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Originally published online as doi:10.1189/jlb.0205070 on May 3, 2005

Published online before print May 3, 2005
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(Journal of Leukocyte Biology. 2005;78:503-514.)
© 2005 by Society for Leukocyte Biology

Functional expression cloning reveals a central role for the receptor for activated protein kinase C 1 (RACK1) in T cell apoptosis

Mirna Mourtada-Maarabouni*,1, Lucy Kirkham*, Farzin Farzaneh{dagger} and Gwyn T. Williams*,1

* School of Life Sciences, Keele University, United Kingdom; and
{dagger} Department of Haematological and Molecular Medicine, Rayne Institute, GKT School of Medicine, London, United Kingdom

1 Correspondence: School of Life Sciences, Keele University, Keele, ST5 5BG, UK. E-mail: bia19{at}biol.keele.ac.uk or g.t.williams{at}keele.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian cDNA expression cloning was used to identify novel genes that regulate apoptosis. Using a functional screen, we identified a partial cDNA for the receptor for activated protein kinase C 1 (RACK1) through selection for resistance to phytohemagglutinin and {gamma}-irradiation. Expression of this partial cDNA in T cell lines using a mammalian expression vector produced an increase in RACK1 expression and resulted in resistance to dexamethasone- and ultraviolet-induced apoptosis. Down-regulation of RACK1 using RNA interference abolished the resistance of the transfected cells to apoptosis. Overexpression of full-length RACK1 also resulted in the suppression of apoptosis mediated by several apoptotic stimuli, and this effect was quantitatively consistent with the effects of the original cDNA isolated on endogenous RACK1 levels. Together, these findings suggest that RACK1 plays an important role in the intracellular signaling pathways that lead to apoptosis in T cells.

Key Words: WD-40 • cell death • forward genetics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is an essential process in the control of many biological functions, playing a critical role in embryonic development, homeostasis, malignant transformation, degenerative and autoimmune diseases, and cytotoxic therapy [1 , 2 ]. The growing number of proteins involved in apoptosis, which has been identified in the human genome, indicates the molecular complexity of the process of apoptosis [3 , 4 ]. Despite the identification of many apoptosis-controlling molecules and the impressive progress made in understanding apoptosis pathways, it is likely that many crucial genes remain to be identified.

Apoptosis is a multistage process, and a growing body of evidence suggests that phosphorylation of specific proteins by several protein kinases is significant in the commitment to and the execution of apoptosis [5 , 6 ]. Indeed, receptor-mediated apoptosis (e.g., that induced by Fas or tumor necrosis factor {alpha}) and mitochondrion-dependent apoptosis pathways are reported to be regulated by many protein kinases such as protein kinase A (PKA) [6 ], phosphatidylinositol-3 kinase/AKT [7 ], members of mitogen-activated protein kinase superfamily [8 ], the tyrosine kinase c-Abl [9 ], and various isoforms of the serine-threonine kinase family PKC [10 ]. Regulation of protein dephosphorylation is, not surprisingly, also important [11 ].

A number of PKC isoforms are activated in response to multiple stimuli and participate in many cellular processes that include growth, differentiation, and apoptosis [12 , 13 ]. Phosphorylation and translocation of PKCs are two essential steps for activation of these enzymes [14 , 15 ]. It is well documented that translocation of activated PKC to the membrane fraction following agonist stimulation can be mediated by the receptors for activated PKC (RACK)-anchoring proteins [16 , 17 ]. These receptors enable PKC isoenzymes to localize appropriately and stabilize their active forms [18 , 19 ].

Using functional expression cloning, we have identified novel molecular components of the apoptosis machinery capable of playing critical roles in cell death. A crucial advantage of this approach is that it is entirely based on the function of the gene itself and requires no prior knowledge of the gene sequence or protein product [20 21 22 ]. The use of this approach has led to the identification of several important genes. These include the novel tumor suppressor gene LUCA15/RNA-binding motif protein 5 [22 23 24 ] and protein phosphatase 4 [11 ]. In addition, variations on this strategy have resulted in the successful identification of other apoptosis-controlling genes, such as human BCL-2 and BCL-x [25 ], apophosis-linked gene (ALG)-2 and ALG-3 [26 ], and the human death-associated proteins [27 , 28 ].

In the present study, we used a retroviral cDNA library derived from FDCP1 haemopoietic cells [29 ] as the source of apoptosis regulators, W7.2c mouse thymoma cells [30 , 31 ] as the screening host and {gamma} radiation followed by exposure to the mitogen phytohemagglutinin (PHA) as the apoptotic stimuli. The use of this approach resulted in the isolation of a cDNA fragment of RACK1, which produced an increase in endogenous RACK1 mRNA and protein levels and inhibited cell death induced by dexamethasone and by ultraviolet (UV) irradiation.

RACK1 is a 36-kDa cytosolic protein, which belongs to the WD-40 family of proteins, characterized by highly conserved, internal WD-40 repeats (Trp-Asp) implicated in protein–protein interactions [32 , 33 ], and is a homologue of the ß subunit of G-proteins. In addition to specifically binding to the active form of PKCß isoforms (ßI and ßII) [16 , 19 , 34 ], RACK1 interacts with several other important signaling proteins including the Src kinase family [35 ], ß1, ß2, ß3, and ß5 integrins [36 ], ß-spectrin and dynamin [37 ], RasGAP [38 ], the androgen receptor [39 ], p73, and pRB [40 ]. This suggests that RACK1 may act as an anchor or adaptor protein, recruiting other proteins to various transmembrane receptors, providing a platform for protein–protein interactions and acting as the focus for several cell-signaling pathways. Accordingly, a number of cellular functions have been attributed to RACK1, e.g., in cell growth [35 ], adhesion, protrusion, and chemotactic migration [41 ]. Our observation of apoptosis suppression by up-regulation of RACK1 indicates that it also plays an important role in the regulation of apoptosis. Two other groups have reported observations consistent with this proposal. First, Sang et al. [42 ] reported that RACK1 inhibited the effects of adenovirus E1a on yeast, Saos osteosarcoma, and HeLa cells, including the induction of apoptosis. Second, Choi et al. [43 ] reported recently that RACK1 overexpression can protect PC-12 cell survival on withdrawal of nerve growth factor. Taken together with our own observations reported below, these data suggest that RACK1 plays a crucial role in regulating apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Host cell line W7.2
The W7.2 mouse cell line [30 , 31 ], originally derived from mouse thymoma line WEHI-105.726, was serially cloned three times in soft agar (method based on Longthorne and Williams [44 ]) to produce a homogeneous cell population with a minimal background of spontaneous, apoptosis-resistant mutants. The apoptosis-sensitive clone W7.2c was selected as the host for these experiments. W7.2c cells were cultured in RPMI 1640 (Sigma, UK) with 10% fetal calf serum (FCS; Hyclone, Logan, UT) and 200 µg/ml gentamycin (Sigma) at 37°C in a 5% CO2 humidified incubator. All experiments were carried out on cells in logarithmic growth phase. Cell viability was determined by vital dye staining using 0.2% nigrosin. Prior to retroviral infection, cells were treated with 90 ng/ml tunicamycin at 3 x 105 cells/ml for 18 h.

Selection of apoptosis-resistant, retrovirally infected W7.2c clones
Frozen aliquots of W7.2c cells infected with the retroviral cDNA library derived from the mouse factor-dependent cell line FDCP1 [29 ], produced as described previously [11 ], were thawed and allowed to recover for several days. The cells were exposed to 500 cGy {gamma}-radiation from a Co60 source, centrifuged, and resuspended in fresh medium for cloning in soft agar (based on Longthorne and Williams [44 ]) with 7.5 µg/ml PHA (HA16, Murex Biotech, UK) and 1 mg/ml G418. Single colonies were picked after 12 days at 37°C and expanded, and genomic DNA was prepared for analysis by polymerase chain reaction (PCR).

Analysis of cDNA inserts
PCR was carried out using vector primers flanking the cloning site [29 ]. PCR products were sequenced by MWG Biotech (Ireland). For subcloning, Pfu proofreading DNA polymerase was used (Promega, Madison, WI), followed by subcloning into pCR-Blunt II-TOPO (Invitrogen, Carlsbad, CA). For further analysis of expression, the insert was subcloned into pRUFneo and transfected into Phoenix-E packaging cells (Garry P. Nolan, Stanford University School of Medicine, CA) using FuGene (Boehringer Mannheim, Germany) to produce mouse ecotropic retrovirus to infect fresh W7.2c cells.

Plasmid DNA transfection
To generate the construct pcDNA3.pc3n3, the pc3n3 insert was amplified from the pRUFneo vector by PCR using vector primers flanking the cloning site [29 ] and Pfu proofreading DNA polymerase (Promega). The PCR product was then sequenced (MWG Biotech) and subcloned directionally into pcDNA3.1/V5-His TOPO (Invitrogen, #K4800-01). To establish W7.2c cell clones stably transfected with pcDNA3.pc3n3 expression construct, W7.2c cells were electroporated with the resulting expression construct or pcDNA3.1 using a gene pulser with a capacitance extender unit (Bio-Rad, Hercules, CA) in 0.4 cm electrode gap cuvettes [44 ]. Briefly, early logarithmic growth phase cells (4x106) in 400 µl RPMI medium without serum were electroporated with 10 µg DNA at 238 V, 1050 µF at room temperature. Following electroporation, cells were resuspended in 10 ml RPMI 1640 containing serum and glutamine. Stable cell lines were established 24 h post-transfection by soft agar cloning in Iscove’s medium (Sigma) containing 2 mM glutamine and 20% heat-inactivated fetal bovine serum in the presence of 0.5 mg/ml G418 (Sigma) for 2–3 weeks. Individual colonies were selected and expanded in complete medium with 1 mg/ml G418. Stable incorporation of the insert was verified by PCR using primers flanking the cloning site of the pRUFneo vector.

To generate stable transfectants of mouse RACK1, the W7.2c cells were cotransfected with expressed sequence tag (EST) clone mouse RACK1 expression construct pcMV-SPORT6-RACK1 (10 µg; dbEST Id: 13833312; GeneBank Acc: BU515312; IMAGE: 6511980) and pcDNA3.1 (1 µg) or with pcMV-SPORT6 and pcDNA3. W7.2 cells were electroporated, and stable clones were selected in 1 mg/ml G418 as described for W7.2-pcDNA3.1.pc3n3 cells. Individual clones were expanded and screened for expression of RACK1 by Western blotting. Clones of W7.2c cells overexpressing RACK1 were maintained in complete RPMI-1640 medium supplemented with 1 mg/ml G418.

RNA interference
RACK1 [small interfering RNA (siRNA) ID #61854; Ref. sequence NM_008143] and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cat. #4605) gene-specific, predesigned siRNA were purchased from Ambion (Austin, TX). RACK1, GAPDH, and control (scrambled siRNA, Cat. #4605) siRNAs were high-pressure liquid chromatography-purified, annealed, and ready to use. To analyze the siRNA transfection efficiency, siRNA duplexes were labeled with Cy3 using the SilencerTM siRNA labeling kit (Ambion, Cat. #1632), following the manufacturer’s instructions, and transfection efficiencies (fluorescently labeled cells after 48 h) were between 70% and 80%.

On the day before transfection, W7.2 cells were split and cultured in RPMI supplemented with 10% FCS. On the day of transfection, 106 cells were spun down and washed once in the Optimem 1 (Invitrogen, #51985-026) before resuspension in 400 µl Optimem. Cells were then incubated with 20 nM or 100 nM siRNA duplex for 10 min at room temperature in a 0.4-cm electroporation gap cuvette. Cells were electroporated for 25 ms at 260 V and 950 µF using a Bio-Rad gene pulser. Following electroporation, cells were incubated at room temperature for 20 min prior to transferring cells to six-well plates containing Iscove’s medium (Sigma) supplemented with 2 mM glutamine and 20% heat-inactivated FCS. The analysis of specific silencing of RACK1 expression was carried out after 48 h by analyzing the expression of RACK1 mRNA using real-time PCR.

Determination of cell viability, clonogenic assay, and detection of apoptosis
Cell viability was determined by nigrosin exclusion analysis and by the MTS assay (Promega, #G5421). Survival of control and treated W7.2c/pcDNA3.1 and W7.2c/pcDNA3.1-pc3n3 stable cell lines was also assessed by the ability of the cells to form colonies in soft agar, using an equal proportion of culture from each experimental condition (1/10; based on Longthorne and Williams [44 ]). The number of colonies formed was counted following 2–3 weeks incubation at 37°C in 5% CO2. The CaspaTagTM fluorescein caspase activity kit (Intergen, Purchase, NY, #S7300-025) was used to detect active caspases in the cells as a marker for apoptosis, according to the manufacturer’s instructions. Detection was performed using a fluorescence plate reader.

Northern blot analysis
Total RNA from 107 W7.2 cells was isolated using Trizol (Gibco-BRL, Grand Island, NY, #15596-026), according to the manufacturer’s instructions. mRNA was extracted from the total RNA using the Oligotex mRNA minikit (Qiagen, Valencia, CA, #70022). mRNA (5 µg) from W7.2c cells was resolved by formaldehyde containing 1% agarose gel electrophoresis, blotted onto a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Little Chalfont, UK), and immobilized by UV cross-linking (Bio-Rad, cross-linker). Hybridization was performed in ULTRAhyb buffer (Ambion) at 42°C with pc3n3 probe. Autoradiographic signals were quantitated using a phosphorimager (Bio-Rad). The blots were subsequently stripped by boiling in 0.5% sodium dodecyl sulfate (SDS) and rehybridized with a mouse ß-actin probe. The radioactive labeling of the pc3n3 and ß-actin probes was carried out by random primer-directed synthesis using the DNA labeling kit (Amersham Pharmacia Biotech) and [{alpha}32P] deoxy-cytidine 5'-triphosphate (dCTP; 3000Ci/mmol, ICN, Cost Mesa, CA). Unincorporated [{alpha}32P] dCTP was removed from the labeled DNA probe using a NICK column containing Sephadex G-50.

Real-time PCR analysis of RACK1
The expression of RACK1 in pc3n3- and RACK1-transfected clones relative to pcDNA3 clones was determined using real-time PCR. RNA (5 µg) was reverse-transcribed using using SuperscriptTM II RNase H– reverse transcriptase (RT) and random primers (Promega), according to the manufacturer’s instructions (Invitrogen, #18064). Real-time PCR was performed using 2 µl cDNA (equivalent to 500 ng total RNA) and Taq Man MGB probes and primers specific to mouse RACK1 (Applied Biosystems, Foster City, CA, Assay ID: Mm00515010_m1) with eukaryotic 18S rRNA as an endogenous control (Applied Biosystems, Assay ID: Hs99999901_s1), according to the manufacturer’s instructions. Note that the assay location of RACK1 is on position 209 and on the boundary of exons 1 and 2 so that the pc3n3 insert, where present, could not interfere with the reaction. Quantitation of the RACK1 gene in pc3n3 and RACK1-transfected clones relative to pcDNA3 clones was determined using the comparative threshold cycle (CT) method using pcDNA3 clones as calibrators. The ABI Prism 7000 sequence detection system was used to measure real-time fluorescence, and data analysis was performed using ABI Prism 7000 SDS software.

Preparation of protein fractions
Transfected W7.2c cells (106) were exposed to dexamethasone for 15, 30, or 60 min in 12-well plates. Subsequently, they were harvested and washed in phosphate-buffered saline (PBS). The cell pellets were then subjected to stepwise extraction of cytosolic and membrane proteins using ProteoExtract subcellular proteome extraction kit and following the manufacturer’s instructions (S-PEK, Calbiochem, San Diego, CA, #539790) [45 ]. Protein concentrations were determined using the Bio-Rad protein assay.

Western blot analysis
Cells (106) were washed twice in PBS and lysed in 50 µl lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 µM pepstatin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The cell suspension was then incubated on ice for 30 min before centrifugation at 10,000 g for 10 min. The protein content in the supernatant was quantified with the Bio-Rad protein assay kit. Protein samples (50 µg) were boiled for 10 min in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (10% glycerol, 0.7 M ß-mercaptoethanol, 3% SDS, 62 mM Tris-HCL, pH 6.8), subjected to 10% SDS-PAGE, and then electrotransferred onto a polyvinylidene difluoride membrane (Bio-Rad), as described previously [11 ]. The blots were then probed with the anti-RACK1 (1:2500 dilution; clone 20, BD Transduction Laboratories, Franklin Lakes, NJ, #R20620), anti-PKCß (dilution 1:250; clone 36, BD Transduction Laboratories, #610127), or anti-ß-actin antibody (dilution 1:5000; Sigma, #A5441), followed by the appropriate horseradish peroxidase-conjugated secondary antibodies, which were anti-goat immunoglobulin G (IgG; diluted 1:10,000; Sigma, #A5420), anti-mouse Ig (diluted 1:800; Dako, Carpinteria, CA, #P0447), and anti-mouse IgM (diluted 1:1000; Santa Cruz Biotechnology, CA, #sc2064). Protein bands were visualized by enhanced chemiluminescence (ECL), according to the manufacturer’s instructions (Amersham Pharmacia Biotech). The bands were quantified by densitometry (Bio-Imaging System, Syngene, Frederick, MD).

Statistical analysis
Data are presented as the mean ± SE. Statistical significance was determined by ANOVA using Origin 6.1. A P value of <0.01 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of cDNAs associated with resistance to apoptosis
Several laboratories have used the mouse T cell line W7.2 in the analysis of apoptosis, as apoptosis can be induced in this cell line by a range of different stimuli [11 , 30 , 31 ]. The apoptosis-sensitive subclone W7.2c was infected with a FDCP1 retroviral cDNA library as described previously [11 ]. A total of 8 x 107 cells were exposed to 500 cGy {gamma}-radiation and grown in soft agar in the presence of PHA. A total of 70 colonies were formed, and 16 well-separated colonies were picked. Colonies were expanded, and the inserts were amplified by PCR and sequenced. A BLAST search showed that pc3n3, the insert from clone PG3.c3, was a partial cDNA [166 base pairs (bp)] of RACK1 (Fig. 1 ).



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  		Figure 1. Sequence of pc3n3. The intronic sequence is printed in bold. The 40 bp from the untranslated region (UTR) are boxed.

 
To exclude the possibility that apoptosis resistance in these cells was the result of a spontaneous mutation, the insert was amplified by PCR using Pfu proofreading polymerase, cloned into pCR-Blunt II-TOPO, subcloned into pRUFneo, and infected into fresh W7.2c cells, producing PG3.c3 2° clones. These clones were tested for cross-resistance to an independent apoptosis inducer, the glucocorticoid analog dexamethasone. PG3.c3 2° cells formed colonies in the continuous presence of 30–100 nM dexamethasone in 96-well plates under conditions where no colonies were found in cells infected with the vector alone. This apoptosis suppression was examined and confirmed as described below.

Overexpression of pc3n3 increases the expression of RACK1 and inhibits dexamethasone- and UV-induced cell death
The effect of pc3n3 was further analyzed using the mammalian expression vector pcDNA3.1. The pc3n3 insert was cloned directionally in pcDNA3.1/V5-His TOPO and transfected into W7.2c cells to generate stable clones, which were then cultured in the absence or presence of 0.1 µM dexamethasone for up to 72 h. The viability of cells overexpressing pc3n3 was increased more than fourfold over that of control cells transfected with vector only (pcDNA3.1), based on vital dye staining (Fig. 2a ) and the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (Fig. 2b) . In addition, the colony-forming ability of these clones after treatment with dexamethasone was significantly protected (Fig. 2c) .



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  		Figure 2. pc3n3 overexpression inhibits dexamethasone-induced cell death. W7.2c were stably transfected with pcDNA3.1 or pcDNA3.1.pc3n3. Viability of clones W7.2c/pcDNA3.1 and W7.2c/pcDNA3.1.pc3n3 at 72 h, following the addition of 0.1 µM dexamethasone, as determined by vital dye staining using 0.2% nigrosin (a) and MTS assay (b). (c) Colony-forming ability of dexamethasone-treated clones. Cells (2x105 cells/ml) were cultured in the presence of dexamethasone 72 h prior to plating an equal volume (20 µl) from each culture in soft agar. Results are expressed as the mean ± SE and represent data obtained from six independent, stable, vector-only clones and 12 independent, stable pc3n3 clones.

 
Overexpression of pc3n3 also suppressed UV-induced apoptosis. Exposure of clones expressing pc3n3 to UV at 254 nm at 40 J/m2 resulted in a marked reduction in apoptosis and cell death relative to control cells, as assessed using the fluorescent caspase inhibitor carboxyfluorescein-valine-alanine-aspartic-fluoromethyl ketone, which allows the detection of active caspases in live cells and by cell counting. After 48 h, the viability of pc3n3-expressing clones was more than threefold higher than that of control cells (Fig. 3a ). In addition, these clones showed a significant reduction in caspase activity relative to irradiated control cells after 24 h (Fig. 3b) .



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  		Figure 3. pc3n3 protects against UV-induced cell death. W7.2c/pcDNA3.1 and W7.2c/pcDNA3.1.pc3n3 were irradiated with UV at 254 nm and 20 J/m2. (a) Viability of clones after 48 h. (b) Caspase detection, as a marker of apoptosis, in UV-treated cells after 24 h using a CaspaTag fluorescein caspase activity kit and fluorescence spectrophotometry.

 
We examined the effect of expression of insert pc3n3 on expression of endogenous RACK1. Northern blotting experiments were carried out on mRNA isolated from pc3n3-transfected clones, and control cells transfected with vector only using the insert pc3n3 as a probe. Figure 4a shows that the endogenous RACK1 (1.1 kb) was expressed in all the clones. Normalized RACK1 expression was increased significantly in pc3n3 clones (Fig. 4b) . The increase in RACK1 expression was also detected by real-time PCR, where results (Fig. 4c) showed a strong, direct correlation with the survival of dexamethasone-treated clones (n=10; P<0.0001; Fig. 4d ), and Western blotting confirmed that the level of RACK1 protein was also increased (Fig. 4e) .



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  		Figure 4. Expression analysis of RACK1 by Northern and Western blotting. (a) Northern analysis of RACK1 after transfection of W7.2c cells with pcDNA3.1 (lanes 1–3) or pcDNA3.1.pc3n3 (lanes 4–8). After stripping, the blot was reprobed with ß-actin probe as a control. (b) Ratios of normalized RACK1 to ß-actin. Measurement of autoradiographic signals was determined using a phosphorimager. The plotted data were averaged from two independent experiments ± SE. *, P < 0.01, compared with vector only. (c) The expression of RACK1 in pc3n3 clones relative to pcDNA3 clones was determined by real-time PCR, using the comparative CT method, normalized with 18S RNA as an internal control. Results are represented as mean ± SE from three separate experiments. (d) Graph of data from individual pc3n3 clones with the fold increase in an endogenous RACK1 message, as determined by real-time PCR plotted against the number of colonies surviving dexamethasone treatment. The correlation is highly significant (R=0.973; P<0.0001). (e) Western blotting of RACK1 protein in pc3n3 and vector only-transfected clones. Lanes 1 and 2 contain total protein from W7.2 cells transfected with pcDNA3.1; lanes 3 (clone 15) and 4 (clone 4) contain W7.2 cells transfected with pcDNA3.1.pc3n3. The corresponding Western blots with anti-ß-actin antibody are shown to demonstrate equivalent loadings.

 
RNA interference down-regulates RACK1 mRNA and RACK1 protein and abolishes apoptosis resistance
The analysis of RACK1 mRNA expression after transfection of the RACK1-specific siRNA by real-time PCR revealed a 77–86% reduction of expression in pc3n3 clones transfected with the RACK1-specific siRNA (Fig. 5a ). Silencing efficiency at 20 nM was comparable with that achieved at 100 nM siRNA (Fig. 5a) . The specificity of the RACK1 siRNA was tested by comparison with the GAPDH siRNA and the scrambled siRNA, which were clearly inefficient in reduction of RACK1 (Fig. 5a) . Down-regulation of RACK1 protein by RACK1-specific siRNA was confirmed by Western blotting (Fig. 5b) .



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  		Figure 5. The effects of RACK1 siRNA on endogenous RACK1 levels, (a) determined by real-time PCR and (b) determined by Western blotting. W7.2/pcDNA3.1.pc3n3 cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, the expression of RACK1 was determined by real-time PCR (a), using the comparative CT method, normalized with 18S RNA as an internal control. The expression of RACK1 in the cells transfected with RACK1 siRNA was compared with that in cells transfected with control (scrambled and GAPDH) siRNAs. Results are represented as mean ± SE (for RACK1 expression relative to untransfected cells) from three separate experiments. Expression of RACK1 protein under the same conditions was determined by Western blotting (b), and equivalent loading was demonstrated using anti-ß-actin antibody.

 
Figure 6 shows that the suppression of dexamethasone-induced apoptosis observed in pc3n3 clones was totally abolished by transfection with RACK1 siRNA. Transfection with GAPDH and control siRNAs had no effect on the inhibition of dexamethasone-induced apoptosis in these clones (Fig. 6) .



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  		Figure 6. RACK1-specific siRNA abolishes the inhibition of dexamethasone-induced apoptosis conferred by pc3n3. W7.2c pcDNA3.1 or pcDNA3.1.pc3n3 transfected with RACK1 siRNA (20 nM) was exposed to 0.1 µM dexamethasone for 72 h. After that time, viability of the cells, as determined by MTS assay, is indicated by the absorbance at 490 nm. Results are expressed as the mean ± SE and represent data obtained from three independent, stable, vector-only clones and four independent, stable pc3n3 clones.

 
RACK1 suppresses dexamethasone-induced apoptosis
In view of the increased level of RACK1 mRNA in the apoptosis-resistant clones, we next determined whether overexpression of full-length RACK1 could affect apoptosis induced by dexamethasone. W7.2c cells were cotransfected with pCMV-Sport.RACK1 (IMAGE: 6511980), and stable clones were selected by growth in G418. Six vector-only, transfected clones and twelve RACK1-transfected clones were cultured in the continuous presence of dexamethasone (0.1 µM). Ten of the RACK1-transfected cells demonstrated resistance to dexamethasone (0.1 µM; Fig. 7a ). The five RACK1-stable clones (clones 1, 2, 4, 5, and 8), which showed the greatest ability to suppress dexamethasone-induced cell death, were selected for further detailed analysis. The level of overexpression of RACK1 was confirmed by Western blotting, which showed a two- to sixfold increase in RACK1 protein in these clones (Fig. 7b and 7c) . Overexpression of RACK1 was also determined using real-time PCR, which revealed a two- to fourfold increase in RACK1 RNA in these clones (Fig. 7d) , which were then cultured in the presence or absence of dexamethasone (0.1 µM) for up to 72 h, after which time, colony-forming ability in soft agar was determined as a measure of long-term survival of the RACK1 clones. Figure 8a shows that the colony-forming ability of RACK1-overexpressing clones was greatly protected compared with those of controls and that the protection of colony-forming activity was strongly correlated with the increase in RACK1 expression as determined by quantitative real-time RT-PCR (Fig. 8b ; P<0.0001). The protection associated with increased RACK1 expression in pc3n3-expressing clones was quantitatively strikingly similar to that found in cells transfected with full-length RACK1 (Fig. 8c) .



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  		Figure 7. Selection of stably transfected RACK1-transfected clones by functional analysis. (a) After G418 selection, six stably transfected, vector-only clones and 12 stably transfected RACK1 clones were examined for their ability to resist cell death in 0.1 µM dexamethasone for 72 h. The five RACK1-stable clones (clones 1, 2, 4, 5, and 8; lanes 5–9), which showed the greatest ability to suppress dexamethasone-induced cell death, and four pcDNA3 clones (lanes 1–4) were then examined by immunoblotting with RACK1 antibody, stripped, and reprobed with ß-actin antibody (b). (c) Ratio of standardized RACK1 expression to ß-actin. The bands observed by ECL detection were quantified by densitometry. The results shown were averaged from three independent experiments ± SE. *, P < 0.01, compared with vector only. (d) The expression of RACK1 in RACK1 transfected clones relative to pcDNA3 clones was determined by real-time PCR, using the comparative CT method, normalized with 18S RNA as an internal control. Results are represented as mean ± SE from three separate experiments.

 


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  		Figure 8. (a) RACK1 overexpression inhibits loss of clonogenicity induced by dexamethasone in W7.2c cells. RACK1-transfected and vector-only clones were treated with 0.1 µM dexamethasone for 72 h prior to plating an equal volume (20 µl) from each culture in soft agar. Results are expressed as the mean ± SE and are obtained from four independent, stable, vector-only clones and five independent, stable, RACK1-transfected clones. (b) Graph of data from individual RACK1 clones with the fold increase in endogenous RACK1 message, as determined by real-time PCR, plotted against the number of colonies surviving dexamethasone treatment. Correlation is highly significant (R=0.939; P<0.0001). (c) Graph of data from pc3n3-transfected, together with RACK1-transfected, clones with the fold increase in an endogenous RACK1 message, as determined by real-time PCR, plotted against the number of colonies surviving dexamethasone treatment.

 
The role of the ß-isoenzyme of PKCß in the antiapoptotic activity of RACK1
RACK1 is a cytosolic protein, one of a group of PKC-interacting proteins, which have been termed RACKs, because of the manner in which they were identified originally [46 ], although they appear to have other important functions in addition (see below). RACKs are reported to enable translocation of PKC isoenzymes and to stabilize their active forms [18 ], and the presence of isoenzyme-specific RACKs has been reported [47 ]. The isoenzyme specifity of RACK1 is reported to be for PKCß [34 , 46 , 48 , 49 ]. Activation of PKC is associated with its translocation from the cytosolic (soluble) fraction to the membrane fraction [50 ]. Therefore, to determine whether the antiapoptotic effect of RACK1 is associated with effects on PKCß, we assessed PKCß translocation in RACK1-expressing cells after dexamethasone treatment. W7.2c/pCMV-SPORT.RACK1 and W7.2c/pCMV-SPORT cells were incubated with 0.1 µM dexamethasone for 0, 15, 30, and 60 min, and the PKCß content of the cytosol and membrane fractions was determined by Western blotting. Figure 9 shows that in RACK1-overexpressing cells only, dexamethasone caused a translocation of PKCß to the membrane fraction, which could be detected after 15 min and was maintained after 1 h, indicating potential involvement of PKCß in the antiapoptotic role of RACK1.



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  		Figure 9. Activation of PKCß in RACK1-transfected clones by dexamethasone. W7.2c/pCMV-SPORT and W7.2c/pCMV-SPORT.RACK1 clones were treated with 0.1 µM dexamethasone for 0 and 60 min prior to stepwise extraction of the cytosolic and organelle/membrane fractions. PKCß was detected by immunoblotting with anti-PKCß antibody. Bands were visualized by ECL (C, cytosol; M, membrane; a). (b) Quantitative data of translocation of PKCß. Intensity of each signal in the cytosol and membrane fractions was quantified by densitometry and presented in a histogram. Data are presented as mean ± SE from three independent experiments. *, P < 0.01, compared with the control.

 
We further investigated whether a direct activation of PKCß could inhibit dexamethasone-induced cell death in W7.2C cell, mimicking the effect of RACK1. To examine this hypothesis, we used the pseudo-RACK1 peptide (TOCRIS, #1790), which is reported to have a greater specificty for PKCß than for other PKC isoenzymes [16 ]. Pseudo-RACK1 binds and activates PKCß and modulates its function in vivo and in vitro [48 ]. Initially, we investigated whether pseudo-RACK1 could activate PKCß in W7.2c cells, which were incubated with 1 µM pseudo-RACK1 for 30 min before assessing PKCß translocation from the cytosol to the membrane by Western blotting. Figure 10 shows that challenging w7.2c cells with pseudo-RACK1 caused an activation of PKCß, as assessed by its translocation from the cytosol to the membrane fraction. W7.2c cells were then exposed to different concentrations (10 nM–1 µM) of the pseudo-RACK1 peptide for 1 h, prior to adding 0.1 µM dexamethasone and further incubation of 72 h. Cell viability assay and viable cell number count showed that the pseudo-RACK1 peptide does not protect W7.2 cells from cell death caused by dexamethasone (Table 1 ). These results may suggest that activation of PKCß alone is not sufficient to protect W7.2c cells from cell death or that pseudo-RACK1 peptide is activating another PKC isoenzyme, which counteracts the effects of PKCß.



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  		Figure 10. Activation of PKCß by pseudo-RACK1 peptide. W7.2 cells were incubated for 30 min with 1 µM pseudo-RACK1 peptide. After that time, cytosolic and membrane fractions were extracted, and PKCß was detected by immunoblotting with anti-PKCß antibody. Bands were visualized by ECL (C, cytosol; M, membrane).

 

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  		Table 1. Activation of PKCß Alone May Not Be Sufficient to Produce the Antiapoptotic Effects of RACK1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
RACK1 was originally identified as a receptor for activated PKCß, which it stabilizes in the active state and anchors to membranes or functional sites [33 , 34 , 48 ]. Indeed, defects in PKC translocation are reported to correlate with a reduced level of RACK1 in the aging rat brain [51 ]. In addition, disruption of the interactions between PKC and RACK1 impaired insulin-induced kinase translocation [16 ] and regulation of cardiac calcium channels [52 ]. Further studies have also implicated RACK1 in binding integrins, Src kinases, and adenoviral oncoprotein E1A and in regulating cytoskeletal organization and integrin-mediated cell migration [35 36 37 , 41 , 53 54 55 ].

Our data demonstrate that up-regulation of RACK1 produces protection from apoptosis. In addition, the close quantitative similarity between the protection from dexamethasone-induced apoptosis and the increase in RACK1 expression in clones expressing pc3n3 and in those expressing full-length RACK1 (Fig. 8) strongly supports the suggestion that pc3n3 does indeed inhibit apoptosis by up-regulation of RACK1. This is further confirmed by the abolition of protection by siRNA-mediated down-regulation of RACK1 in the resistant clones (Fig. 6) . The exact mechanism by which pc3n3 could affect RACK1 mRNA levels is not clear. It is unlikely that Pc3n3 could have a direct effect on RACK1 mRNA, as it is in the sense orientation and does not have the full sequence. However, pc3n3 does contain part of the RACK1 3'-UTR, and an increasing body of evidence indicates that a number of genes can be subject to regulation through interactions with 3'-UTR regions [56 ].

As described earlier, RACK1 is reported to interact with the active form of PKCß. Our results show that overexpression of RACK1 led to a translocation of PKCß in response to dexamethasone, suggesting that the PKCß activation could be involved in the antiapoptotic effects of RACK1. Although pseudo-RACK1, a PKCß agonist, failed to protect W7.2c cells from cell death mediated by dexamethasone, the role of PKCß in RACK1 antiapoptotic effects cannot be excluded, as pseudo-RACK1 is reported to bind to other PKC ({delta} and {varepsilon}) isoforms in a less-specific manner [48 ], and activation of other PKC isoforms could be obscuring the effects of PKCß.

RACK1 effects can be mediated through a number of other targets, and the reported interaction with the transcription factor p73 is of particular interest. RACK1 is reported to be a negative modulator of p73, and overexpression of RACK1 in human osteosarcoma SAOS-2 directly suppresses p73-mediated transcription and inhibits p73-dependent apoptosis [40 ]. Accordingly, RACK1 inhibition of dexamethasone and UV-mediated cell death in W7.2c cell lines could involve the inhibition of p73-mediated apoptosis. It is interesting that the inhibitory effect of RACK1 on p73 is counteracted by pRB, suggesting a functional link amongst RACK1, p73, and pRB [40 ]. Examination of the effects of siRNA-mediated inactivation of endogenous pRB and/or RACK1 in W7.2 cells is required to analyze the functional interactions amongst p73, RACK1, and pRB. In addition, RACK1 is reported to interact with transcription factor nuclear factor of activated T cells and to repress its transactivation [57 ].

RACK1 is composed of seven WD domains, and these motifs have been implicated in protein–protein interactions [40 , 55 , 56 ]. It is therefore possible that the antiapoptotic effects of RACK1 could be mediated through interactions with other, as yet unrecognized, molecules important in apoptosis.

Much remains unknown concerning the functional role of RACK1; however, studies have indicated that it is up-regulated in angiogenesis and in human carcinomas [58 ]. RACK1 has also been found to interact directly with Epstein-Barr virus transactivator protein BZLF1 [59 ]. In addition, the involvement of RACK1 in the regulation of Src function and cell growth has been reported [38 , 41 ]. It is interesting that RACK1 appears to function as scaffold for the androgen receptor upon activation of PKCß and in the absence of ligand, enabling the translocation of the androgen receptor to the nucleus but rendering it unable to activate transcription of its target genes [39 ]. Most recently, two laboratories have shown that at least in fungi, RACK1 (yeast Asc1p) associates with ribosomes to regulate the translation of specific mRNAs [60 , 61 ].

We have identified RACK1 as a potential regulator of apoptosis, using functional cDNA expression cloning, and shown that overexpression of RACK1 in W7.2 cells inhibits apoptosis induced by a number of stimuli. Identification of signaling pathways involving RACK1 may therefore define novel means of regulating apoptosis in pathological situations. Further investigations will focus on determining the downstream mechanisms by which RACK1 regulates apoptosis.


   ACKNOWLEDGEMENTS
 
We thank the Wellcome Trust, the Leukaemia Research Fund, and The Grand Charity of the National Order of Freemasons for financial support. We also thank the Dead Cell Lab, Dr. Robyn Starr, and our other collaborators at WEHI, Melbourne, and Dr. Tom Gonda and colleagues at the Hanson Institute, Adelaide, for warm hospitality, help, and advice and Dr. Janet Meredith for subcloning pc3n3.

Received February 4, 2005; revised March 18, 2005; accepted April 1, 2005.


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 DISCUSSION
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