SET levels with the inhibition of phosphatase activity. Journal of Proteome Research • Vol. 3, No. 6, 2004 1181

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Figure 6. Inhibition of nuclear phosphatase activity in GR12QLNIH3T3 cells. Nuclear phosphatase activity in 24 h serum starved pcDNA3-NIH3T3 cells and GR12QL-NIH3T3 cells were determined as described under experimental procedures using pNPP as a substrate. The phosphatase activity is presented as % of control value (n ) 3; mean ( SEM).

Discussion Results presented here unravel several novel aspects of GR12mediated oncogenic signaling. PCNA was originally identified as a 36 kDa nuclear protein whose expression is often associated with proliferating and oncogenic cellular phenotypes.9,10 PCNA has been identified to play a key role in DNA replication through its interaction with DNA polymerase-δ and -. DNA replication in eukaryotes involves stringent coordination and sequential switching between PolR and Polδ/Pol in the repliosome.31,32 PCNA plays a central role in coordinating these events by forming a ring around linear DNA to tether the 3’end of the leading strand of the template DNA to Polδ or Pol to initiate processive DNA-replication.31 Expression of PCNA is primarily cell cycle regulated in which the expression of PCNA increases in late G1-phase, reaching its maximum in S-phase, and declining from then onward.31-33 Although growth factors and serum have been shown to stimulate the expression of PCNA during S-phase through transcriptional as well as posttranscriptional mechanisms, it has been observed PCNA is constitutively expressed all through the cycle in actively growing cells.31 At present, it is not clear whether the increase in the expression of PCNA is due to a specific PCNA-targeted signaling pathway regulated by GR12QL or a default mechanism in response to the progression of more cells to S phase by GR12QL. Nevertheless, taking into consideration that PCNA is being used as a proliferation and prognostic marker in a variety of tumors,34,35 the increased PCNA expression in GR12QL is quite significant that it further confirms the strong oncogenic potential of GR12QL. Our results showing GR12QL-mediated increased expression SET gain more importance in light of the potential role of SET in leukemogenesis and other cancers. Although, the normal function of SET remains to be elucidated, oncogenesis associated with SET has been ascribed to its association with different nuclear proteins leading to the disruption of normal acetylation or demethylation of nuclear proteins both of which can result in asynchronous transcriptional activation.36-38 Furthermore, it has been identified that SET promotes cell cycle progression by modulating the activities of CDKs such as CDK1 (ref 27), CDK2 (ref 29), and CDK5 (ref 28) through its interaction with 1182

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different activators and/or inhibitors of CDKs.27-30 The ability of purified SET to inhibit PP2A in vitro13 and the coimmunoprecipitation of SET with PP2A-HRX-leukemic fusion protein complex14 suggests a critical role for SET-regulated PP2A activity in leukemogenesis. In present study, we have observed a 55% inhibition in the phosphatase activity in GR12QL expressing cells which correlates well with increase SET expression in these cells. Taken together, our findings suggest the interesting possibility that SET is a key player in the genesis and/or maintenance of GR12QL-transformed phenotype. Our immunoblot analysis identifies a slow migrating immuno-reactive band above the major SET-protein band (Figure 5A). A similar slow-migrating protein band can also be seen upon transient expression of GR12QL by exposing the autoradigraphic film for a longer time (data not shown). It has been identified that SET is expressed as two splice variants namely a 41 kDa SET/TAF-1R and 39 kDa SET/TAF-1β,19 of which the 39 kDa SET/TAF-1β is the major isoform in many different cell types.38 Consistent with these observations, the 39 kDa SET/ TAF-1β is expressed in these cells in response to GR12QL (Figure 5A, B). Through its interactions with different proteins, SET appears to play a crucial role in different nuclear activities including acetylation, methylation, transcription, chromosome remodeling, and cell cycle regulation.36-41 Together with the observation that the SET-fusion proteins and SET-HRX complexes are involved in leukemogenesis13-19 great interest has been evinced to identify the mechanism(s) through which SET is normally regulated in the cell.36-41 Our findings that the transient expression of GR12QL can stimulate the expression of SET together with the observation that GR12 is ubiquitously expressed leads to the interesting proposition that GR12-mediated signaling pathways play a role in the regulation of SET expression even in normal cells. Thus, our results indicating that the activated mutant of GR12 stimulates the expression of SET can provide valuable clues to the possible upstream signaling events involved in the regulation of SET. Further studiesswhich are being intensely pursued in the laboratorys are focused on defining the mechanism through which GR12 regulates SET levels and the role of such regulation in myeloid cell differentiation, growth, and leukemogenesis. Abbreviations: G-Proteins, GTP-binding Protein; PCNA, Proliferating Cell Nuclear Antigen; PP2A, Protein Phosphatase Type 2A; CDK, Cyclin Dependent Kinase; MALDI-MS, MatrixAssisted Laser Desorption/Ionization Mass Spectrometry.

Acknowledgment. This work was supported by a grant from the National Institutes of Health (GM49897). The generous gift of SET-antibodies by Dr. T. D. Copeland (NIH) and the critical reading of the manuscript by Ms. Kimia Kashef are gratefully acknowledged. References (1) Hanahan, D.; Weinberg, R. A. The hallmarks of cancer. Cell 2000, 100, 57-70. (2) Chan, A. M.; Fleming, T. P.; McGovern, E. S.; Chedid, M.; Miki, T.; Aaronson, S. A. Expression cDNA cloning of a transforming gene encoding the wild-type GR12 gene product. Mol. Cell. Biol. 1993, 2, 762-768. (3) Xu, N.; Bradley, L.; Ambdukar, I.; Gutkind, J. S. A mutant R subunit of G12 potentiates the eicosanoid pathway and is highly oncogenic in NIH 3T3 cells. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6741-6745. (4) Vara Prasad, M. V.; Shore, S. K.; Dhanasekaran, N. Activated mutant of GR13 induces Egr-1, c-fos, and transformation in NIH 3T3 cells. Oncogene 1994, 8, 2425-2429.

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