Correspondence to J.-L. Guan: jg19{at}cornell.edu
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Introduction |
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FIP200 is also identified by Chano et al. (2002a) independently as a potential regulator of the RB1 gene (designated by this group as RB1CC1 for RB1-inducible coiled-coil 1, but will be referred to here as FIP200 for convenience). The expression levels of FIP200 correlated with those of RB1 in various cancer cell lines and normal human tissues. In addition, FIP200 and RB1 are preferentially coexpressed and contributed to the maturation of human embryonic musculoskeletal cells and may regulate the proliferative activity and maturation of tumor cells derived from these tissues (Chano et al., 2002d). Lastly, it was found that 20% of primary breast cancers that were screened contained large deletion mutations in FIP200 that are predicted to generate markedly truncated proteins (Chano et al., 2002c). These studies are consistent with our findings showing negative regulation of cell cycle progression by FIP200 (Abbi et al., 2002).
TSC1 and TSC2 (or hamartin and tuberin, respectively) are both tumor suppressor genes and mutation in either gene causes tuberous sclerosis (TSC) that occurs in 1 in 6,000 of the population and is defined by the formation of hamartomas in a wide range of tissues. Both TSC1 and TSC2 have coiled-coil regions and they exist as heterodimers (Plank et al., 1998; van Slegtenhorst et al., 1998; Kwiatkowski, 2003). Although TSC1 has no known enzymatic activity, TSC2 contains a COOH-terminal GAP domain for the small G protein Rheb (Garami et al., 2003; Inoki et al., 2003a; Saucedo et al., 2003; Stocker et al., 2003; Tee et al., 2003; Y. Zhang et al., 2003). Recent studies have indicated that the TSC1TSC2 complex regulates cellular functions mainly by their inhibition of mTOR and its targets S6 kinase (S6K) and 4E-BP1 (Potter et al., 2001; Tapon et al., 2001; Gao et al., 2002; Goncharova et al., 2002; Inoki et al., 2002; Manning et al., 2002; Tee et al., 2003). Increased S6K activity is observed in TSC mutations in D. melanogaster, cells derived from TSC1 or TSC2 knockout mice, or cells treated with TSC1 or TSC2 small interfering RNA. Consistent with its function as a negative regulator of mTOR and its targets, the TSC complex has been found to regulate various cellular functions such as cell cycle progression, cell size control, cell survival, and apoptosis (Hengstschlager et al., 2001; Inoki et al., 2003b; Shamji et al., 2003).
To investigate the molecular mechanisms by which FIP200 regulates intracellular signaling pathways and cellular functions, we used yeast two-hybrid screening to identify other proteins that interact with FIP200. Here, we report identification of FIP200 interaction with the TSC1TSC2 complex and show that this interaction leads to inhibition of TSC1TSC2 complex function resulting in increased S6K activity and cell growth. These studies suggest a novel function for FIP200 in the regulation of cell size control in addition to its functioning as an inhibitor for FAK and regulator of RB1 expression.
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Results |
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Association of FIP200 with the TSC1TSC2 complex
TSC1 and TSC2 have been shown to function as a complex in vivo (Plank et al., 1998; van Slegtenhorst et al., 1998; Kwiatkowski, 2003). Therefore, we tested whether FIP200 could associate with the TSC1TSC2 complex in cells. pKH3-FIP200 was cotransfected into 293T cells with plasmids encoding Myc-TSC1 and GST-TSC2. Lysates were prepared from the transfected cells and immunoprecipitated by anti-HA, and the associated proteins were analyzed by Western blotting by respective antibodies. Fig. 1 D shows that both TSC1 and TSC2 were coprecipitated with FIP200, but not in pKH3 control vectortransfected cells. Consistent with these transfection studies, we could also detect the interaction of endogenous FIP200 with the endogenous TSC1TSC2 complex in 293T cells (Fig. 1 E). Both TSC1 and TSC2 were found in anti-FIP200 immunoprecipitates, but not in the control immunoprecipitates with an irrelevant antibody anti-HA. Likewise, FIP200 is also detected in both anti-TSC1 and -TSC2 immunoprecipitates, but not control immunoprecipitates. Lastly, coimmunoprecipitation of endogenous FIP200 with TSC1, but not vinculin, was also observed in mouse embryonic fibroblasts (MEFs; Fig. 1 F). Together, these results demonstrate that FIP200 could associate with the TSC1TSC2 complex in mammalian cells such as 293T cells and MEFs.
FIP200 interaction with the TSC1TSC2 complex is not involved in FIP200 regulation of cell cycle progression
Our previous studies showed that FIP200 plays an important role in the regulation of cell cycle progression (Abbi et al., 2002). Some studies also showed that the TSC1TSC2 complex is capable of regulating cell proliferation (Soucek et al., 1998, 2001; Miloloza et al., 2000; Hengstschlager and Rosner, 2003). Thus, the identification of the interaction between FIP200 and the TSC1TSC2 complex raises the possibility that this interaction may play a role in the regulation of cell cycle progression by FIP200. Indeed, the FIP200 N1-859 fragment, which can interact with the TSC1TSC2 complex (Fig. 2 A), showed similar activity as the full-length FIP200 in the inhibition of cell cycle progression as measured by BrdU incorporation (not depicted). However, the smaller segment of FIP200 (N1-638; Fig. 1 A), which did not associate with the TSC1TSC2 complex (Fig. 2 A), could also inhibit cell cycle progression (Abbi et al., 2002). These results suggest that FIP200 interaction with the TSC1TSC2 complex might not be required for FIP200 regulation of cell cycle progression.
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FIP200 function in cell size control
Recent studies have identified the TSC1TSC2 complex as a key regulator of cell size control (Inoki et al., 2003b; Shamji et al., 2003). Therefore our identification of the interaction between FIP200 and the TSC1TSC2 complex raises the interesting possibility that FIP200 may regulate cell size through interaction with the TSC1TSC2 complex. To investigate this possibility directly, 293T cells were transiently transfected with an expression vector encoding FIP200 or an empty vector control and the effects on cell size were then measured by determining the mean forward scatter (mean FSC-H) with a flow cytometer. Fig. 3 A shows that overexpression of FIP200 in these cells led to a right-shift of the mean FSC-H distribution compared with the empty vectortransfected cells, which corresponds to an increase in the size of the FIP200-transfected populations of cells. Quantification of the data indicated an 5% increase in the average cell size in the FIP200-transfected 293T cells (Fig. 3 B). This is likely to be an underestimation of the FIP200 effect on cell size as the transfection efficiency is <100% for these cells. Similar studies indicated that the N1-859 segment of FIP200, but not the N1-638 segment, which does not bind to TSC1TSC2, also increased the average cell size of the transfected 293T cells (Fig. 3, A and B). Furthermore, the increase in cell size was observed in 293T cells in either G1 or G2/M phase (Fig. 3 C). Together, these results suggested a novel function for FIP200 in the regulation of cell size, possibly through its interaction with the TSC1TSC2 complex.
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Similar studies were performed to test a potential role of FAK in FIP200 regulation of S6K phosphorylation because previous studies showed that FIP200 could inhibit FAK, which may play a role in the activation of S6K (Malik and Parsons, 1996). Interestingly, we found that down-regulation of FAK by the RNAi vector pBS-U6-FAK #1 did not affect the reduction of S6K phosphorylation induced by pBS-U6-FIP200 #7, although it clearly down-regulated expression of FAK in these cells (Fig. 7 A). The specificity of the vectors was also demonstrated by analysis of parallel samples with anti-FIP200 and anti-vinculin. Consistent with the RNAi results, we also found that overexpression of FIP200 increased S6K phosphorylation in both FAK+/+ and FAK/ cells (Fig. 7 B). Together, these results suggest that FIP200 regulation of S6K phosphorylation is through its inhibition of the TSC1TSC2 complex but not FAK.
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Discussion |
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Most other proteins known to play roles in both cell proliferation and cell size usually regulate these two cellular processes in a similar manner. For example, PTEN can inhibit cell proliferation and negatively regulate cell size (Groszer et al., 2001), whereas Myc can promote cell proliferation and increase cell size (Iritani and Eisenman, 1999). In contrast, our studies indicated that FIP200 regulates cell proliferation and cell size in a differential manner, which inhibits cell proliferation but increases cell size. Interestingly, recent studies also suggested the critical cell cycle regulator pRB also plays a role in cell size control, but in a differential manner as its role in cell cycle regulation. It was shown that Rb triple knockout MEFs (triple knockout MEFs lacking all three Rb family proteins pRb, p107, and p130) have increased cell proliferation, but a significantly reduced cell size compared with control MEFs (Sage et al., 2000). It is interesting to note that FIP200 has been reported to up-regulate RB1 expression in recent studies by Chano and co-workers (Chano et al., 2002a; Kontani et al., 2003), although it remains to be determined whether the pRB pathway is involved in the regulation of cell size by FIP200. Nevertheless, data presented in this study strongly suggest that FIP200 regulates cell size through its interaction with the TSC1TSC2 complex.
Our previous study showed that FIP200 plays an important role in cell proliferation; however, little is known about the targets and molecular mechanisms by which FIP200 regulates cell proliferation. Several studies also suggest the important role of the TSC1TSC2 complex in cell cycle control. Overexpression of TSC1 or TSC2 can inhibit cell cycle progression (Soucek et al., 1998, 2001; Miloloza et al., 2000; Hengstschlager and Rosner, 2003). TSC2 has been shown to interact with several cell cycle regulators such as cdk1, cyclin A, and cyclin B (Catania et al., 2001). Furthermore, TSC2 was shown to stabilize p27 and negatively regulate cdk2 function (Soucek et al., 1998). However, recent studies using TSC1 and TSC2 null MEFs showed decreased proliferation (instead of increased proliferation as would be predicted from the overexpression studies) compared with control MEFs (Y. Zhang et al., 2003). Furthermore, both TSC1 and TSC2 have been shown to be required for serum stimulation of Akt activation, which plays critical roles in cell proliferation and cell survival (Kwiatkowski et al., 2002; Y. Zhang et al., 2003). Therefore, the function of TSC in the regulation of cell proliferation is complicated. We initially hypothesized that the interaction between FIP200 and the TSC1TSC2 complex would play a role in FIP200-mediated cell cycle progression. However, we found that FIP200 can inhibit cell cycle progression in TSC1/ MEFs as well as control MEFs. In addition, the FIP200 N1-638 segment does not interact with the TSC1TSC2 complex but still inhibits cell cycle progression. Together, these data suggest that interaction between FIP200 and the TSC1TSC2 complex is not involved in FIP200-mediated cell cycle progression.
Our results demonstrated that FIP200 functions to positively regulate cell size and S6K phosphorylation. Although we cannot exclude completely the possible role of other as yet unidentified proteins that interact with FIP200, current evidence suggests that these effects are through FIP200 interaction with the TSC1TSC2 complex. We found that association of FIP200 or its segments N1-859 and N1-638 with the TSC1TSC2 complex correlated with their ability to increase cell size, up-regulate S6K phosphorylation, and decrease TSC1TSC complex formation. Conversely, knockdown of endogenous FIP200 by RNAi reduced S6K phosphorylation and cell size. We also observed that FIP200 RNAi has no effect on S6K phosphorylation in TSC1 knockdown cells but can decrease S6K phosphorylation in control cells. Consistent with this, FIP200 failed to stimulate S6K phosphorylation and increase cell size in TSC1-null MEFs in contrast to control MEFs and that the stimulatory effect in control MEFs was abolished by rapamycin. These results suggested that the TSC1TSC2 complex and its downstream target mTOR are required for FIP200 regulation of S6K phosphorylation. Using both RNAi approach and FAK/ cells, we showed that FIP200 inhibition of FAK is not involved in the regulation of S6K phosphorylation by FIP200.
Recent studies have established the TSC1TSC2 complex as a key regulator of cell size control, and rapid progress has been made in delineating the downstream biochemical pathways by which TSC regulates cell size as well as their roles in the regulation of other cellular functions (Hengstschlager et al., 2001; Inoki et al., 2003b; Kwiatkowski, 2003; Shamji et al., 2003). In contrast, relatively little is known about the molecular mechanisms by which TSC is regulated by upstream regulators. Several proteins have been shown to interact with TSC2 and thus regulate the TSC1TSC2 complex function. Protein kinases AKT and AMPK have been identified to phosphorylate TSC2 and negatively and positively regulate TSC1TSC2 complex function, respectively (Inoki et al., 2002, 2003b). 14-3-3 was shown to interact with and negatively regulate TSC2 without affecting TSC1TSC2 interaction (Li et al., 2002; Shumway et al., 2003). ERM family proteins and neurofilament-light chain were shown to interact with TSC1, but it is not clear whether these interactions regulate TSC1TSC2 complex function (Lamb et al., 2000; Haddad et al., 2002). Here, we identified a novel interaction between FIP200 and TSC1 and provided evidence suggesting that this interaction could negatively regulate TSC1TSC2 complex function to increase S6K phosphorylation and cell size. This is the first paper suggesting a regulatory mechanism through protein interaction with TSC1 of the complex.
Our results suggested that FIP200 is important for nutrient stimulation-induced, but not energy- or serum-induced, S6K activation (Fig. 9). Nutrient-induced S6K phosphorylation was lower in FIP200/ MEFs compared with FIP200+/+ MEFs as well as in 293T cells treated with FIP200 RNAi compared with cells treated with control RNAi (Fig. 9, C and F). However, we noted that the decrease was moderate in the FIP200/ MEFs, whereas it was greater in the short-term RNAi knockdown experiments. This could be due to differences in the cells used in these two different sets of experiments. Alternatively, FIP200/ MEFs may have adapted to the FIP200 null condition during the culture (thus becoming less dependent on FIP200 for nutrient-induced S6K phosphorylation). However, we tested the expression levels of TSC1 and TSC2 in these cells and found that their expression levels are similar to those in FIP200+/+ MEFs, suggesting that there is no compensatory changes in either TSC1 or TSC2 expression in the FIP200/MEFs. Future studies will be necessary to determine whether expression of other genes is altered, which could partially compensate for the loss of FIP200 in nutrient-stimulated S6K phosphorylation. Nevertheless, these results suggest that FIP200 functions in the nutrient input to the TSC1TSC2 complex rather than being a general component of the TSC1TSC2 signaling pathway.
The molecular mechanisms by which FIP200 interaction with TSC1 inhibits TSC1TSC2 functions are not clear at present. We found that overexpression of FIP200 reduced TSC1TSC2 complex formation (Fig. 8, A and B), raising the possibility that FIP200 could inhibit TSC1TSC2 function by disrupting the complex formation. However, several considerations would argue against such a possible mechanism. First, knockdown of endogenous FIP200 by RNAi did not significantly promote endogenous TSC1TSC2 complex formation (Fig. 8 C). Second, although our binding studies suggested that TSC1 coiled-coil region mediates its interaction with TSC2 (unpublished data), the relevant functional segment of FIP200 (N1-859) does not contain its COOH-terminal coiled-coil region. Furthermore, the FIP200 binding region in TSC1 resides in residues 403787, which overlaps little with the putative coiled-coil region of TSC1 (residues 719998; also see Fig. 1 A). Thus, it is unlikely that FIP200 could disrupt TSC1TSC2 interaction simply by its coiled-coil domain binding to the coiled-coil region of TSC1 and displace TSC2 from its association with TSC1. Lastly, we observed that FIP200 could coimmunoprecipitate both endogenous (Fig. 1 E) and transfected (Fig. 2 A) TSC2, which would not be possible if FIP200 functioned to disrupt the complex by competing with TSC2 for TSC1 binding. Although we cannot completely exclude the possibility that FIP200 could function to reduce the TSC1TSC2 complex formation under some conditions, the aforementioned consideration would suggest that FIP200 may inhibit TSC1TSC2 complex function through mechanisms other than disruption of the complex formation. It is possible that FIP200 binding to TSC1 may also inhibit TSC1TSC2 function by inducing conformational change in TSC2 (indirectly through TSC1) to inhibit its GAP activity toward Rheb. Future studies will be necessary to investigate such a possible mechanism.
In summary, our studies identified FIP200 as a novel interacting protein with TSC1 and suggested that FIP200 could negatively regulate TSC function. These results indicate that, in addition to its function in cell cycle progression, FIP200 also plays a role in cell size control. Furthermore, they provide new insight into the molecular mechanism of TSC regulation. These studies raise interesting implications regarding the molecular mechanisms by which cell proliferation and cell size are coordinately regulated and, potentially, how dysregulation of cell cycle progression and cell size control lead to diseases such as cancer.
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Materials and methods |
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Yeast two-hybrid assays
The vector pLexA-FIP200 N1-859 was used to screen a human heart MATCHMAKER LexA cDNA library (>106 independent clones; gift from C. Wu, University of Pittsburgh, Pittsburgh, PA). The yeast two-hybrid screen was performed essentially as described previously (Tu et al., 1999).
Cell culture
The FIP200/ and control MEFs were isolated from E12.5 embryos. The early passage (P1P3) MEFs were used for studies here. The TSC1/ and control MEFs were a gift of D. Kwiatkowski (Brigham and Women's Hospital, Boston, MA) and were maintained in DME supplemented with 10% FBS as described previously (Kwiatkowski et al., 2002). The FAK/ fibroblasts derived from the FAK-null mouse embryos were a gift of D. Ilic (University of California, San Francisco, San Francisco, CA) and were maintained in DME supplemented with 10% FBS. 293T cells were cultured in DME supplemented with 10% FBS. NIH3T3 cells were maintained in DME supplemented with 10% CS.
Plasmid DNA construction
The vectors used in the yeast two-hybrid screen, pLexA, pLexA-Lamin C, and pB42AD, have been described previously (Tu et al., 1999). pKH3-FIP200 was used as a template to generate pLexA-FIP200 N1-859: the PCR fragment amplified by primers 5'-gaattcatgaagttatatgtatttctgg-3' (forward) and 5'-ctcgagtagtgttatttccagagaaca-3' (reverse) was first inserted into pGEMT (BD Biosciences), then digested with XhoI and EcoRI, and the fragment was then inserted into pLexA (BD Biosciences) digested with the same enzymes to generate pLexA-FIP200 N1-859.
The mammalian cell expression vectors pKH3, pKH3-FIP200, and pKH3- FIP200 N1-638 have been described previously (Abbi et al., 2002). pKH3-FIP200 was used as a template to produce pKH3-FIP200 N1-859: the PCR product with primers 5'-cgcggatccatgaagttatatgtatttctgg-3' (forward) and 5'-cccatcgattcatagtcttatttccagagaacattt-3' (reverse) was digested with BamHI and ClaI. The fragment was then ligated to a linearized pKH3 vector digested with the same enzymes. The same PCR product was digested with BamHI and ClaI and then inserted into linearized pMAL at the corresponding sites to generate pMAL-FIP200-N859, which is then used to express MBP-N1-859 fusion protein from bacteria.
The expression vectors encoding HA-S6K, Flag-4EBP1, HA-TSC2, GST-TSC2, Myc-TSC1, and Myc-TSC1 1402 aa have been described previously (Inoki et al., 2002, 2003a,b). Myc-TSC1 was used as template to generate the following construct: the TSC1 7891165 aa fragment was amplified by primers 5'-cgcggatccgaattcgacaaccagagccaggaa-3' (forward) and 5'-cccatcgatttagctgttttcatgatgagtctc-3' (reverse), digested with BamHI and ClaI, and then inserted into linearized myc-tagged vector pHAN digested with BamHI and ClaI, resulting in Myc-TSC1 7891165 aa. The TSC1 403787 aa fragment was amplified by primers 5'-cgcggatccgatgactacgtgcacatttca-3' (forward) and 5'-cccatcgatctggctctggttgtagaatcc-3' (reverse), digested with BamHI and ClaI, and then inserted into linearized myc-tagged vector pHAN digested with the same enzymes, resulting in Myc-TSC1 403787 aa. The same PCR product was digested with BamHI and ClaI and then inserted into linearized pGEX2T at the corresponding sites to generate pGEX2T-TSC1 403787 aa, which is then used to express the GST-TSC1 403787 aa fusion protein from bacteria. The enzymes used in cloning were all obtained from New England Biolabs, Inc. Nucleotide sequences of all constructs were confirmed by DNA sequencing.
Immunoprecipitation and Western blotting
Preparation of whole cell lysates, immunoprecipitation, and Western blotting were performed as previously described (Abbi et al., 2002).
Preparation of GST, MBP fusion proteins, and in vitro binding assays
GST fusion proteins were produced and purified as described previously (Abbi et al., 2002). MBP fusion proteins were purified according to the manufacturer's instructions (New England Biolabs, Inc.). MBP and MBP-N1-859 fusion proteins (10 µg) were immobilized on amylose resin and then incubated at 4°C with 2 µg GST-TSC1 403787 in binding buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1 mM MgCl2, and 1% Triton X-100) overnight at 4°C with rotation. The samples were then washed five times with binding buffer, boiled in SDS buffer, resolved by SDS-PAGE, and analyzed by Western blotting anti-GST antibody.
RNAi constructs and transfection
The RNAi vector pBS-U6 is a gift from Y. Shi (Harvard Medical School, Boston, MA) and has been described previously (Sui et al., 2002). Construction of RNAi vector was achieved in two separate steps: a 22-nt oligo (oligo 1) was first inserted into the pBS-U6 vector digested with ApaI (blunted) and HindIII. The inverted motif that contains the 6-nt spacer and 5 Ts (oligo 2) was then subcloned into the HindIII and EcoRI sites of the intermediate plasmid to generate pBS-U6-FIP200. For pBS-U6-FIP200 #7, the sequence of oligo1 is 5'-GGAGATTTGGTACTCATCATCA-3' (forward) and 5'-AGCTTGATGATGAGTACCAAATCTCC-3' (reverse); the sequence of oligo2 is 5'-AGCTTGATGATGAGTACCAAATCTCCCTTTTTG-3' (forward) and 5'-AATTCAAAAAGGGAGATTTGGTACTCATCATCA-3' (reverse). For pBS-U6-TSC1 #3, the sequence of oligo1 is 5'-GGGAGGTCAACGAGCTCTATTA (forward) and 5'-AGCTTAATAGAGCTCGTTGACCTCCC-3' (reverse); the sequence of oligo2 is 5'-AGCTTAATAGAGCTCGTTGACCTCCCCTTTTTG-3' (forward) and 5'-AATTCAAAAAG-GGGAGGTCAACGAGCTCTATTA (reverse). For pBS-U6-FAK #1, the sequence of oligo1 is 5'-GGCCAGTATTATCAGGCATGGA-3' (forward) and 5'-AGCTTCCATGCCTGATAATACTGGCC-3' (reverse); the sequence of oligo2 is 5'-AGCTTCCATGCCTGATAATACTGGCCCTTTTTG-3' (forward) and 5'-AATTCAAAAAGGGCCAGTATTATCAGGCATGGA-3' (reverse). The RNAi targeting sequences were all analyzed by BLAST search to ensure that they did not have significant sequence homology with other genes.
For RNAi knockdown experiments, 293T cells grown in 6-well plates were transfected with 1 µg RNAi vectors and 50 ng HA-S6K constructs using Lipofectamine reagent (Invitrogen) in accordance with the manufacturer's instructions. For double knockdown experiments, 293T cells were transfected with two RNAi vectors, each 500 ng, and 50 ng HA-S6K constructs. To detect endogenous S6K phosphorylation, cells grown in 6-well plates were transfected with 1 µg RNAi vectors without HA-S6K construct. After 3 d, growing cells were either directly lysed for Western blotting or used for growth factor and nutrient stimulation experiments as mentioned in the corresponding legends.
Cell size assay
To determine cell size and DNA content, FACS analysis with Cell Quest software was performed as previously described (Inoki et al., 2003b).
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Acknowledgments |
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This research was supported by National Institutes of Health grant GM52890 to J.-L. Guan.
Submitted: 17 November 2004
Accepted: 30 June 2005
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References |
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