Abstract
Earlier we showed that RACK1 regulates growth of human colon cells by suppressing Src activity at G1 and mitotic checkpoints. Here, we show that RACK1 also induces apoptosis of the cells, partly by inhibiting Src. In the intrinsic pathway, RACK1 inhibits expression of anti-apoptotic Bcl-2 and Bcl-XL, induces expression of pro-apoptotic Bim, targets Bim and Bax to the mitochondria, induces oligomerization of Bax (which requires Bim and inhibition of Src), depolarizes mitochondria membranes, releases cytochrome c, and activates caspases-9 and -3 and death substrates. Bax and Bim are required for RACK1-mediated mitochondrial cell death. RACK1-induced oligomerization of Bax is required for staurosporine-mediated cell death. RACK1 also induces apoptosis by blocking Src activation of the Akt cell survival pathway. This leads to activation of the transcription factor FOXO3, a potent inducer of apoptosis and G1 arrest. Collectively, our results show that RACK1, partly by inhibiting Src, promotes mitochondrial cell death and blocks Akt-mediated cell survival. Thus, RACK1 inhibits growth and induces death of colon cells. Exploitation of these dual functions could lead to novel colon cancer therapies that mimic RACK1 function.
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References
Antonsson B, Conti F, Ciavatta FA, Montessuit S, Lewis S, Martinou I et al. (1997). Inhibition of Bax channel-forming activity by Bcl-2. Science 277: 370–372.
Arimoto K, Fukuda H, Imajoh-Ohmi S, Saito H, Takekawa M . (2008). Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways. Nature Cell Biol 10: 1324–1332.
Buensuceso CS, Woodside D, Huff JL, Plopper GE, O’Toole TE . (2001). The WD Rack1 protein mediates protein kinase C and integrin-dependent cell migration. J Cell Sci 114: 1691–1698.
Chandra D, Choy G, Deng X, Bhatia B, Daniel P, Tang DG . (2004). Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death. Mol Cell Biol 24: 6592–6607.
Chang BY, Chiang M, Cartwright CA . (2001). The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1. J Biol Chem 276: 20346–20356.
Chang BY, Conroy KB, Machleder EM, Cartwright CA . (1998). RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells. Mol Cell Biol 18: 3245–3256.
Chang BY, Harte R, Cartwright CA . (2002). RACK1: a novel substrate for the Src protein-tyrosine kinase. Oncogene 21: 7619–7629.
Chen R, Kim O, Yang J, Sato K, Eisenmann KM, McCarthy J et al. (2001). Regulation of Akt/PKB activation by tyrosine phosphorylation. J Biol Chem 34: 31858–31862.
Cheng JQ, Lindsley CW, Cheng GZ, Yang H, Nicosia SV . (2005). The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 24: 7482–7492.
Chipuk JE, Green DR . (2008). How do BCL-2 proteins induce mitochondrial outer membrane permeabilization? Trends Cell Biol 18: 157–164.
Cox EA, Bennin D, Doan AT, O’Toole T, Huttenlocher A . (2003). RACK1 regulates integrin-mediated adhesion, protrusion, and chemotactic cell migration via its Src-binding site. Mol Biol Cell 14: 658–669.
Danial NN, Korsmeyer SJ . (2004). Cell death: critical control points. Cell 116: 205–219.
Dorn GW, Mochly-Rosen D . (2002). Intracellular transport mechanisms of signal transducers. Annu Rev Physiol 64: 407–429.
Galluzzi L, Joza N, Tasdemir E, Maiuri MC, Hengartner M, Abrams JM et al. (2008). No death without life: vital functions of apoptotic effectors. Cell Death Diff 15: 1113–1123.
Gilley J, Coffer PJ, Ham J . (2003). FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. J Cell Biol 162: 613–622.
Golubovskaya VM, Gross S, Kaur AS, Wilson RI, Xu L, Yang XH et al. (2003). Simultaneous inhibition of focal adhesion kinase and Src enhance detachment and apoptosis in colon cancer cell lines. Mol Cancer Res 1: 755–764.
Gottlob K, Fulco M, Levrero M, Graessmann A . (1998). The hepatitis B virus HBx protein inhibits caspase 3 activity. J Biol Chem 273: 33347–33353.
Green DR . (2005). Apoptotic pathways: ten minutes to dead. Cell 121: 671–674.
Griffiths GJ, Koh MY, Brunton VG, Cawthorne C, Reeves NA, Greaves M et al. (2004). Expression of kinase defective mutants of c-Src in human metastatic colon cancer cells decreases Bcl-XL and increases Oxaliplatin and Fas-induced apoptosis. J Biol Chem 279: 46113–46121.
Hermanto U, Zong CS, Li W, Wang L-H . (2002). RACK1, an insulin-like growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with extracellular matrix. Mol Cell Biol 22: 2345–2365.
Inagaki K, Churchill E, Mochly-Rosen D . (2006). Epsilon protein kinase C as a potential therapeutic target for the ischemic heart. Cardiovasc Res 70: 222–230.
Jiang T, Qiu Y . (2003). Interaction between Src and a C-terminal proline-rich motif of Akt is required for Akt activation. J Biol Chem 278: 15789–15793.
Kang BP, Urbonas A, Baddoo A, Baskin S, Malhotra A, Meggs LG . (2003). IGF-1 inhibits the mitochondrial apoptosis program in mesangial cells exposed to high glucose. Am J Physiol Renal Physiol 285: F1013–F1024.
Kiely PA, Leahy M, O’Gorman D, O’Connor R . (2005). RACK1-mediated integration of adhesion and insulin-like growth factor I (IGF-I) signaling and cell migration are defective in cells expressing an IGF-I receptor mutated at tyrosines 1250 and 1251. J Biol Chem 280: 7624–7633.
Kiely PA, Sant A, O’Connor R . (2002). RACK1 is an insulin-like growth factor 1 (IGF-1) receptor-interacting protein that can regulate IGF-1-mediated Akt activation and protection from cell death. J Biol Chem 277: 22581–22589.
Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S et al. (2006). Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 5: 375–388.
Liou J-Y, Aleksic N, Chen S-F, Han T-J, Shyue S-K, Wu KK . (2005). Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance. Exp Cell Res 306: 75–84.
Lipsich LA, Lewis AJ, Brugge JS . (1983). Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol 48: 352–360.
Makin G, Dive C . (2003). Recent advances in understanding apoptosis: new therapeutic opportunities in cancer chemotherapy. Trends Mol Med 9: 251–255.
Mamidipudi V, Chang BY, Harte RA, Lee KC, Cartwright CA . (2004b). RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells. FEBS Lett 567: 321–326.
Mamidipudi V, Dhillon NK, Parman T, Miller LD, Lee KC, Cartwright CA . (2007). RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints. Oncogene 26: 2914–2924.
Mamidipudi V, Zhang J, Lee KC, Cartwright CA . (2004a). RACK1 regulates G1/S progression by suppressing Src kinase activity. Mol Cell Biol 24: 6788–6798.
Meggio F, Donella-Deana A, Ruzzene M, Brunati AM, Cesaro L, Guerra B et al. (1995). Different susceptibility of protein kinases to staurosporine inhibition - kinetic studies and molecular bases for the resistance of protein kinase CK2. Eur J Biochem 234: 317–322.
Miyazaki T, Neff L, Tanaka S, Horne WC, Baron R . (2003). Regulation of cytochrome c oxidase activity by c-Src in osteoclasts. J Cell Biol 160: 709–718.
Muslin AJ, Xing H . (2000). 14-3-3 proteins: regulation of subcellular localization by molecular interference. Cell Signal 12: 703–709.
Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, Sellers WR . (2000). Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Biol 20: 8969–8982.
Oberst A, Bender C, Green DR . (2008). Living with death: the evolution of the mitochondrial pathway of apoptosis in animals. Cell Death Differ 15: 1139–1146.
Ramaswamy S, Nakamura N, Sansal I, Bergeron L, Sellers WR . (2002). A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell 2: 81–91.
Reginato MJ, Mills KR, Becker EB, Lynch DK, Bonni A, Muthuswamy SK et al. (2005). Bim regulation of lumen formation in cultured mammary epithelial acini is targeted by oncogenes. Mol Cell Biol 25: 4591–4601.
Reginato MJ, Mills KR, Paulus JK, Lynch DK, Sgroi DC, Debnath J et al. (2003). Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis. Nat Cell Biol 5: 733–740.
Saito M, Korsmeyer SJ, Schlesinger PH . (2000). BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat Cell Biol 2: 553–555.
Sharma SV, Gajowniczek P, Way IP, Lee DY, Jiang J, Yuza Y et al. (2006). A common signaling cascade may underlie ‘addiction’ to the Src, BCR-ABL, and EGF receptor oncogenes. Cancer Cell 5: 425–435.
Shi Y . (2004). Caspase activation: revisiting the induced proximity model. Cell 117: 855–858.
Souroujon MC, Mochly-Rosen D . (1998). Peptide modulators of protein-protein interactions in intracellular signaling. Nat Biotechnol 16: 919–924.
Sun Y, Tang XM, Half E, Kuo T, Sinicrope FA . (2002). Cyclooxygenase-2 overexpression reduces apoptotic susceptibility by inhibiting the cytochrome c-dependent apoptotic pathway in human colon cancer cells. Cancer Res 21: 6323–6328.
Trotman LC, Alimonti A, Scaglioni PP, Koutcher JA, Cordon-Cardo C, Pandolfi PP . (2006). Identification of a tumour suppressor network opposing nuclear Akt function. Nature 441: 523–527.
van Delft MF, Wei AH, Mason KD, Vandenberg CJ, Chen L, Czabotar PE et al. 2006. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 10: 389–399.
Vomastek T, Iwanicki MP, Schaeffer H-J, Tarcsafalvi A, Parsons JT, Weber MJ . (2007). RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility. Mol Cell Biol 27: 8296–8305.
Willis SN, Fletcher JI, Kaufmann T, van Delft MF, Chen L, Czabotar PE et al. (2007). Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315: 856–859.
Windham TC, Parikh NU, Siwak DR, Summy JM, McConkey DJ, Kraker AJ et al. (2002). Src activation regulates anoikis in human colon tumor cell lines. Oncogene 21: 7797–7807.
Yethon JA, Epand RF, Leber B, Epand RM . (2003). Interaction with a membrane surface triggers a reversible conformational change in Bax normally associated with induction of apoptosis. J Biol Chem 278: 48935–48941.
Yip KW, Reed JC . (2008). Bcl-2 family proteins and cancer. Oncogene 27: 6398–6406.
Zhang H, Kim JK, Edwards CA, Xu Z, Taichman R, Wang CY . (2005). Clusterin inhibits apoptosis by interacting with activated Bax. Nat Cell Biol 7: 909–915.
Zhou H, Li XM, Meinkoth J, Pittman RN . (2000). Akt regulates cell survival and apoptosis at a postmitochondrial level. J Cell Biol 151: 483–494.
Acknowledgements
We thank Jenny Cheng and Gayathri Swaminathan for many helpful discussions and critical review of the data and manuscript. This work was supported by a grant from the National Institutes of Health to CAC (DK43743). Preliminary studies were supported by a pilot grant from the Broad Medical Research Program (IBD-0068).
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Mamidipudi, V., Cartwright, C. A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways. Oncogene 28, 4421–4433 (2009). https://doi.org/10.1038/onc.2009.293
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DOI: https://doi.org/10.1038/onc.2009.293
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