PRR5, a Novel Component of mTOR Complex 2, Regulates Platelet-derived Growth Factor Receptor β Expression and Signaling
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m704343200
ISSN1083-351X
AutoresSo-Yon Woo, Donghwan Kim, Chang-Bong Jun, Young Mi Kim, Emilie Vander Haar, Seong-il Lee, James W. Hegg, Sricharan Bandhakavi, Timothy J. Griffin, Do‐Hyung Kim,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Functions of mTORC2, relative to mTORC1, have remained little understood. Here we report identification of a novel component of mTORC2 named PRR5 (PRoline-Rich protein 5), a protein encoded by a gene located on a chromosomal region frequently deleted during breast and colorectal carcinogenesis (Johnstone, C. N., Castellvi-Bel, S., Chang, L. M., Sung, R. K., Bowser, M. J., Pique, J. M., Castells, A., and Rustgi, A. K. (2005) Genomics 85, 338–351). PRR5 interacts with rictor, but not raptor, and the interaction is independent of mTOR and not disturbed under conditions that disrupt the mTOR-rictor interaction. PRR5, unlike Sin1, another component of mTORC2, is not important for the mTOR-rictor interaction and mTOR activity toward Akt phosphorylation. Despite no significant effect of PRR5 on mTORC2-mediated Akt phosphorylation, PRR5 silencing inhibits Akt and S6K1 phosphorylation and reduces cell proliferation rates, a result consistent with PRR5 roles in cell growth and tumorigenesis. The inhibition of Akt and S6K1 phosphorylation by PRR5 knock down correlates with reduction in the expression level of platelet-derived growth factor receptor β (PDGFRβ). PRR5 silencing impairs PDGF-stimulated phosphorylation of S6K1 and Akt but moderately reduces epidermal growth factor- and insulin-stimulated phosphorylation. These findings propose a potential role of mTORC2 in the cross-talk with the cellular machinery that regulates PDGFRβ expression and signaling. The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Functions of mTORC2, relative to mTORC1, have remained little understood. Here we report identification of a novel component of mTORC2 named PRR5 (PRoline-Rich protein 5), a protein encoded by a gene located on a chromosomal region frequently deleted during breast and colorectal carcinogenesis (Johnstone, C. N., Castellvi-Bel, S., Chang, L. M., Sung, R. K., Bowser, M. J., Pique, J. M., Castells, A., and Rustgi, A. K. (2005) Genomics 85, 338–351). PRR5 interacts with rictor, but not raptor, and the interaction is independent of mTOR and not disturbed under conditions that disrupt the mTOR-rictor interaction. PRR5, unlike Sin1, another component of mTORC2, is not important for the mTOR-rictor interaction and mTOR activity toward Akt phosphorylation. Despite no significant effect of PRR5 on mTORC2-mediated Akt phosphorylation, PRR5 silencing inhibits Akt and S6K1 phosphorylation and reduces cell proliferation rates, a result consistent with PRR5 roles in cell growth and tumorigenesis. The inhibition of Akt and S6K1 phosphorylation by PRR5 knock down correlates with reduction in the expression level of platelet-derived growth factor receptor β (PDGFRβ). PRR5 silencing impairs PDGF-stimulated phosphorylation of S6K1 and Akt but moderately reduces epidermal growth factor- and insulin-stimulated phosphorylation. These findings propose a potential role of mTORC2 in the cross-talk with the cellular machinery that regulates PDGFRβ expression and signaling. Cell growth relies on coordinated regulation of signaling pathways that integrate cellular physiological status in response to nutrient levels, growth factor signals, and environmental stress. 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Recognizing the complex relationship between mTOR, S6K1, and Akt and knowing that we have not yet identified mammalian homologues of AVO2 and BIT61, we hypothesized that other unidentified mTOR-binding effector proteins may provide clues to the mechanism underlying mTORC2 signaling. In this study, we identified a novel component of mTORC2 named PRR5, a protein having an implicative function in tumorigenesis (1Johnstone C.N. Castellvi-Bel S. Chang L.M. Sung R.K. Bowser M.J. Pique J.M. Castells A. Rustgi A.K. Genomics. 2005; 85: 338-351Crossref PubMed Scopus (25) Google Scholar). We determined that PRR5 specifically interacts with rictor, but not raptor, and the interaction is tighter than the rictor-mTOR interaction and independent of mTOR. We identified PRR5 and rictor residues crucial for the PRR5-rictor interaction and determined that PRR5 is important for PDGFRβ expression and PDGF signaling to Akt and S6K1. Reagents and Antibodies—Anti-mTOR (sc-1549), epidermal growth factor receptor (EGFR) (sc-03), Fas (sc-20140), p21 (sc-397), tubulin (sc-12462), PDFGR (sc-432), glyceraldehyde-3-phosphate dehydrogenase (sc-25778), and 14-3-3 (sc-732) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies specific to human PRR5 were generated against full-length PRR5 fused with glutathione S-transferase and an epitope peptide near the C terminus containing a sequence RGSGMSDLEGSGGR from YenZym antibodies (Burlingame, CA). Raptor- and rictor-specific antibodies were described in our previous report (14Vander Haar E. Lee S-I. Bandhakavi S. Griffin T.J. Kim D.-H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (936) Google Scholar). Antibodies against S6K1 (9202), phospho-S6K1 Thr-389 (p-S6K1; 9205), Akt (9272), phospho-Akt Ser-372 (p-Akt; 9271), 4E-BP1 (9452), phospho-4E-BP1 (2855), and insulin receptor β (3025) antibodies were from Cell Signaling Technology (Danvers, MA). Rabbit IgG TrueBlot (18–8816) used to detect PRR5 in immunoprecipitates was obtained from eBioscience (San Diego, CA). Anti-HA antibody (HA.11) was from Covance (Berkeley, CA). Anti-Myc 9E10 and growth factors EGF and PDGF were purchased from EMD Biosciences (San Diego, CA). Porcine insulin was purchased from Sigma. Glutathione 4B beads were from GE Healthcare. Identification of PRR5—The strategy that we described in our previous study (14Vander Haar E. Lee S-I. Bandhakavi S. Griffin T.J. Kim D.-H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (936) Google Scholar) was used with modifications in steps of immunoprecipitation and preparation of trypsinized samples. mTOR immunoprecipitate was prepared from HEK293T cells as described previously (14Vander Haar E. Lee S-I. Bandhakavi S. Griffin T.J. Kim D.-H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (936) Google Scholar) using a lysis buffer containing 40 mm Hepes, pH 7.4, 120 mm NaCl, 1 mm EDTA, 50 mm NaF, 1.5 mm Na3VO4, 10 mm β-glycerophosphate, 0.3% Chaps, and EDTA-free protease inhibitors (Roche Applied Science). mTOR immunoprecipitates were washed four times with the lysis buffer and twice with the lysis buffer without the detergent. mTOR-binding proteins were eluted from the immunoprecipitate in a buffer containing 0.075% SDS. The eluate was diluted with a trypsin digestion buffer (25 mm ammonium bicarbonate, 2.5 mm CaCl2, pH 8.0) and incubated with trypsin (2 μg) overnight. The trypsinized sample was diluted with 0.1% formic acid to obtain a pH below 3.0 and loaded onto a mixed mode cation exchange cartridge (MCX cartridge; Waters Inc., Milford, MA) to remove salt and detergent from the samples. Peptides bound to the resin were eluted with 5% ammonium hydroxide in methanol and lyophilized. Lyophilized samples were dissolved in 0.1% formic acid and analyzed by microcapillary electrospray tandem mass spectrometry on an electrospray linear ion trap mass spectrometer (ThermoElectron, Waltham, MA). Tandem mass spectrometry spectral data were analyzed as described in our previous study (14Vander Haar E. Lee S-I. Bandhakavi S. Griffin T.J. Kim D.-H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (936) Google Scholar). Peptide sequence matches were filtered using a probabilistic scoring algorithm called Peptide Prophet (26Eng J.K. McCormack A.L. Yates J.R. J. Am. Soc. Mass. Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5472) Google Scholar, 27Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3912) Google Scholar) that assigns a value between 0 and 1 to peptide sequence matches, with a score of 1 representing the highest confidence match. Plasmid Constructions and Mutagenesis—PRR5 cDNAs for isoforms 1, 2, and 3 of human origin kindly provided by Dr. C. Johnstone and Dr. A. Rustgi at the University of Pennsylvania were cloned into prk5-myc and prk5-HA expression vectors by use of a PCR amplification kit (Roche Applied Science). The PRR5 and rictor DNA fragments used in Fig. 4, C and D, were generated by PCR amplification and subcloned into mammalian expression vector prk5-myc, and all the clones were confirmed by sequencing. pLKO shRNA vector (provided by Dr. S. Stewart, Washington University) was used for knockdown experiments. Target sequences were 5′-catgctgcaggccatcttcta-3′ (sh-PRR5 4), 5′-ggacaagattcgcttctatga-3′ (sh-PRR5 15), 5′-aaccctgcctttgtcatgcct-3′ (sh-mTOR), 5′-caccaccaaagcaacctatag-3′ (sh-rictor), and 5′-aacgtacgcggaatacttcga-3′ (scrambled shRNA). All other constructs used in the experiments have been previously described (10Kim D-H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2391) Google Scholar, 11Kim D-H. Sarbassov D.D. Ali S.M. Latek R.R. Kalyani V.P. Erdjument-Bromage H. Tempst P. Sabatini D.M. Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (774) Google Scholar, 14Vander Haar E. Lee S-I. Bandhakavi S. Griffin T.J. Kim D.-H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (936) Google Scholar). Cell Culture and Transfection—HEK293T, HeLa, HT1080, HepG2 cells, and other cancer cell lines were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin/streptomycin at 37 °C in 5% CO2. For transient expression, HEK293T cells were transfected with recombinant DNAs or shRNA plasmids using FuGENE 6 (Roche Applied Science) following the manufacturer's protocol. Cells were harvested 2 days post-transfection for co-immunoprecipitation assay. Recombinant Protein Production—GST-tagged PRR5 isoforms 1, 2, and 3 cloned in pGEX6T-2 (Amersham Biosciences) were expressed in BL21(DE3) cells (EMD Biosciences) by induction with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside for 16 h and purified with glutathione-Sepharose 4B beads according to a standard protocol. Co-immunoprecipitation and Western Blotting—For co-immunoprecipitation studies, whole-cell extracts were prepared in 0.3% Chaps buffer and immunoprecipitated with the anti-mTOR, anti-raptor, anti-rictor, anti-PRR5, anti-HA, or anti-Myc antibodies. Precipitated proteins were washed four times in 0.3% Chaps buffer, loaded onto 8% Tris-glycine gels (Invitrogen), transferred for 4 h onto immunoblot polyvinylidene difluoride membranes (Bio-Rad), and detected with ECL Western blotting detection reagents (Perkin-Elmer). Lentiviral Preparation, Viral Infection, and Stable Cell Generation—A pLKO-shRNA plasmid encoding an shRNA that targets PRR5 or a scrambled sequence was transduced into HEK293T cells with lentiviral packaging vectors pHR′8.2ΔR and pCMV-VSV-G (provided by Dr. S. Stewart, Washington University) using FuGENE 6. Viruses were collected from the medium 60 h after transfection, and target cells were infected with the collected viruses four times over 15 h in the presence of polybrene. Cells were harvested 3 to 5 days post-infection or selected under puromycin for several days. Cell Proliferation Assay—HeLa cells transduced with lentiviral shRNAs were split into 6-cm plates at 20% confluence; the next day cells were trypsinized and diluted ten times with Dulbecco's modified Eagle's medium. One ml of diluted cell culture was loaded on a ViCell analyzer (Beckman Coulter Inc., Fullerton, CA). Real-time PCR Analysis—Total RNA was prepared from HeLa cells transduced by lentiviral shRNAs using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Single-stranded cDNA was synthesized from 5 μg of total RNA using the iScript cDNA synthesis kit for real-time PCR (Bio-Rad) and resuspended in diethylpyrocarbonate-treated water. PCR products were generated by PCR amplification using Lightcycler Faststart DNA Masterplus SYBR Green 1 (Bio-Rad). Amplification of human PDGFRβ cDNA was performed using a forward primer, 5′-tgtgacggagagtgtgaatgac-3′, paired with a reverse primer, 5′-agggtgcggttgtctttgaac-3′. Amplification of TATA box-binding protein cDNA was performed using a forward primer, 5′-taatcccaagcggtttgctg-3′, paired with a reverse primer, 5′-gcacaccattttcccagaactg-3′. Identification of PRR5 as an mTOR-binding Protein—In our recent study, we described an approach combining the electrospray linear ion trap mass spectrometer and mTOR immunoprecipitation to identify mTOR-binding proteins (14Vander Haar E. Lee S-I. Bandhakavi S. Griffin T.J. Kim D.-H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (936) Google Scholar). This approach, without relying on SDS-PAGE separation of proteins, increased the sensitivity of detection and led us to identify PRAS40 and Sin1 that were barely detectable on Coomassie-stained gels. We modified sample preparative conditions for mass spectrometry as detailed under “Experimental Procedures.” In the new preparation, we identified three peptides of high scores of P value, a parameter of fidelity for MS/MS matches, that were detected from proteins isolated specifically in mTOR immunoprecipitate but not in control immunoprecipitates (supplemental Table 1) (14Vander Haar E. Lee S-I. Bandhakavi S. Griffin T.J. Kim D.-H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (936) Google Scholar). The three identified peptides were derived from PRR5, a proline-rich protein that has an implicative role in tumorigenesis (1Johnstone C.N. Castellvi-Bel S. Chang L.M. Sung R.K. Bowser M.J. Pique J.M. Castells A. Rustgi A.K. Genomics. 2005; 85: 338-351Crossref PubMed Scopus (25) Google Scholar). The PRR5 gene is located on chromosome 22q13.31, a region that is frequently deleted during human breast and colorectal carcinogenesis. PRR5 was previously shown to exist as several isoforms of splicing variants (1Johnstone C.N. Castellvi-Bel S. Chang L.M. Sung R.K. Bowser M.J. Pique J.M. Castells A. Rustgi A.K. Genomics. 2005; 85: 338-351Crossref PubMed Scopus (25) Google Scholar). PRR5, containing a high content of proline residues (28 among 388 amino acids, 7.2%), is conserved in higher eukaryotes. BLAST search revealed that human PRR5 shares 33–86% identity with genes from amphibians, fishes, rodents, and primates but does not show similarity to genes from Drosophila melanogaster and Caenorhabditis elegans as well as BIT61 and AVO2, two TOR2-interacting proteins whose mammalian homologues have not been found (supplemental Fig. S1). PRR5 residues 91–169 share sequence similarity with the HbrB domain, a domain found in proteins involved in hyphal growth and polarity (28Gatherar I.M. Pollerman S. Dunn-Coleman N. Turner G. Fungal Genet. Biol. 2004; 41: 463-471Crossref PubMed Scopus (22) Google Scholar). The longest isoform (isoform 1) contains 388 amino acids with a proline-rich region near the C terminus (Fig. 1A). Isoforms 2 and 3 are 9 and 95 amino acids shorter than isoform 1 at the N terminus, respectively. To confirm that mTOR specifically interacts with PRR5, mTOR immunoprecipitate was obtained from HEK293T cells and the amount of transiently expressed PRR5 isoform 2, a highly expressed form of the three isoforms (Fig. 1A), was analyzed by Western blotting. Supporting the specific interaction between mTOR and PRR5, PRR5 was detected only in mTOR immunoprecipitate purified in the absence of an mTOR antibody-blocking peptide but not in the presence of the blocking peptide or in immunoprecipitates obtained using control antibodies (Fig. 1B). Consistent with the specific interaction between mTOR and PRR5, endogenous mTOR was isolated in immunoprecipitates of recombinant PRR5 but not in control immunoprecipitates (Fig. 1C). We generated polyclonal antibodies specific to human PRR5 using GST fusion PRR5 full-length protein or an epitope peptide near the C terminus as an antigen. The latter antibody was able to pull down endogenous PRR5 that is associated with endogenous mTOR (Fig. 1D). Using the PRR5-specific antibody, we confirmed that endogenous PRR5 is purified specifically by anti-mTOR immunoprecipitation but not control antibodies (Fig. 1E). Tissue distribution of human PRR5 mRNA had been reported previously (1Johnstone C.N. Castellvi-Bel S. Chang L.M. Sung R.K. Bowser M.J. Pique J.M. Castells A. Rustgi A.K. Genomics. 2005; 85: 338-351Crossref PubMed Scopus (25) Google Scholar). PRR5 mRNA is most abundant in kidney and liver. It is also highly detected in brain, spleen, testis, and placenta. Northern blot analysis had shown multiple different-sized bands evident in tissues including spleen, testis, and heart. We observed that PRR5 is expressed in different amounts in several human cell lines such as 293T, HeLa, HepG2, human fibrosarcoma cell line HT1080, and human breast cancer cell lines MCF-7, T47D, and MDA-MB-231 (Fig. 1F). In these cells, we observed that PRR5 is immunoprecipitated by mTOR antibody (Fig. 1G). MDA-MB-231 expresses little amount of isoforms 1 and 2, a result consistent with reverse transcription PCR data (1Johnstone C.N. Castellvi-Bel S. Chang L.M. Sung R.K. Bowser M.J. Pique J.M. Castells A. Rustgi A.K. Genomics. 2005; 85: 338-351Crossref PubMed Scopus (25) Google Scholar). PRR5 is most highly expressed in 293T cells, a cell line derived from the kidney where PRR5 mRNA level is most abundant (1Johnstone C.N. Castellvi-Bel S. Chang L.M. Sung R.K. Bowser M.J. Pique J.M. Castells A. Rustgi A.K. Genomics. 2005; 85: 338-351Crossref PubMed Scopus (25) Google Scholar). PRR5 Is a Component of mTORC2—Knowing that PRR5 is an interacting protein of mTOR, we questioned which mTOR complex contains PRR5. Importantly, PRR5 was detected in mTOR and rictor immunoprecipitates, but not in raptor immunoprecipitates, suggesting that PRR5 specifically targets mTORC2 (Fig. 2A). Supporting that a large proportion of the mTOR-rictor complex contains PRR5, a higher amount of Myc-tagged PRR5 was recovered bound to rictor than Myc-mTOR, although both Myc-tagged proteins were expressed at similar levels (Fig. 2B). We thought that a stronger association of rictor with PRR5 than mTOR might support a role of rictor in the mediation of the PRR5-mTOR interaction. To test the possibility that PRR5 binding to mTOR requires rictor, we knocked down rictor in 293T cells through a lentiviral shRNA transduction and determined the amount of PRR5 associated with mTOR in mTOR immunoprecipitate. Rictor silencing led to a significant reduction in the amount of PRR5 not only in mTOR immunoprecipitate but also in cell lysate, indicating that rictor is important for the stability of PRR5 (Fig. 3A). Sin1, another component of mTORC2, has been shown to be important for the mTOR-rictor interaction (14Vander Haar E. Lee S-I. Bandhakavi S. Griffin T.J. Kim D.-H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (936) Google Scholar, 29Frias M.A. Thoreen C.C. Jaffe J.D. Schroder W. Sculley T. Carr S.A. Sabatini D.M. Curr. Biol. 2006; 16: 1-6Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 30Jacinto E. Facchinetti V. Liu D. Soto N. Wei S. Jung S.Y. Huang Q. Qin J. Su B. Cell. 2006; 126: 1-13Abstract Full Text Full Text PDF Google Scholar, 31Yang Q. Inoki K. Ikenoue T. Guan K.L. Genes Dev. 2006; 20: 2820-2832Crossref PubMed Scopus (410) Google Scholar). Unlike Sin1 silencing, PRR5 silencing did not lead to a change in the affinity of the interaction between mTOR and rictor, supporting that PRR5 is not important for the rictor-mTOR interaction (Fig. 3A). Consistent with this result, overexpression of PRR5 did not alter the affinity of the mTOR-rictor interaction, an interaction stabilized by Sin1 overexpression (Fig. 3B). Furthermore, the PRR5-mTOR interaction, but not the PRR5-rictor interaction, was destabilized in a lysis buffer containing Triton X-100 (Fig. 3, C and D), indicating that the PRR5-rictor interaction is resistant to the detergent condition that disrupts the mTOR-rictor interaction (10Kim D-H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2391) Google Scholar). These results demonstrate that PRR5 binds rictor preferentially and independently of mTOR and rictor is important for the mTOR-PRR5 interaction. PRR5 Residues 10–95 and 188–218 Are Crucial for Binding Rictor—Knowing that PRR5 interacts with mTOR and rictor, we questioned whether all the isoforms interact with mTOR and rictor isoforms. We expressed HA-tagged isoforms in 293T cells and analyzed the amount of endogenous mTOR and rictor recovered with HA-PPR5 isoforms in HA immunoprecipitate. Supporting that isoforms 1 and 2 interact with mTOR and rictor, we observed that endogenous mTOR and rictor are immunoprecipitated with HA-tagged isoforms 1 and 2, but not with isoform 3 (Fig. 4A). Confirming the specific interaction of isoforms 1 and 2 with mTOR and rictor, only isoforms 1 and 2, but not isoform 3, expressed as GST fusion proteins in Escherichia coli pulled down endogenous mTOR and rictor (Fig. 4B). These results suggest that the N-terminal 95 amino acids contain residues important for binding mTOR and rictor. The N-terminal region of PRR5 overlaps with the residues conserved among higher eukaryotic genes (supplemental Fig. S1), supporting that the interaction with rictor is likely important during the evolution of higher eukaryotes. To search for C-terminal residues important for binding mTOR and rictor, we made C-terminal-truncated mutants of PRR5 and tested the mutants for their ability to bi
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