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Melanoma differentiation-associated gene-7 protein physically associates with the double-stranded RNA-activated protein kinase PKR
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doi:10.1016/j.ymthe.2005.01.018
Melanoma Differentiation-Associated Gene-7 Protein Physically Associates with the Double-Stranded RNA-Activated Protein Kinase PKR Abujiang Pataer,1,* Stephan A. Vorburger,2 Sunil Chada,3 Siddharth Balachandran,4 Glen N. Barber,4 Jack A. Roth,1 Kelly K. Hunt,2 and Stephen G. Swisher1,* 1
Department of Thoracic and Cardiovascular Surgery and 2Department of Surgical Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 445, Houston, TX 77030, USA 3 Introgen Therapeutics, Inc., Houston, TX 77030, USA 4 Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136, USA *To whom correspondence and reprint requests should be addressed. Fax: +1 713 794 4901. E-mail: [email protected] or [email protected].
Available online 3 March 2005
We previously reported that adenoviral-mediated overexpression of the melanoma differentiation-associated gene-7 (Ad-mda7; approved gene symbol IL24) leads to the rapid induction of PKR and activation of its downstream targets, resulting in apoptosis induction in human lung cancer cells. To evaluate the mechanism by which Ad-mda7 activates PKR, we studied the interaction between MDA-7 and PKR proteins. Following Ad-mda7 transduction of lung cancer cells, intracellular and extracellular MDA-7 protein was generated, leading to dose- and timedependent PKR induction. Purified MDA-7 protein administered extracellularly did not induce PKR or apoptosis, suggesting that Ad-mda7-mediated PKR activation and apoptosis were not dependent on extracellular MDA-7 protein. Following Ad-mda7 transduction, RT-PCR demonstrated no increase in PKR mRNA levels despite increased levels of PKR protein, suggesting posttranscriptional regulation of PKR by MDA-7. Immunofluorescence and coimmunoprecipitation studies demonstrated that MDA-7 protein physically interacts with PKR. Transduction of PKR+/+ and PKR / transformed MEFs with Ad-mda7 demonstrated phosphorylated MDA-7 and PKR proteins in the lysates of PKR+/+ but not PKR / cells. These findings identify the first binding partner for MDA-7 and suggest that direct interaction between PKR and MDA-7 may be important for PKR activation and apoptosis induction, possibly through MDA-7 phosphorylation or activation of other downstream targets. Key Words: MDA-7 (IL-24), PKR, apoptosis, gene therapy, adenovirus
INTRODUCTION Melanoma differentiation-associated gene-7 (mda-7; approved gene symbol IL24) was identified by a subtraction hybridization approach in human HO-1 melanoma cells treated with the combination of recombinant human fibroblast interferon (IFN)-h and the protein kinase C activator mezerein [1]. The mda-7 cDNA encodes an evolutionarily conserved protein of 206 amino acids with a predicted size of 23.8 kDa and exhibits greatest homology to the interleukin-10 (IL-10) subfamily [2,3]. Adenoviral-mediated overexpression of melanoma differentiation-associated gene-7 (Ad-mda7) induces apoptosis in a wide range of cancer cells through the activation of multiple signal transduction pathways [4–7]. We have reported that Ad-mda7-
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mediated apoptosis in lung cancer cells occurs through activation of the dsRNA-dependent protein kinase (PKR) [5]. In this study, we further investigated the relationship between MDA-7 expression and PKR activation. Immunofluorescence and coimmunoprecipitation studies demonstrated that MDA-7 and PKR proteins interact directly, leading to phosphorylation of MDA-7 and PKR. Phosphorylation of MDA-7 was observed on both threonine and serine residues and was dependent on the presence of PKR as demonstrated by transduction of PKR+/+ and PKR / transformed MEFs with Ad-mda7. These findings identify PKR as the first binding partner for MDA-7 and suggest that the direct interaction between PKR and MDA-7 may be important for PKR activation and the induction of
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apoptosis, possibly through MDA-7 phosphorylation or activation of other downstream targets.
RESULTS Ad-mda7 Induces Apoptosis and PKR on Human Lung Cancer Cells We performed flow cytometric analysis on the A549 (wt p53) and H1299 (null p53) human lung cancer cells at 48 h following infection with Ad-mda7, Ad-Luc, or PBS (Fig. 1A). Ad-mda7 treatment resulted in a dosedependent induction of PKR in A549 cells (Fig. 1B) and H1299 cells (data not shown). PKR levels continued to increase with Ad-mda7 dose. Ad-mda7 (2500 vp/cell) treatment of lung cancer cells also showed a temporal induction of PKR, with robust induction at 24 h posttreatment and maximal levels noted at 48 h (Fig. 1C). We performed real-time PCR to determine if Admda7-mediated induction of PKR protein (Figs. 1B and 1C) occurred through a transcriptional or a posttranscriptional mechanism. As opposed to Ad-E2F-1, Admda7 did not increase PKR mRNA levels, suggesting a posttranscriptional mechanism (Fig. 1D). Intracellular MDA-7 Protein Plays a Role in Ad-mda7-Induced Cell Death We evaluated the anti-tumor effects of secreted MDA-7 on A549 (wt p53) and H1299 (null p53) human lung cancer cells to determine if extracellular MDA-7 could induce apoptosis and PKR induction. Figs. 2A and 2B demonstrate that MDA-7 secreted protein is not able to inhibit cell growth (Fig. 2A) or cause cell death (2B) in lung cancer cells. MDA-7 secreted protein also failed to induce PKR or activate STAT3 in A549 cells (Fig. 2C). In contrast, Ad-mda7-treated A549 cells show induction of PKR and activated STAT3, consistent with our previous observations that intracellular MDA-7 is critical for Admda7-mediated apoptosis in lung cancer cells [7]. MDA-7 Protein Physically Interacts with the dsRNA-Dependent Protein Kinase PKR We next assessed subcellular localization of PKR and MDA-7. We saw PKR expression (green) in cytoplasmic and nuclear compartments, while MDA-7 expression (red) was predominantly in the cytosol. Using confocal microscopy, we noted cytoplasmic colocalization of MDA-7 and PKR (Fig. 3A). We then looked for a direct interaction between MDA-7 and PKR. As shown in Fig. 3B, anti-PKR antibodies did not immunoprecipitate MDA-7 protein in the PBS- or Ad-Luc-treated cells; however, in Ad-mda7-transduced cells, MDA-7 was detected after immunoprecipitation with antibodies against PKR (Fig. 3B). The negative control (IgG) did not immunoprecipitate MDA-7 protein in the Ad-mda7treated cells (Fig. 3B). To confirm the protein–protein interaction, we performed the reciprocal experiment
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FIG. 1. Adenoviral overexpression of MDA-7 in lung cancer cells results in apoptosis and posttranscriptional PKR induction. (A) Percentage of apoptotic cell death as determined by PI and flow cytometry in A549 (wild-type p53) and H1299 (null p53) cells 48 h following treatment with PBS, Ad-Luc (2500 vp), or Ad-mda7 (2500 vp). Triplicate experiments were performed for each cell line. (B) Western blot analysis of the dose-dependent expression of PKR after Ad-mda7 transduction. (C) Western blot analysis of the timedependent expression of PKR after Ad-mda7 (2500 vp) transduction. (D) Realtime PCR analysis of PKR mRNA in Ad-mda7-, Ad-luc-, Ad-E2F-1-, or PBStreated cells.
with PBS-, Ad-Luc-, and Ad-mda7-treated cell lysates immunoprecipitated with antibody against MDA-7. We detected PKR only in Ad-mda7-treated cell lysates (Fig. 3B). These immunoprecipitation studies suggest that MDA-7 binds to the dsRNA-dependent protein kinase PKR.
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of MDA-7 and PKR (Fig. 4C). Tyrosine phosphorylation of MDA-7 and PKR was not demonstrated (data not shown). These studies demonstrate serine and threonine phosphorylation of MDA-7 and PKR following Ad-mda7 transduction and suggest that MDA-7 phosphorylation may be dependent on PKR or another downstream serine/ threonine kinase.
DISCUSSION
FIG. 2. Intracellular MDA-7 protein plays an important role in Ad-mda7induced cell death and PKR induction. (A) Cell number following PBS, Admda7, or secreted MDA-7 protein treatment. (B) Apoptosis induction following treatment with PBS, Ad-mda7, and secreted MDA-7 protein. (C) Western blot analysis of the expression of PKR, STAT3, and phosphoSTAT3 in A549 cell lysates 48 h after treatment with PBS, Ad-mda7 (2500 vp), or secreted MDA-7. Results of one representative experiment of three are shown.
PKR and MDA-7 Proteins Are Phosphorylated Following Ad-mda7 Transduction Following Ad-mda7 transduction, we demonstrated phosphorylated forms of both PKR and MDA-7 in both human lung cancer cells (Fig. 4A). We demonstrated phosphorylation of PKR and MDA-7 on threonine and serine residues but not tyrosine residues. We performed Ad-mda7 transduction of PKR+/+ and PKR / cells to determine whether phosphorylation was dependent on PKR. Only PKR+/+ cells demonstrated apoptosis and phosphorylation of PKR and MDA-7 (Figs. 4B and 4C). We detected both serine and threonine phosphorylation
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mda-7 is a tumor suppressor gene first identified by subtraction hybridization of a melanoma cell line induced to terminally differentiate with IFN-h and mezerein [1]. Ad-mda7 appears to mediate anti-tumor activity through the activation of multiple signal transduction pathways leading to growth inhibition and apoptosis induction [4–8]. We have previously demonstrated that Ad-mda7-mediated apoptosis of lung cancer cells appears to be dependent on PKR induction although the mechanism of PKR activation has not been defined [5]. In the present investigation we have further explored the intracellular relationship between MDA-7 and PKR proteins. Following Ad-mda7 transduction of lung cancer cells, MDA-7 protein exists both intracellularly and in a secreted extracellular form [6]. The secreted extracellular form of MDA-7/IL-24 can bind to two different receptors, the type 1 IL-20 heterodimeric receptor complex comprising IL-20R1/IL-20R2 and the type 2 complex comprising IL-22R1/IL-20R2, which lead to activation of the STAT signaling pathways [9]. Fisher’s group has recently shown that treatment of malignant gliomas with bacterially expressed GST–MDA-7 protein can reproduce the cancer cell specificity seen with Ad-mda7. This required supraphysiologic levels of protein, and receptor–ligand interactions were not evaluated [10]. We evaluated whether extracellular recombinant human MDA-7/IL-24 protein could induce PKR activation and cell death in human lung cancer cells. Our results suggest that, in lung cancer cells, Ad-mda7-induced apoptosis and PKR induction are dependent on intracellular MDA-7 protein production since extracellular MDA-7 was unable to induce apoptosis, PKR induction, or STAT3 activation (Figs. 2A, 2B, and 2C). This observation is consistent with our report that A549 and H1299 lung cancer cells lack MDA-7 receptors [11] and provides an explanation as to why secreted MDA-7 fails to activate STAT3 or PKR when delivered extracellularly to these cells. We analyzed PKR mRNA levels by real-time PCR to determine whether Ad-mda7-induced PKR activation was due to mRNA upregulation. We found that PKR mRNA accumulation was not increased following Admda7 transduction and was not responsible for the elevated levels of PKR protein (Fig. 1D). The increase in PKR protein following Ad-mda7 transduction therefore appears to be due to posttranscriptional mecha-
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FIG. 3. MDA-7 colocalizes and interacts directly with PKR. (A) Immunofluorescence microscopy with antibodies against MDA-7 stained red and PKR stained green demonstrated upregulation of both MDA-7 and PKR proteins in Ad-mda7treated cells, but not in control-treated cells after 48 h. (B) Protein fractions, obtained from A549 and H1299 cells treated with PBS, Ad-Luc (2500 vp), or Ad-mda7 (2500 vp) for 48 h, were immunoprecipitated with anti-human PKR (left) or anti-human MDA-7 (right) and assayed for MDA-7 or PKR proteins.
nisms rather than mRNA induction. This mechanism of PKR induction is in contrast to that of Ad-E2F1, in which E2F-1 increases PKR expression at the mRNA level [12]. PKR is known to bind to several proteins, including eIF-2a, HCV E2, E3L, NFAR-1 and -2, and PACT/RAX [13– 17]. Our study identifies MDA-7 as a novel binding partner for PKR. The cellular activator PACT is known to induce PKR activation through direct protein–protein interaction with the dsRNA binding regions of PKR. We, therefore, examined whether MDA-7 might also interact and induce PKR through a similar interaction. Our immunofluorescence studies show that MDA-7 colocalizes with PKR protein in Ad-mda7-transduced lung cancer cells and immunoprecipitation demonstrates that MDA-7 and PKR proteins interact directly (Fig. 3B). We previously demonstrated that MDA-7 regulation of the hcatenin and PI3K signaling pathways does not require direct binding of MDA-7 to the bdestruction complexQ of GSK3-h/h-catenin/axin/APC in lung and breast cancer cells [18]. The current study, however, demonstrates the first evidence of a specific binding partner for MDA-
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7. Whether MDA-7 binds to the dsRNA binding region on PKR, like PACT, or another area remains to be determined. Human PKR contains at least 15 autophosphorylation sites that are involved in PKR activation [13–15]. Once activated, PKR becomes autophosphorylated and acquires the capacity to phosphorylate its downstream targets, including eIF-2a, leading to cessation of protein synthesis and cell death [13–15]. We found that, following Admda7 transduction, both PKR and MDA-7 proteins are phosphorylated at threonine and serine residues in lung cancer cells and PKR+/+ cells, but not in PKR / cells. These findings support the concept that MDA-7 interacts directly with PKR and that MDA-7 binding result in PKR activation and autophosphorylation. MDA-7 phosphorylation may then occur due to PKR kinase activity or through phosphorylation by another kinase. PKR is activated by various stimuli, including dsRNA, heparin, and the dsRNA-binding protein RAX/PACT, which is induced following cellular stress. Interestingly, activation of PKR by RAX/PACT appears to be dependent on the phosphorylation of RAX/PACT that occurs after binding
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FIG. 4. MDA-7 and PKR protein phosphorylated following Ad-mda7 transduction. (A) Protein fractions obtained from A549 cells treated with Ad-Luc (2500 vp) or Ad-mda7 (2500 vp) for 48 h were immunoprecipitated with anti-phospho-threonine or anti-phospho-serine and assayed for MDA-7 and PKR proteins. Results of one representative experiment of three are shown. (B) Western blot analysis of the expression of PKR, MDA-7, and actin in PKR+/+ and PKR / cell lysates 48 h after treatment with Ad-mda7 (2500 vp) demonstrating similar levels of transduced MDA-7. (C) Protein fractions, obtained from PKR+/+ and PKR / cells treated with PBS, AdLuc (2500 vp), or Ad-mda7 (2500 vp) for 48 h were immunoprecipitated with anti-phosphothreonine or anti-phospho-serine and assayed for MDA-7 and PKR proteins. Percentage of cell death in PKR+/+ and PKR / cells following treatment with PBS, Ad-luc (2500 vp), and Ad-mda7 (2500 vp) is indicated at the bottom. The cells were analyzed by flow cytometry 48 h after transduction. Results of one representative experiment of three are shown.
to PKR [19]. A nonphosphorylated form of RAX is still able to bind to PKR but is unable to activate PKR following stress, presumably because it cannot be phosphorylated. MDA-7 may follow a similar pattern of PKR activation by initially binding to PKR in a nonphosphorylated state that is unable to activate PKR until it is phosphorylated by another cellular kinase. Alternatively, the direct binding of MDA-7 to PKR may induce a conformational change in PKR leading to PKR activation, autophosphorylation, and MDA-7 phosphorylation by PKR itself. Further studies will be performed to determine the sequence of events and the importance of MDA-7 phosphorylation in PKR activation. The current study cannot separate the kinetics of MDA-7 and PKR phosphorylation; however, induction of apoptosis occurs only when both proteins are phosphorylated on serine and threonine. The importance of MDA-7 phosphorylation in the anti-tumor effects of Ad-mda7 remains in question. The phosphorylation of proteins often leads to diverse functions, including kinase activation, protein stabilization, growth stimulation, proteasome regulation, and apopto-
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sis induction [20]. The MDA-7 sequence has six potential phosphorylation sites (four serines, 88, 101, 161, and 164, and two threonines, 111 and 133) [20]. The phosphorylated MDA-7 sites appear to be serine and threonine: it is not known which serine or threonine sites are phosphorylated (Figs. 4A and 4C). Additionally, phosphorylation on tyrosine residues has not been detected, suggesting the kinase responsible for MDA-7 phosphorylation is a Ser/Thr kinase. PKR is a Ser/Thr kinase, allowing it to be a candidate kinase for MDA-7 phosphorylation. Further support for PKR being the kinase responsible for MDA-7 activity is given by our previous studies using inhibitors of PKR, MEKK, PI3K, and MAPK: only 2-aminopurine, which blocks PKR function, was able to abrogate tumor cell killing by Ad-mda7 [5,6]. The functional consequences of MDA-7 phosphorylation are not currently known but may include control of apoptosis induction. We observed MDA-7 phosphorylation in lung cancer cells and PKR+/+ cells (Figs. 4A and 4C) induced to undergo apoptosis by Ad-mda7, but not in PKR / cells, which are resistant to Ad-mda7 apoptosis. After Ad-mda7 treatment, normal human cells also
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express high levels of MDA-7, but do not undergo apoptosis. Further studies will determine whether the resistance to killing in normal cells correlates with MDA7 and PKR phosphorylation status. In conclusion, we have extended our initial observation that Ad-mda7-induced apoptosis is dependent on PKR to assess the mechanisms of PKR and MDA-7 interaction. We demonstrate that following Ad-mda7 transduction, MDA-7 binds PKR, documenting the first known binding partner for MDA-7 and defining a novel binding partner for PKR. Additionally, we show that both MDA-7 and PKR are phosphorylated following Ad-mda7 transduction of lung cancer cells. The possibility exists that MDA-7 phosphorylation is due to PKR kinase activity or a downstream PKR target since only PKR+/+ cells demonstrate phosphorylation of MDA-7. The function and role of MDA-7 phosphorylation in apoptosis induction and PKR activation remain to be elucidated but may in the future provide an additional therapeutic target to enhance further Ad-mda7 antitumoral activity.
MATERIALS
AND
METHODS
Cell lines and reagents. A549 and H1299 human lung cancer cell lines were obtained from the American Type Culture Collection. PKR+/+ and PKR / isogenic mouse embryonic fibroblasts were obtained from Dr. Glen N. Barber (University of Miami School of Medicine). Recombinant MDA-7 protein was obtained from Introgen Therapeutics (Houston, TX, USA). Adenovirus production. Construction of the Ad-mda7, Ad-bak, Ad-lacZ, and Ad-Luc vectors has been previously reported [5]. Adenoviral vectors were used at titers needed to transduce at least 70% of the cells. Flow cytometry analysis. We measured apoptotic cells by propidium iodide staining and FACS analysis (Becton–Dickinson FACScan, Mountain View, CA, USA; FL-3 channel) as previously described [5]. Real-time PCR. RT-PCR was carried out using the Thermoscript RT-PCR system kit according to the instructions provided by the manufacturer (Life Technologies, Gaithersburg, MD, USA). Total RNA was isolated according to the protocol for TRIzol extraction (Life Technologies), and real-time PCR was performed using the protocol reported previously [12]. To quantify the PKR mRNA expression, we used TaqMan PCR probes (Perkin–Elmer Applied Biosystems, Foster, CA, USA) and the 7700 sequence detector (Perkin–Elmer Applied Biosystems). Reactions contained 1V TaqMan Universal PCR Master Mix (Perkin–Elmer Applied Biosystems), 900 nM forward primers (5V-CCTGTCCTCTGGTTCTTTTGCT-3V) and reverse primers (5V-GATGATTCAGAAGCGAGTGTGC-3V), and 200 nM TaqMan probes (5V-ACGTGTGAGTCCCAAAGCAACTCTTTAGTGAC-3V). GAPDH primers and probes were added at 50 nM. Thermal cycling proceeded with 50 cycles of 958C for 15 s and 608C for 1 min. Input RNA amounts were calculated with relative standard curves for both the mRNAs of interest and GAPDH. Western blot analysis. Forty-eight hours after transfection, cell extracts were prepared and immunoblot assays performed as previously described [5]. Anti-PKR (K-17), anti-h-actin, anti-STAT3 (F-2), and phospho-specific anti-STAT3 (B-7) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the polyclonal or monoclonal antibody to MDA-7 was obtained from Introgen Therapeutics. Coimmunoprecipitation analysis. Cells were treated with Ad-mda7 or Ad-Luc for 48 h and then lysed in RIPA buffer (1 PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). The primary antibody was
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incubated overnight at 48C. Protein A/G agarose was added to the mix and incubated for 4 h. Centrifugation and washing of pellets were performed and the samples were assayed by Western blot analysis. Cellular localization studies. A549 and H1299 cells (5 104 cells/well) were grown on chamber slides until 70% confluence and then transfected with Ad-luc, Ad-mda7, or PBS. Forty-eight hours later, cells were washed with PBS and fixed with 4% paraformaldehyde/PBS for confocal analysis as previously described [12]. Rabbit polyclonal anti-PKR (K-17) and mouse monoclonal anti-MDA-7 were utilized and assessed by immunofluorescence microscopy. Statistical analysis. The data reported represent the means of three or more independent experiments and the bars show the standard deviation. ANOVA and two-tailed Student’s t test were used for statistical analysis of multiple groups and pair-wise comparison, respectively, with P b 0.05 considered significant.
ACKNOWLEDGMENTS This work was supported by grants from the National Cancer Institute and the National Institutes of Health [P01 CA78778-01A1 (J.A.R., S.G.S.), SBIR S/C 1R43 CA86587-1 (S.G.S., S.C.), SPORE 2P50-CA70970-04, and CA97598 and CA89778 (S.C.)], by gifts to the Division of Surgery from Tenneco and Exxon for the Core Laboratory Facility, by a UT M. D. Anderson Cancer Center Support Core Grant (CA 16672), by donations from the Charles Rogers Memorial and support from the Homer Flower Research Fund, by a grant from the Tobacco Settlement Funds as appropriated by the Texas State Legislature (Project 8), by the W. M. Keck Foundation, and by a sponsored research agreement with Introgen Therapeutics, Inc. (SR93-004-1). We thank Debbie Smith for her assistance in the preparation of the manuscript. RECEIVED FOR PUBLICATION DECEMBER 2, 2004; ACCEPTED JANUARY 27, 2005.
REFERENCES 1. Jiang, H., Lin, J. J., Su, Z. A., Goldstein, N. I., and Fisher, P. B. (1995). Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 11: 2477 – 2486. 2. Fickenscher, H., Hor, S., Kupers, H., Knappe, A., Wittmann, S., and Sticht, H. (2002). The interleukin-10 family of cytokines. Trends Immunol. 23: 89 – 96. 3. Caudell, E. G., et al. (2002). The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24. J. Immunol. 168: 6041 – 6046. 4. Sarkar, D., et al. (2002). mda-7 (IL-24): signaling and functional roles. Biotechniques Suppl. 30 – 39. 5. Pataer, A., et al. (2002). Adenoviral transfer of the melanoma differentiationassociated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA dependent protein kinase (PKR). Cancer Res. 62: 2239 – 2243. 6. Mhashilkar, A. M., et al. (2003). MDA-7 negatively regulates the h-catenin and PI3K signaling pathways in breast and lung tumor cells. Mol. Ther. 8: 207 – 219. 7. Pataer, A., Chada, S., Hunt, K. K., Roth, J. A., and Swisher, S. G. (2003). Adenoviral melanoma differentiation-associated gene 7 induces apoptosis in lung cancer cells through mitochondrial permeability transition-independent cytochrome c release. J. Thoracic Cardiovasc. Surg. 125: 1328 – 1335. 8. Mhashilkar, A. M., et al. (2001). Melanoma differentiation associated gene (mda7): a novel anti-tumor gene for cancer gene therapy. Mol. Med. 7: 271 – 282. 9. Wang, M., Tan, Z., Zhang, R., Kotenko, S. V., and Liang, P. (2002). Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL20R1/IL-20R2. J. Biol. Chem. 277: 7341 – 7347. 10. Su, Z. Z., et al. (2003). Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in a p53-independent manner. Oncogene 22: 1164 – 1180. 11. Chada, S., et al. (2004). Bystander activity of Ad-mda7: MDA-7 protein kills melanoma cells via an IL-20 receptor-dependent but STAT3-independent mechanism. Mol. Ther. 10: 1085 – 1095. 12. Vorburger, S. A., et al. (2002). Role for the double-stranded RNA activated protein kinase PKR in E2F-1-induced apoptosis. Oncogene 21: 6278 – 6288. 13. Williams, B. R. (2001). Signal integration via PKR. Sci. STKE 89: RE2. 14. Barber, G. N. (2001). Host defense, viruses and apoptosis. Cell Death Differ. 8: 113 – 126.
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15. Jagus, R., Joshi, B., and Barber, G. N. (1999). PKR, apoptosis and cancer. Int. J. Biochem. Cell. Biol. 31: 123 – 138. 16. Taylor, D. R., Shi, S. T., Romano, P. R., Barber, G. N., and Lai, M. M. (1999). Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285: 107 – 110. 17. Yin, Z., Haynie, J., Williams, B. R., and Yang, Y. C. (2003). C114 is a novel IL-11inducible nuclear double-stranded RNA-binding protein that inhibits protein kinase R. J. Biol. Chem. 278: 22838 – 22845.
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18. Sieger, K. A., et al. (2004). The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells. Mol. Ther. 9: 355 – 367. 19. Bennett, R.L., Blalock, W.L., and May, W.S. (2004). Serine 18 phosphorylation of RAX, the PKR activator, is required for PKR activation and consequent translation inhibition. J. Biol. Chem. 279: 42687 – 42693. 20. Zhang, H.-G., Wang, J., Yang, X., Hsu, H.-C., and Mountz, J. D. (2004). Regulation of apoptosis proteins in cancer cells by ubiquitin. Oncogene 23: 2009 – 2015.
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Melanoma Differentiation-Associated Gene-7 Protein Physically Associates with the Double-Stranded RNA-Activated Protein Kinase PKR Abujiang Pataer,1,* Stephan A. Vorburger,2 Sunil Chada,3 Siddharth Balachandran,4 Glen N. Barber,4 Jack A. Roth,1 Kelly K. Hunt,2 and Stephen G. Swisher1,* 1
Department of Thoracic and Cardiovascular Surgery and 2Department of Surgical Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 445, Houston, TX 77030, USA 3 Introgen Therapeutics, Inc., Houston, TX 77030, USA 4 Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136, USA *To whom correspondence and reprint requests should be addressed. Fax: +1 713 794 4901. E-mail: [email protected] or [email protected].
Available online 3 March 2005
We previously reported that adenoviral-mediated overexpression of the melanoma differentiation-associated gene-7 (Ad-mda7; approved gene symbol IL24) leads to the rapid induction of PKR and activation of its downstream targets, resulting in apoptosis induction in human lung cancer cells. To evaluate the mechanism by which Ad-mda7 activates PKR, we studied the interaction between MDA-7 and PKR proteins. Following Ad-mda7 transduction of lung cancer cells, intracellular and extracellular MDA-7 protein was generated, leading to dose- and timedependent PKR induction. Purified MDA-7 protein administered extracellularly did not induce PKR or apoptosis, suggesting that Ad-mda7-mediated PKR activation and apoptosis were not dependent on extracellular MDA-7 protein. Following Ad-mda7 transduction, RT-PCR demonstrated no increase in PKR mRNA levels despite increased levels of PKR protein, suggesting posttranscriptional regulation of PKR by MDA-7. Immunofluorescence and coimmunoprecipitation studies demonstrated that MDA-7 protein physically interacts with PKR. Transduction of PKR+/+ and PKR / transformed MEFs with Ad-mda7 demonstrated phosphorylated MDA-7 and PKR proteins in the lysates of PKR+/+ but not PKR / cells. These findings identify the first binding partner for MDA-7 and suggest that direct interaction between PKR and MDA-7 may be important for PKR activation and apoptosis induction, possibly through MDA-7 phosphorylation or activation of other downstream targets. Key Words: MDA-7 (IL-24), PKR, apoptosis, gene therapy, adenovirus
INTRODUCTION Melanoma differentiation-associated gene-7 (mda-7; approved gene symbol IL24) was identified by a subtraction hybridization approach in human HO-1 melanoma cells treated with the combination of recombinant human fibroblast interferon (IFN)-h and the protein kinase C activator mezerein [1]. The mda-7 cDNA encodes an evolutionarily conserved protein of 206 amino acids with a predicted size of 23.8 kDa and exhibits greatest homology to the interleukin-10 (IL-10) subfamily [2,3]. Adenoviral-mediated overexpression of melanoma differentiation-associated gene-7 (Ad-mda7) induces apoptosis in a wide range of cancer cells through the activation of multiple signal transduction pathways [4–7]. We have reported that Ad-mda7-
MOLECULAR THERAPY Vol. 11, No. 5, May 2005 Copyright C The American Society of Gene Therapy 1525-0016/$30.00
mediated apoptosis in lung cancer cells occurs through activation of the dsRNA-dependent protein kinase (PKR) [5]. In this study, we further investigated the relationship between MDA-7 expression and PKR activation. Immunofluorescence and coimmunoprecipitation studies demonstrated that MDA-7 and PKR proteins interact directly, leading to phosphorylation of MDA-7 and PKR. Phosphorylation of MDA-7 was observed on both threonine and serine residues and was dependent on the presence of PKR as demonstrated by transduction of PKR+/+ and PKR / transformed MEFs with Ad-mda7. These findings identify PKR as the first binding partner for MDA-7 and suggest that the direct interaction between PKR and MDA-7 may be important for PKR activation and the induction of
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apoptosis, possibly through MDA-7 phosphorylation or activation of other downstream targets.
RESULTS Ad-mda7 Induces Apoptosis and PKR on Human Lung Cancer Cells We performed flow cytometric analysis on the A549 (wt p53) and H1299 (null p53) human lung cancer cells at 48 h following infection with Ad-mda7, Ad-Luc, or PBS (Fig. 1A). Ad-mda7 treatment resulted in a dosedependent induction of PKR in A549 cells (Fig. 1B) and H1299 cells (data not shown). PKR levels continued to increase with Ad-mda7 dose. Ad-mda7 (2500 vp/cell) treatment of lung cancer cells also showed a temporal induction of PKR, with robust induction at 24 h posttreatment and maximal levels noted at 48 h (Fig. 1C). We performed real-time PCR to determine if Admda7-mediated induction of PKR protein (Figs. 1B and 1C) occurred through a transcriptional or a posttranscriptional mechanism. As opposed to Ad-E2F-1, Admda7 did not increase PKR mRNA levels, suggesting a posttranscriptional mechanism (Fig. 1D). Intracellular MDA-7 Protein Plays a Role in Ad-mda7-Induced Cell Death We evaluated the anti-tumor effects of secreted MDA-7 on A549 (wt p53) and H1299 (null p53) human lung cancer cells to determine if extracellular MDA-7 could induce apoptosis and PKR induction. Figs. 2A and 2B demonstrate that MDA-7 secreted protein is not able to inhibit cell growth (Fig. 2A) or cause cell death (2B) in lung cancer cells. MDA-7 secreted protein also failed to induce PKR or activate STAT3 in A549 cells (Fig. 2C). In contrast, Ad-mda7-treated A549 cells show induction of PKR and activated STAT3, consistent with our previous observations that intracellular MDA-7 is critical for Admda7-mediated apoptosis in lung cancer cells [7]. MDA-7 Protein Physically Interacts with the dsRNA-Dependent Protein Kinase PKR We next assessed subcellular localization of PKR and MDA-7. We saw PKR expression (green) in cytoplasmic and nuclear compartments, while MDA-7 expression (red) was predominantly in the cytosol. Using confocal microscopy, we noted cytoplasmic colocalization of MDA-7 and PKR (Fig. 3A). We then looked for a direct interaction between MDA-7 and PKR. As shown in Fig. 3B, anti-PKR antibodies did not immunoprecipitate MDA-7 protein in the PBS- or Ad-Luc-treated cells; however, in Ad-mda7-transduced cells, MDA-7 was detected after immunoprecipitation with antibodies against PKR (Fig. 3B). The negative control (IgG) did not immunoprecipitate MDA-7 protein in the Ad-mda7treated cells (Fig. 3B). To confirm the protein–protein interaction, we performed the reciprocal experiment
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FIG. 1. Adenoviral overexpression of MDA-7 in lung cancer cells results in apoptosis and posttranscriptional PKR induction. (A) Percentage of apoptotic cell death as determined by PI and flow cytometry in A549 (wild-type p53) and H1299 (null p53) cells 48 h following treatment with PBS, Ad-Luc (2500 vp), or Ad-mda7 (2500 vp). Triplicate experiments were performed for each cell line. (B) Western blot analysis of the dose-dependent expression of PKR after Ad-mda7 transduction. (C) Western blot analysis of the timedependent expression of PKR after Ad-mda7 (2500 vp) transduction. (D) Realtime PCR analysis of PKR mRNA in Ad-mda7-, Ad-luc-, Ad-E2F-1-, or PBStreated cells.
with PBS-, Ad-Luc-, and Ad-mda7-treated cell lysates immunoprecipitated with antibody against MDA-7. We detected PKR only in Ad-mda7-treated cell lysates (Fig. 3B). These immunoprecipitation studies suggest that MDA-7 binds to the dsRNA-dependent protein kinase PKR.
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of MDA-7 and PKR (Fig. 4C). Tyrosine phosphorylation of MDA-7 and PKR was not demonstrated (data not shown). These studies demonstrate serine and threonine phosphorylation of MDA-7 and PKR following Ad-mda7 transduction and suggest that MDA-7 phosphorylation may be dependent on PKR or another downstream serine/ threonine kinase.
DISCUSSION
FIG. 2. Intracellular MDA-7 protein plays an important role in Ad-mda7induced cell death and PKR induction. (A) Cell number following PBS, Admda7, or secreted MDA-7 protein treatment. (B) Apoptosis induction following treatment with PBS, Ad-mda7, and secreted MDA-7 protein. (C) Western blot analysis of the expression of PKR, STAT3, and phosphoSTAT3 in A549 cell lysates 48 h after treatment with PBS, Ad-mda7 (2500 vp), or secreted MDA-7. Results of one representative experiment of three are shown.
PKR and MDA-7 Proteins Are Phosphorylated Following Ad-mda7 Transduction Following Ad-mda7 transduction, we demonstrated phosphorylated forms of both PKR and MDA-7 in both human lung cancer cells (Fig. 4A). We demonstrated phosphorylation of PKR and MDA-7 on threonine and serine residues but not tyrosine residues. We performed Ad-mda7 transduction of PKR+/+ and PKR / cells to determine whether phosphorylation was dependent on PKR. Only PKR+/+ cells demonstrated apoptosis and phosphorylation of PKR and MDA-7 (Figs. 4B and 4C). We detected both serine and threonine phosphorylation
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mda-7 is a tumor suppressor gene first identified by subtraction hybridization of a melanoma cell line induced to terminally differentiate with IFN-h and mezerein [1]. Ad-mda7 appears to mediate anti-tumor activity through the activation of multiple signal transduction pathways leading to growth inhibition and apoptosis induction [4–8]. We have previously demonstrated that Ad-mda7-mediated apoptosis of lung cancer cells appears to be dependent on PKR induction although the mechanism of PKR activation has not been defined [5]. In the present investigation we have further explored the intracellular relationship between MDA-7 and PKR proteins. Following Ad-mda7 transduction of lung cancer cells, MDA-7 protein exists both intracellularly and in a secreted extracellular form [6]. The secreted extracellular form of MDA-7/IL-24 can bind to two different receptors, the type 1 IL-20 heterodimeric receptor complex comprising IL-20R1/IL-20R2 and the type 2 complex comprising IL-22R1/IL-20R2, which lead to activation of the STAT signaling pathways [9]. Fisher’s group has recently shown that treatment of malignant gliomas with bacterially expressed GST–MDA-7 protein can reproduce the cancer cell specificity seen with Ad-mda7. This required supraphysiologic levels of protein, and receptor–ligand interactions were not evaluated [10]. We evaluated whether extracellular recombinant human MDA-7/IL-24 protein could induce PKR activation and cell death in human lung cancer cells. Our results suggest that, in lung cancer cells, Ad-mda7-induced apoptosis and PKR induction are dependent on intracellular MDA-7 protein production since extracellular MDA-7 was unable to induce apoptosis, PKR induction, or STAT3 activation (Figs. 2A, 2B, and 2C). This observation is consistent with our report that A549 and H1299 lung cancer cells lack MDA-7 receptors [11] and provides an explanation as to why secreted MDA-7 fails to activate STAT3 or PKR when delivered extracellularly to these cells. We analyzed PKR mRNA levels by real-time PCR to determine whether Ad-mda7-induced PKR activation was due to mRNA upregulation. We found that PKR mRNA accumulation was not increased following Admda7 transduction and was not responsible for the elevated levels of PKR protein (Fig. 1D). The increase in PKR protein following Ad-mda7 transduction therefore appears to be due to posttranscriptional mecha-
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FIG. 3. MDA-7 colocalizes and interacts directly with PKR. (A) Immunofluorescence microscopy with antibodies against MDA-7 stained red and PKR stained green demonstrated upregulation of both MDA-7 and PKR proteins in Ad-mda7treated cells, but not in control-treated cells after 48 h. (B) Protein fractions, obtained from A549 and H1299 cells treated with PBS, Ad-Luc (2500 vp), or Ad-mda7 (2500 vp) for 48 h, were immunoprecipitated with anti-human PKR (left) or anti-human MDA-7 (right) and assayed for MDA-7 or PKR proteins.
nisms rather than mRNA induction. This mechanism of PKR induction is in contrast to that of Ad-E2F1, in which E2F-1 increases PKR expression at the mRNA level [12]. PKR is known to bind to several proteins, including eIF-2a, HCV E2, E3L, NFAR-1 and -2, and PACT/RAX [13– 17]. Our study identifies MDA-7 as a novel binding partner for PKR. The cellular activator PACT is known to induce PKR activation through direct protein–protein interaction with the dsRNA binding regions of PKR. We, therefore, examined whether MDA-7 might also interact and induce PKR through a similar interaction. Our immunofluorescence studies show that MDA-7 colocalizes with PKR protein in Ad-mda7-transduced lung cancer cells and immunoprecipitation demonstrates that MDA-7 and PKR proteins interact directly (Fig. 3B). We previously demonstrated that MDA-7 regulation of the hcatenin and PI3K signaling pathways does not require direct binding of MDA-7 to the bdestruction complexQ of GSK3-h/h-catenin/axin/APC in lung and breast cancer cells [18]. The current study, however, demonstrates the first evidence of a specific binding partner for MDA-
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7. Whether MDA-7 binds to the dsRNA binding region on PKR, like PACT, or another area remains to be determined. Human PKR contains at least 15 autophosphorylation sites that are involved in PKR activation [13–15]. Once activated, PKR becomes autophosphorylated and acquires the capacity to phosphorylate its downstream targets, including eIF-2a, leading to cessation of protein synthesis and cell death [13–15]. We found that, following Admda7 transduction, both PKR and MDA-7 proteins are phosphorylated at threonine and serine residues in lung cancer cells and PKR+/+ cells, but not in PKR / cells. These findings support the concept that MDA-7 interacts directly with PKR and that MDA-7 binding result in PKR activation and autophosphorylation. MDA-7 phosphorylation may then occur due to PKR kinase activity or through phosphorylation by another kinase. PKR is activated by various stimuli, including dsRNA, heparin, and the dsRNA-binding protein RAX/PACT, which is induced following cellular stress. Interestingly, activation of PKR by RAX/PACT appears to be dependent on the phosphorylation of RAX/PACT that occurs after binding
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FIG. 4. MDA-7 and PKR protein phosphorylated following Ad-mda7 transduction. (A) Protein fractions obtained from A549 cells treated with Ad-Luc (2500 vp) or Ad-mda7 (2500 vp) for 48 h were immunoprecipitated with anti-phospho-threonine or anti-phospho-serine and assayed for MDA-7 and PKR proteins. Results of one representative experiment of three are shown. (B) Western blot analysis of the expression of PKR, MDA-7, and actin in PKR+/+ and PKR / cell lysates 48 h after treatment with Ad-mda7 (2500 vp) demonstrating similar levels of transduced MDA-7. (C) Protein fractions, obtained from PKR+/+ and PKR / cells treated with PBS, AdLuc (2500 vp), or Ad-mda7 (2500 vp) for 48 h were immunoprecipitated with anti-phosphothreonine or anti-phospho-serine and assayed for MDA-7 and PKR proteins. Percentage of cell death in PKR+/+ and PKR / cells following treatment with PBS, Ad-luc (2500 vp), and Ad-mda7 (2500 vp) is indicated at the bottom. The cells were analyzed by flow cytometry 48 h after transduction. Results of one representative experiment of three are shown.
to PKR [19]. A nonphosphorylated form of RAX is still able to bind to PKR but is unable to activate PKR following stress, presumably because it cannot be phosphorylated. MDA-7 may follow a similar pattern of PKR activation by initially binding to PKR in a nonphosphorylated state that is unable to activate PKR until it is phosphorylated by another cellular kinase. Alternatively, the direct binding of MDA-7 to PKR may induce a conformational change in PKR leading to PKR activation, autophosphorylation, and MDA-7 phosphorylation by PKR itself. Further studies will be performed to determine the sequence of events and the importance of MDA-7 phosphorylation in PKR activation. The current study cannot separate the kinetics of MDA-7 and PKR phosphorylation; however, induction of apoptosis occurs only when both proteins are phosphorylated on serine and threonine. The importance of MDA-7 phosphorylation in the anti-tumor effects of Ad-mda7 remains in question. The phosphorylation of proteins often leads to diverse functions, including kinase activation, protein stabilization, growth stimulation, proteasome regulation, and apopto-
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sis induction [20]. The MDA-7 sequence has six potential phosphorylation sites (four serines, 88, 101, 161, and 164, and two threonines, 111 and 133) [20]. The phosphorylated MDA-7 sites appear to be serine and threonine: it is not known which serine or threonine sites are phosphorylated (Figs. 4A and 4C). Additionally, phosphorylation on tyrosine residues has not been detected, suggesting the kinase responsible for MDA-7 phosphorylation is a Ser/Thr kinase. PKR is a Ser/Thr kinase, allowing it to be a candidate kinase for MDA-7 phosphorylation. Further support for PKR being the kinase responsible for MDA-7 activity is given by our previous studies using inhibitors of PKR, MEKK, PI3K, and MAPK: only 2-aminopurine, which blocks PKR function, was able to abrogate tumor cell killing by Ad-mda7 [5,6]. The functional consequences of MDA-7 phosphorylation are not currently known but may include control of apoptosis induction. We observed MDA-7 phosphorylation in lung cancer cells and PKR+/+ cells (Figs. 4A and 4C) induced to undergo apoptosis by Ad-mda7, but not in PKR / cells, which are resistant to Ad-mda7 apoptosis. After Ad-mda7 treatment, normal human cells also
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express high levels of MDA-7, but do not undergo apoptosis. Further studies will determine whether the resistance to killing in normal cells correlates with MDA7 and PKR phosphorylation status. In conclusion, we have extended our initial observation that Ad-mda7-induced apoptosis is dependent on PKR to assess the mechanisms of PKR and MDA-7 interaction. We demonstrate that following Ad-mda7 transduction, MDA-7 binds PKR, documenting the first known binding partner for MDA-7 and defining a novel binding partner for PKR. Additionally, we show that both MDA-7 and PKR are phosphorylated following Ad-mda7 transduction of lung cancer cells. The possibility exists that MDA-7 phosphorylation is due to PKR kinase activity or a downstream PKR target since only PKR+/+ cells demonstrate phosphorylation of MDA-7. The function and role of MDA-7 phosphorylation in apoptosis induction and PKR activation remain to be elucidated but may in the future provide an additional therapeutic target to enhance further Ad-mda7 antitumoral activity.
MATERIALS
AND
METHODS
Cell lines and reagents. A549 and H1299 human lung cancer cell lines were obtained from the American Type Culture Collection. PKR+/+ and PKR / isogenic mouse embryonic fibroblasts were obtained from Dr. Glen N. Barber (University of Miami School of Medicine). Recombinant MDA-7 protein was obtained from Introgen Therapeutics (Houston, TX, USA). Adenovirus production. Construction of the Ad-mda7, Ad-bak, Ad-lacZ, and Ad-Luc vectors has been previously reported [5]. Adenoviral vectors were used at titers needed to transduce at least 70% of the cells. Flow cytometry analysis. We measured apoptotic cells by propidium iodide staining and FACS analysis (Becton–Dickinson FACScan, Mountain View, CA, USA; FL-3 channel) as previously described [5]. Real-time PCR. RT-PCR was carried out using the Thermoscript RT-PCR system kit according to the instructions provided by the manufacturer (Life Technologies, Gaithersburg, MD, USA). Total RNA was isolated according to the protocol for TRIzol extraction (Life Technologies), and real-time PCR was performed using the protocol reported previously [12]. To quantify the PKR mRNA expression, we used TaqMan PCR probes (Perkin–Elmer Applied Biosystems, Foster, CA, USA) and the 7700 sequence detector (Perkin–Elmer Applied Biosystems). Reactions contained 1V TaqMan Universal PCR Master Mix (Perkin–Elmer Applied Biosystems), 900 nM forward primers (5V-CCTGTCCTCTGGTTCTTTTGCT-3V) and reverse primers (5V-GATGATTCAGAAGCGAGTGTGC-3V), and 200 nM TaqMan probes (5V-ACGTGTGAGTCCCAAAGCAACTCTTTAGTGAC-3V). GAPDH primers and probes were added at 50 nM. Thermal cycling proceeded with 50 cycles of 958C for 15 s and 608C for 1 min. Input RNA amounts were calculated with relative standard curves for both the mRNAs of interest and GAPDH. Western blot analysis. Forty-eight hours after transfection, cell extracts were prepared and immunoblot assays performed as previously described [5]. Anti-PKR (K-17), anti-h-actin, anti-STAT3 (F-2), and phospho-specific anti-STAT3 (B-7) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the polyclonal or monoclonal antibody to MDA-7 was obtained from Introgen Therapeutics. Coimmunoprecipitation analysis. Cells were treated with Ad-mda7 or Ad-Luc for 48 h and then lysed in RIPA buffer (1 PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). The primary antibody was
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incubated overnight at 48C. Protein A/G agarose was added to the mix and incubated for 4 h. Centrifugation and washing of pellets were performed and the samples were assayed by Western blot analysis. Cellular localization studies. A549 and H1299 cells (5 104 cells/well) were grown on chamber slides until 70% confluence and then transfected with Ad-luc, Ad-mda7, or PBS. Forty-eight hours later, cells were washed with PBS and fixed with 4% paraformaldehyde/PBS for confocal analysis as previously described [12]. Rabbit polyclonal anti-PKR (K-17) and mouse monoclonal anti-MDA-7 were utilized and assessed by immunofluorescence microscopy. Statistical analysis. The data reported represent the means of three or more independent experiments and the bars show the standard deviation. ANOVA and two-tailed Student’s t test were used for statistical analysis of multiple groups and pair-wise comparison, respectively, with P b 0.05 considered significant.
ACKNOWLEDGMENTS This work was supported by grants from the National Cancer Institute and the National Institutes of Health [P01 CA78778-01A1 (J.A.R., S.G.S.), SBIR S/C 1R43 CA86587-1 (S.G.S., S.C.), SPORE 2P50-CA70970-04, and CA97598 and CA89778 (S.C.)], by gifts to the Division of Surgery from Tenneco and Exxon for the Core Laboratory Facility, by a UT M. D. Anderson Cancer Center Support Core Grant (CA 16672), by donations from the Charles Rogers Memorial and support from the Homer Flower Research Fund, by a grant from the Tobacco Settlement Funds as appropriated by the Texas State Legislature (Project 8), by the W. M. Keck Foundation, and by a sponsored research agreement with Introgen Therapeutics, Inc. (SR93-004-1). We thank Debbie Smith for her assistance in the preparation of the manuscript. RECEIVED FOR PUBLICATION DECEMBER 2, 2004; ACCEPTED JANUARY 27, 2005.
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