Depletion of Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatases 1 and 2 by Bcr-Abl Promotes Chronic Myelogenous Leukemia Cell Proliferation through Continuous Phosphorylation of Akt Isoforms
2009; Elsevier BV; Volume: 284; Issue: 33 Linguagem: Inglês
10.1074/jbc.m808182200
ISSN1083-351X
AutoresIsao Hirano, Satoki Nakamura, Daisuke Yokota, Takaaki Ono, Kazuyuki Shigeno, Shinya Fujisawa, Kaori Shinjo, Kazunori Ohnishi,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoThe constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells. The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells. Chronic myelogenous leukemia (CML) 2The abbreviations used are: CMLchronic myelogenous leukemiaPHLPPpleckstrin homology domain leucine-rich repeat protein phosphataseCFU-GEMMcolony-forming unit-granulocyte, erythroid, macrophage, megakaryocyteCFU-GMcolony-forming unit-granulocyte, macrophageBFU-Eburst-forming unit-erythroidPP2Athe serine/threonine protein phosphatase 2API3Kphosphatidylinositol 3-kinasePTENphosphatase and tensin homolog deleted on chromosome 10siRNAshort interfering RNAAMLacute myeloblastic leukemiaALDHaldehyde dehydrogenaseRT-PCRreal time PCRG3PDHglyceraldehyde-3-phosphate dehydrogenaseMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideE3ubiquitin-protein isopeptide ligase. 2The abbreviations used are: CMLchronic myelogenous leukemiaPHLPPpleckstrin homology domain leucine-rich repeat protein phosphataseCFU-GEMMcolony-forming unit-granulocyte, erythroid, macrophage, megakaryocyteCFU-GMcolony-forming unit-granulocyte, macrophageBFU-Eburst-forming unit-erythroidPP2Athe serine/threonine protein phosphatase 2API3Kphosphatidylinositol 3-kinasePTENphosphatase and tensin homolog deleted on chromosome 10siRNAshort interfering RNAAMLacute myeloblastic leukemiaALDHaldehyde dehydrogenaseRT-PCRreal time PCRG3PDHglyceraldehyde-3-phosphate dehydrogenaseMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideE3ubiquitin-protein isopeptide ligase. is a hematopoietic stem cell disorder that is characterized by the Philadelphia chromosome (1Kurzrock R. Gutterman J.U. Talpaz M. N. Engl. J. Med. 1988; 319: 990-998Crossref PubMed Scopus (720) Google Scholar, 2Rudkin C.T. Nowell P.C. Hungerford D.A. Science. 1964; 144: 1229-1231Crossref PubMed Scopus (56) Google Scholar), a shortened chromosome 22 that is a by-product of a reciprocal chromosomal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11), resulting in a chimeric Bcr-Abl oncoprotein with highly deregulated, constitutive tyrosine kinase activity (3Shtivelman E. Lifshitz B. Gale R.P. Ganaani E. Nature. 1985; 315: 550-554Crossref PubMed Scopus (1249) Google Scholar, 4Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1115) Google Scholar). The most commonly occurring form of Bcr-Abl is a 210-kDa protein that is a critical role in the pathogenesis of CML (5Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1913) Google Scholar). As a result of its elevated tyrosine kinase activity, Bcr-Abl activates a multitude of signaling pathways, including the Ras (6Sawyers C.L. McLaughlin J. Witte O.N. J. Exp. Med. 1995; 181: 307-313Crossref PubMed Scopus (245) Google Scholar), PI3K/Akt (7Skorski T. Bellacosa A. Nieborowska-Skorska M. Majewski M. Martinez R. Choi J.K. Trotta R. Wlodarski P. Perrotti D. Chan T.O. Wasik M.A. Tsichlis P.N. Calabretta B. EMBO J. 1997; 16: 6151-6161Crossref PubMed Scopus (552) Google Scholar, 8Varticovski L. Daley G.Q. Jackson P. Baltimore D. Cantley L.C. Mol. Cell. Biol. 1991; 11: 1107-1113Crossref PubMed Scopus (180) Google Scholar), Janus kinase/signal transducer and activator of transcription (9Carlesso N. Frank D.A. Griffin J.D. J. Exp. Med. 1996; 183: 811-820Crossref PubMed Scopus (429) Google Scholar), and NF-κB (10Reuther J.Y. Reuther G.W. Cortez D. Pendergast A.M. Baldwin Jr., A.S. Genes Dev. 1998; 12: 968-981Crossref PubMed Scopus (350) Google Scholar) signaling pathways. Furthermore the serine/threonine protein phosphatase PP2A is also functionally inactivated by Bcr-Abl (11Neviani P. Santhanam R. Trotta R. Notari M. Blaser B.W. Liu S. Mao H. Chang J.S. Galietta A. Uttam A. Roy D.C. Valtieri M. Bruner-Klisovic R. Caligiuri M.A. Bloomfield C.D. Marcucci G. Perrotti D. Cancer Cell. 2005; 8: 355-368Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). These signaling pathways, especially the Bcr-Abl-induced PI3K/Akt activation, play a role in Bcr-Abl-mediated leukemogenesis.PP2A is inactivated in blast crisis CML through Bcr-Abl-mediated transcriptional up-regulation of the PP2A inhibitor SET. Hyperphosphorylation and inactivation of proapoptotic PP2A substrate kinases, such as phospho-Akt and phospho-ERK (extracellular signal-regulated kinase), lead to their prolonged activation and ability to drive the survival and proliferative signaling pathway (11Neviani P. Santhanam R. Trotta R. Notari M. Blaser B.W. Liu S. Mao H. Chang J.S. Galietta A. Uttam A. Roy D.C. Valtieri M. Bruner-Klisovic R. Caligiuri M.A. Bloomfield C.D. Marcucci G. Perrotti D. Cancer Cell. 2005; 8: 355-368Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). The inactivation of PP2A is essential for Bcr-Abl-mediated leukemogenesis and blastic transformation.Another serine/threonine protein phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP), terminates Akt signaling by dephosphorylating the hydrophobic motif on Akt (12Gao T. Brognard J. Newton A.C. J. Biol. Chem. 2008; 283: 6300-6311Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The PHLPP family of phosphatases includes PHLPP1α (1205 amino acids), PHLPP1β (1717 amino acids), and PHLPP2 (1323 amino acids) (14Gao T. Furnari F. Newton A.C. Mol. Cell. 2005; 18: 13-24Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar, 15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). PHLPP1α and -β are variants spliced from the same gene located at chromosome 18q21.33 (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar), and they differ by a 56-kDa N-terminal extension (13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The gene encoding PHLPP2 resides at 16q22.3 (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). PHLPP1 and PHLPP2 own a pleckstrin homology domain with a region of leucine-rich repeats, a PP2C phosphatase domain, and a C-terminal PDZ ligand (13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The genes encoding PHLPP1 and PHLPP2 were frequently lost in various cancers such as colon (16Goel A. Arnold C.N. Niedzwiecki D. Chang D.K. Ricciardiello L. Carethers J.M. Dowell J.M. Wasserman L. Compton C. Mayer R.J. Bertagnolli M.M. Boland C.R. Cancer Res. 2003; 63: 1608-1614PubMed Google Scholar), breast (17Rakha E.A. Genes Chromosomes Cancer. 2006; 45: 527-535Crossref PubMed Scopus (69) Google Scholar), and ovarian cancers (18Patael-Karasik Y. Daniely M. Gotlieb W.H. Ben-Baruch G. Schiby J. Barakai G. Goldman B. Aviram A. Friedman E. Cancer Genet. Cytogenet. 2000; 121: 26-32Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), Wilms tumors (19Safford S.D. Goyeau D. Freemerman A.J. Bentley R. Everett M.L. Grundy P.E. Skinner M.A. Ann. Surg. Oncol. 2003; 10: 136-143Crossref PubMed Scopus (26) Google Scholar), prostate cancer (20Torring N. Borre M. Sorensen K.D. Andersen C.L. Wiuf C. Orntoft T.F. Br. J. Cancer. 2007; 96: 499-506Crossref PubMed Scopus (48) Google Scholar), and hepatocellular carcinomas (21Tsuda H. Zhang W.D. Shimosato Y. Yokota J. Terada M. Sugimura T. Miyamura T. Hirohashi S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6791-6794Crossref PubMed Scopus (281) Google Scholar). PHLPP1 and PHLPP2 are present in the cytosolic, nuclear, and membrane fraction of cells and are expressed in cell lines including brain, breast, lung, prostate, and ovarian cancer cell lines (15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). PHLPP1 and PHLPP2 decrease activity of Akt and increase apoptosis and inhibition of cell proliferation through the dephosphorylation of the hydrophobic motif (Ser-473) in Akt. Depletion of either PHLPP1 or PHLPP2 causes a 30-fold increase in Akt phosphorylation after EGF stimulation in a normal breast cell line (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). Knockdown studies have revealed that PHLPP1 influences the phosphorylation state of Akt2 and Akt3, whereas PHLPP2 affects the phosphorylation state of Akt1 and Akt3.In the PI3K/Akt signaling pathway, defective PTEN activates Akt signaling by preventing conversion of phosphatidylinositol 3,4,5-trisphosphate back to phosphatidylinositol 4,5-bisphosphate and contributes to retaining Akt phosphorylation. Nonetheless there are many examples of elevated Akt phosphorylation in cancer cells that have intact PTEN expression. PHLPP levels are markedly reduced in some cancer cell lines that have elevated Akt phosphorylation, and the reintroduction of PHLPP reduces cell growth (23Mendoza M.C. Blenis J. Mol. Cell. 2007; 25: 798-800Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, in Bcr-Abl-mediated leukemogenesis, the role of PLHPP remains unclear.In the present study, we found that Bcr-Abl suppressed the expression of PHLPP1 and PHLPP2. We analyzed the role of PHLPP1 and PHLPP2 in CML cell lines and the hematopoietic progenitor cells derived from CML patients by inhibiting the expression of PHLPP1 and PHLPP2. We detected a change in Ser-473 upon Akt phosphorylation with CML-derived hematopoietic progenitor cells exhibiting more Akt phosphorylation than normal hematopoietic progenitor cells.DISCUSSIONWe investigated the role of PHLPP expression on the induction of apoptosis or growth inhibition of CML cells treated with Abl kinase inhibitors. We demonstrated that Bcr-Abl inhibited the expression of PHLPP1 and PHLPP2 and promoted the CML cell progression through continuous phospho-Akt activation.The PI3K/Akt pathway is a central node in determining cellular fate. Hyperactivation or continuous activation of this pathway promotes cell survival, growth, and proliferation (24Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5073) Google Scholar). This pathway is activated in cancer by mechanisms including amplification or gain-of-function mutations in upstream receptor protein-tyrosine kinases (25Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3097) Google Scholar, 26Marx J.L. Science. 1989; 244: 654-655Crossref PubMed Scopus (4) Google Scholar), activating mutations in PI3K and Akt (27Carpten J.D. Faber A.L. Horn C. Donoho G.P. Briggs S.L. Robbins C.M. Hostetter G. Boguslawski S. Moses T.Y. Savage S. Uhlik M. Lin A. Du J. Qian Y.W. Zeckner D.J. Tucker-Kellogg G. Touchman J. Patel K. Mousses S. Bittner M. Schevitz R. Lai M.H. Blanchard K.L. Thomas J.E. Nature. 2007; 448: 439-444Crossref PubMed Scopus (990) Google Scholar), and loss-of-function mutations in the regulatory phosphatase PTEN (28Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4232) Google Scholar). In addition, PP2A or PHLPPs play key roles in tumorigenesis.Growth factors and cytokines activate PI3K to initiate Akt signaling. PI3K phosphorylates phosphatidylinositol to generate the lipid phosphatidylinositol 3,4,5-trisphosphate, which binds the pleckstrin homology domains of the three Akt isoforms (Akt1, -2, and -3) and phosphoinositide-dependent kinase 1, recruiting them to the membrane. The Akt isoforms are activated by phosphoinositide-dependent kinase 1 phosphorylation of their activation loop (Thr-308) and phosphoinositide-dependent kinase 2 phosphorylation of their hydrophobic motif (Ser-473). Akt1 is ubiquitously expressed; controls organism size, adipogenesis, and skeletal muscle differentiation; and inhibits cell motility. The Akt2 expression is elevated in insulin-responsive tissues, such as muscle and liver. Akt2 is required for glucose homeostasis and promotes cell motility. Akt3 expression is more specific to neural tissue and is required for the development to the normal neuronal cell size (29Stambolic V. Woodgett J.R. Trends. Cell Biol. 2006; 16: 461-466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Akt activity is directly repressed by PHLPP1 and PHLPP2 (14Gao T. Furnari F. Newton A.C. Mol. Cell. 2005; 18: 13-24Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar), which specifically dephosphorylate the hydrophobic motif (Ser-473) of Akt isoforms. This dephosphorylation decreases the activity of Akt isoforms, increases apoptosis, and inhibits cell proliferation. Coimmunoprecipitation studies have shown that an interaction between PHLPP1 and Akt2 or Akt3 influences their phosphorylation state, whereas PHLPP2 affects the phosphorylation of Akt1 and Akt3 (13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Depletion of PHLPP1 not only increases the phosphorylation state of many Akt substrates (glycogen synthase kinase-3β, tuberin (TSC2), and forkhead box O) but also increases the phosphorylation state of a unique set of Akt substrates including the E3 ligase, HDM2 (human homologue to murine double minute 2), and glycogen synthase kinase-3α (15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Moreover depletion of PHLPP2 results in increased phosphorylation of glycogen synthase kinase-3β, TSC2, forkhead box O, and p27Kip1 (15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Thus, PHLPP1 and PHLPP2 inactivate multiple cancer-related signaling pathways. In the present study, we found that treatment with Abl kinase inhibitors or depletion of Bcr-Abl induced the expression of both PHLPP1 and PHLPP2 in CML cells and that this expression of PHLPP1 and PHLPP2 in CML cells inhibited their proliferation.The development of Abl kinase inhibitors, such as STI571, AMN107, and BMS354825, has provided a beneficial treatment for CML patients as well as a new tool for studying the effect of inhibition of the Abl kinase activity in cells harboring the endogenous Bcr-Abl gene (30Andreu E.J. Lledo E. Poch E. Ivorra C. Albero M.P. Martinez-Climent J.A. Montiel-Duarte C. Rifon J. Perez-Calvo J. Arbona C. Prosper F. Perez-Roger I. Cancer Res. 2005; 65: 3264-3272Crossref PubMed Scopus (104) Google Scholar). In both Bcr-Abl+ cells and primary CML CD34+ cells, STI571 inhibition of Bcr-Abl tyrosine kinase activity results in a G1 cell cycle arrest mediated by the PI3K pathway. A study that used inhibitors of both Bcr-Abl and PI3K found that the decrease in the p27Kip1 protein levels in Bcr-Abl+ cells is due to regulation at the levels of transcription and degradation by activating the PI3K pathway (31Klejman A. Rushen L. Morrione A. Slupianek A. Skorski T. Oncogene. 2002; 21: 5868-5876Crossref PubMed Scopus (138) Google Scholar).The PI3K signaling pathway is deregulated in many human cancers and is considered an attractive target for the development of novel chemotherapeutic agents. The PI3K pathway contributes to transformation by Bcr-Abl, and PI3K inhibitors synergize with Abl kinase inhibitors (imatinib) to greatly increase apoptosis of CML cells from chronic phase and blast crisis patients (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). The PI3K effecter most closely associated with cell transformation is Akt, and the substrates of activated Akt regulate the cell cycle, growth, metabolism, and survival. Our study may indicate that Akt isoforms phosphorylated by activated PI3K are maintained by the Bcr-Abl-mediated suppression of PHLPP1 and PHLPP2 in CML cells. These mechanisms were clarified by our study. We found that the expression of PHLPP1 and PHLPP2 mRNA and protein was increased by Abl kinase inhibitors or depletion of Bcr-Abl expression. Moreover in K562 and Meg01 cells, the three Akt isoforms were constitutively phosphorylated on Ser-473, and the Abl kinase inhibitors reduced the phosphorylation of Ser-473 on Akt1, -2, and -3. We also showed that the depletion of PHLPP1 inhibited the phosphorylation of Ser-473 on AKt2 and Akt3, and the depletion of PHLPP2 inhibited the phosphorylation of Ser-473 on Akt1 and Akt3.We investigated the effects of depletion of PHLPP1 and PHLPP2 in CML cells. In both K562 and Meg01 cells, the cell proliferation was remarkably inhibited by treatment with STI571, AMN107, and BMS354825, whereas cell proliferation was moderately inhibited when these cells transfected with PHLPP1 and/or PHLPP2 siRNA were treated with STI571, AMN107, and BMS354825. These results reveal that PHLPP1 and PHLPP2 have an important role in the inhibition of CML cell proliferation by the Abl kinase inhibitors.The CFU-GEMM, CFU-GM, and BFU-E derived from normal progenitor cells were moderately reduced when they were treated with STI571, AMN107, or BNM354825 (data not shown). In contrast, the CFU-GEMM, CFU-GM, and BFU-E derived from CML progenitor cells were significantly reduced when they were treated with STI571, AMN107, and BMS354825 or transfected with Bcr-Abl siRNA. Moreover the relative expression of PHLPP1 and PHLPP2 mRNA increased in CFU-GEMM, CFU-GM, and BFU-E treated with the Abl kinase inhibitors or transfected with Bcr-Abl siRNA, respectively. In CFU-GEMM, CFU-GM, and BFU-E transfected with PHLPP1 and/or PHLPP2 siRNA, the inhibition effects of CFU by the Abl kinase inhibitors were weakened. These results indicate that the increased expression of PHLPP1 and PHLPP2 induced by the Abl kinase reduced the committed colony-forming cells derived from CML progenitor cells. These findings indicate that Bcr-Abl inhibited the expression of PHLPP1 and PHLPP2, resulting in continuously phosphorylated Ser-473 on Akt1, -2, and -3 and the promotion of Bcr-Abl+ progenitor cell proliferation.In conclusion, this study shows for the first time that Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and that the Abl kinase inhibitors induced both PHLPPs. These induced PHLPPs play an important role in cell growth inhibition in CML cells in vitro. Chronic myelogenous leukemia (CML) 2The abbreviations used are: CMLchronic myelogenous leukemiaPHLPPpleckstrin homology domain leucine-rich repeat protein phosphataseCFU-GEMMcolony-forming unit-granulocyte, erythroid, macrophage, megakaryocyteCFU-GMcolony-forming unit-granulocyte, macrophageBFU-Eburst-forming unit-erythroidPP2Athe serine/threonine protein phosphatase 2API3Kphosphatidylinositol 3-kinasePTENphosphatase and tensin homolog deleted on chromosome 10siRNAshort interfering RNAAMLacute myeloblastic leukemiaALDHaldehyde dehydrogenaseRT-PCRreal time PCRG3PDHglyceraldehyde-3-phosphate dehydrogenaseMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideE3ubiquitin-protein isopeptide ligase. 2The abbreviations used are: CMLchronic myelogenous leukemiaPHLPPpleckstrin homology domain leucine-rich repeat protein phosphataseCFU-GEMMcolony-forming unit-granulocyte, erythroid, macrophage, megakaryocyteCFU-GMcolony-forming unit-granulocyte, macrophageBFU-Eburst-forming unit-erythroidPP2Athe serine/threonine protein phosphatase 2API3Kphosphatidylinositol 3-kinasePTENphosphatase and tensin homolog deleted on chromosome 10siRNAshort interfering RNAAMLacute myeloblastic leukemiaALDHaldehyde dehydrogenaseRT-PCRreal time PCRG3PDHglyceraldehyde-3-phosphate dehydrogenaseMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideE3ubiquitin-protein isopeptide ligase. is a hematopoietic stem cell disorder that is characterized by the Philadelphia chromosome (1Kurzrock R. Gutterman J.U. Talpaz M. N. Engl. J. Med. 1988; 319: 990-998Crossref PubMed Scopus (720) Google Scholar, 2Rudkin C.T. Nowell P.C. Hungerford D.A. Science. 1964; 144: 1229-1231Crossref PubMed Scopus (56) Google Scholar), a shortened chromosome 22 that is a by-product of a reciprocal chromosomal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11), resulting in a chimeric Bcr-Abl oncoprotein with highly deregulated, constitutive tyrosine kinase activity (3Shtivelman E. Lifshitz B. Gale R.P. Ganaani E. Nature. 1985; 315: 550-554Crossref PubMed Scopus (1249) Google Scholar, 4Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1115) Google Scholar). The most commonly occurring form of Bcr-Abl is a 210-kDa protein that is a critical role in the pathogenesis of CML (5Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1913) Google Scholar). As a result of its elevated tyrosine kinase activity, Bcr-Abl activates a multitude of signaling pathways, including the Ras (6Sawyers C.L. McLaughlin J. Witte O.N. J. Exp. Med. 1995; 181: 307-313Crossref PubMed Scopus (245) Google Scholar), PI3K/Akt (7Skorski T. Bellacosa A. Nieborowska-Skorska M. Majewski M. Martinez R. Choi J.K. Trotta R. Wlodarski P. Perrotti D. Chan T.O. Wasik M.A. Tsichlis P.N. Calabretta B. EMBO J. 1997; 16: 6151-6161Crossref PubMed Scopus (552) Google Scholar, 8Varticovski L. Daley G.Q. Jackson P. Baltimore D. Cantley L.C. Mol. Cell. Biol. 1991; 11: 1107-1113Crossref PubMed Scopus (180) Google Scholar), Janus kinase/signal transducer and activator of transcription (9Carlesso N. Frank D.A. Griffin J.D. J. Exp. Med. 1996; 183: 811-820Crossref PubMed Scopus (429) Google Scholar), and NF-κB (10Reuther J.Y. Reuther G.W. Cortez D. Pendergast A.M. Baldwin Jr., A.S. Genes Dev. 1998; 12: 968-981Crossref PubMed Scopus (350) Google Scholar) signaling pathways. Furthermore the serine/threonine protein phosphatase PP2A is also functionally inactivated by Bcr-Abl (11Neviani P. Santhanam R. Trotta R. Notari M. Blaser B.W. Liu S. Mao H. Chang J.S. Galietta A. Uttam A. Roy D.C. Valtieri M. Bruner-Klisovic R. Caligiuri M.A. Bloomfield C.D. Marcucci G. Perrotti D. Cancer Cell. 2005; 8: 355-368Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). These signaling pathways, especially the Bcr-Abl-induced PI3K/Akt activation, play a role in Bcr-Abl-mediated leukemogenesis. chronic myelogenous leukemia pleckstrin homology domain leucine-rich repeat protein phosphatase colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte colony-forming unit-granulocyte, macrophage burst-forming unit-erythroid the serine/threonine protein phosphatase 2A phosphatidylinositol 3-kinase phosphatase and tensin homolog deleted on chromosome 10 short interfering RNA acute myeloblastic leukemia aldehyde dehydrogenase real time PCR glyceraldehyde-3-phosphate dehydrogenase 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ubiquitin-protein isopeptide ligase. chronic myelogenous leukemia pleckstrin homology domain leucine-rich repeat protein phosphatase colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte colony-forming unit-granulocyte, macrophage burst-forming unit-erythroid the serine/threonine protein phosphatase 2A phosphatidylinositol 3-kinase phosphatase and tensin homolog deleted on chromosome 10 short interfering RNA acute myeloblastic leukemia aldehyde dehydrogenase real time PCR glyceraldehyde-3-phosphate dehydrogenase 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ubiquitin-protein isopeptide ligase. PP2A is inactivated in blast crisis CML through Bcr-Abl-mediated transcriptional up-regulation of the PP2A inhibitor SET. Hyperphosphorylation and inactivation of proapoptotic PP2A substrate kinases, such as phospho-Akt and phospho-ERK (extracellular signal-regulated kinase), lead to their prolonged activation and ability to drive the survival and proliferative signaling pathway (11Neviani P. Santhanam R. Trotta R. Notari M. Blaser B.W. Liu S. Mao H. Chang J.S. Galietta A. Uttam A. Roy D.C. Valtieri M. Bruner-Klisovic R. Caligiuri M.A. Bloomfield C.D. Marcucci G. Perrotti D. Cancer Cell. 2005; 8: 355-368Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). The inactivation of PP2A is essential for Bcr-Abl-mediated leukemogenesis and blastic transformation. Another serine/threonine protein phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP), terminates Akt signaling by dephosphorylating the hydrophobic motif on Akt (12Gao T. Brognard J. Newton A.C. J. Biol. Chem. 2008; 283: 6300-6311Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The PHLPP family of phosphatases includes PHLPP1α (1205 amino acids), PHLPP1β (1717 amino acids), and PHLPP2 (1323 amino acids) (14Gao T. Furnari F. Newton A.C. Mol. Cell. 2005; 18: 13-24Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar, 15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). PHLPP1α and -β are variants spliced from the same gene located at chromosome 18q21.33 (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar), and they differ by a 56-kDa N-terminal extension (13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The gene encoding PHLPP2 resides at 16q22.3 (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). PHLPP1 and PHLPP2 own a pleckstrin homology domain with a region of leucine-rich repeats, a PP2C phosphatase domain, and a C-terminal PDZ ligand (13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The genes encoding PHLPP1 and PHLPP2 were frequently lost in various cancers such as colon (16Goel A. Arnold C.N. Niedzwiecki D. Chang D.K. Ricciardiello L. Carethers J.M. Dowell J.M. Wasserman L. Compton C. Mayer R.J. Bertagnolli M.M. Boland C.R. Cancer Res. 2003; 63: 1608-1614PubMed Google Scholar), breast (17Rakha E.A. Genes Chromosomes Cancer. 2006; 45: 527-535Crossref PubMed Scopus (69) Google Scholar), and ovarian cancers (18Patael-Karasik Y. Daniely M. Gotlieb W.H. Ben-Baruch G. Schiby J. Barakai G. Goldman B. Aviram A. Friedman E. Cancer Genet. Cytogenet. 2000; 121: 26-32Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), Wilms tumors (19Safford S.D. Goyeau D. Freemerman A.J. Bentley R. Everett M.L. Grundy P.E. Skinner M.A. Ann. Surg. Oncol. 2003; 10: 136-143Crossref PubMed Scopus (26) Google Scholar), prostate cancer (20Torring N. Borre M. Sorensen K.D. Andersen C.L. Wiuf C. Orntoft T.F. Br. J. Cancer. 2007; 96: 499-506Crossref PubMed Scopus (48) Google Scholar), and hepatocellular carcinomas (21Tsuda H. Zhang W.D. Shimosato Y. Yokota J. Terada M. Sugimura T. Miyamura T. Hirohashi S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6791-6794Crossref PubMed Scopus (281) Google Scholar). PHLPP1 and PHLPP2 are present in the cytosolic, nuclear, and membrane fraction of cells and are expressed in cell lines including brain, breast, lung, prostate, and ovarian cancer cell lines (15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). PHLPP1 and PHLPP2 decrease activity of Akt and increase apoptosis and inhibition of cell proliferation through the dephosphorylation of the hydrophobic motif (Ser-473) in Akt. Depletion of either PHLPP1 or PHLPP2 causes a 30-fold increase in Akt phosphorylation after EGF stimulation in a normal breast cell line (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). Knockdown studies have revealed that PHLPP1 influences the phosphorylation state of Akt2 and Akt3, whereas PHLPP2 affects the phosphorylation state of Akt1 and Akt3. In the PI3K/Akt signaling pathway, defective PTEN activates Akt signaling by preventing conversion of phosphatidylinositol 3,4,5-trisphosphate back to phosphatidylinositol 4,5-bisphosphate and contributes to retaining Akt phosphorylation. Nonetheless there are many examples of elevated Akt phosphorylation in cancer cells that have intact PTEN expression. PHLPP levels are markedly reduced in some cancer cell lines that have elevated Akt phosphorylation, and the reintroduction of PHLPP reduces cell growth (23Mendoza M.C. Blenis J. Mol. Cell. 2007; 25: 798-800Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, in Bcr-Abl-mediated leukemogenesis, the role of PLHPP remains unclear. In the present study, we found that Bcr-Abl suppressed the expression of PHLPP1 and PHLPP2. We analyzed the role of PHLPP1 and PHLPP2 in CML cell lines and the hematopoietic progenitor cells derived from CML patients by inhibiting the expression of PHLPP1 and PHLPP2. We detected a change in Ser-473 upon Akt phosphorylation with CML-derived hematopoietic progenitor cells exhibiting more Akt phosphorylation than normal hematopoietic progenitor cells. DISCUSSIONWe investigated the role of PHLPP expression on the induction of apoptosis or growth inhibition of CML cells treated with Abl kinase inhibitors. We demonstrated that Bcr-Abl inhibited the expression of PHLPP1 and PHLPP2 and promoted the CML cell progression through continuous phospho-Akt activation.The PI3K/Akt pathway is a central node in determining cellular fate. Hyperactivation or continuous activation of this pathway promotes cell survival, growth, and proliferation (24Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5073) Google Scholar). This pathway is activated in cancer by mechanisms including amplification or gain-of-function mutations in upstream receptor protein-tyrosine kinases (25Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3097) Google Scholar, 26Marx J.L. Science. 1989; 244: 654-655Crossref PubMed Scopus (4) Google Scholar), activating mutations in PI3K and Akt (27Carpten J.D. Faber A.L. Horn C. Donoho G.P. Briggs S.L. Robbins C.M. Hostetter G. Boguslawski S. Moses T.Y. Savage S. Uhlik M. Lin A. Du J. Qian Y.W. Zeckner D.J. Tucker-Kellogg G. Touchman J. Patel K. Mousses S. Bittner M. Schevitz R. Lai M.H. Blanchard K.L. Thomas J.E. Nature. 2007; 448: 439-444Crossref PubMed Scopus (990) Google Scholar), and loss-of-function mutations in the regulatory phosphatase PTEN (28Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4232) Google Scholar). In addition, PP2A or PHLPPs play key roles in tumorigenesis.Growth factors and cytokines activate PI3K to initiate Akt signaling. PI3K phosphorylates phosphatidylinositol to generate the lipid phosphatidylinositol 3,4,5-trisphosphate, which binds the pleckstrin homology domains of the three Akt isoforms (Akt1, -2, and -3) and phosphoinositide-dependent kinase 1, recruiting them to the membrane. The Akt isoforms are activated by phosphoinositide-dependent kinase 1 phosphorylation of their activation loop (Thr-308) and phosphoinositide-dependent kinase 2 phosphorylation of their hydrophobic motif (Ser-473). Akt1 is ubiquitously expressed; controls organism size, adipogenesis, and skeletal muscle differentiation; and inhibits cell motility. The Akt2 expression is elevated in insulin-responsive tissues, such as muscle and liver. Akt2 is required for glucose homeostasis and promotes cell motility. Akt3 expression is more specific to neural tissue and is required for the development to the normal neuronal cell size (29Stambolic V. Woodgett J.R. Trends. Cell Biol. 2006; 16: 461-466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Akt activity is directly repressed by PHLPP1 and PHLPP2 (14Gao T. Furnari F. Newton A.C. Mol. Cell. 2005; 18: 13-24Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar), which specifically dephosphorylate the hydrophobic motif (Ser-473) of Akt isoforms. This dephosphorylation decreases the activity of Akt isoforms, increases apoptosis, and inhibits cell proliferation. Coimmunoprecipitation studies have shown that an interaction between PHLPP1 and Akt2 or Akt3 influences their phosphorylation state, whereas PHLPP2 affects the phosphorylation of Akt1 and Akt3 (13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Depletion of PHLPP1 not only increases the phosphorylation state of many Akt substrates (glycogen synthase kinase-3β, tuberin (TSC2), and forkhead box O) but also increases the phosphorylation state of a unique set of Akt substrates including the E3 ligase, HDM2 (human homologue to murine double minute 2), and glycogen synthase kinase-3α (15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Moreover depletion of PHLPP2 results in increased phosphorylation of glycogen synthase kinase-3β, TSC2, forkhead box O, and p27Kip1 (15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Thus, PHLPP1 and PHLPP2 inactivate multiple cancer-related signaling pathways. In the present study, we found that treatment with Abl kinase inhibitors or depletion of Bcr-Abl induced the expression of both PHLPP1 and PHLPP2 in CML cells and that this expression of PHLPP1 and PHLPP2 in CML cells inhibited their proliferation.The development of Abl kinase inhibitors, such as STI571, AMN107, and BMS354825, has provided a beneficial treatment for CML patients as well as a new tool for studying the effect of inhibition of the Abl kinase activity in cells harboring the endogenous Bcr-Abl gene (30Andreu E.J. Lledo E. Poch E. Ivorra C. Albero M.P. Martinez-Climent J.A. Montiel-Duarte C. Rifon J. Perez-Calvo J. Arbona C. Prosper F. Perez-Roger I. Cancer Res. 2005; 65: 3264-3272Crossref PubMed Scopus (104) Google Scholar). In both Bcr-Abl+ cells and primary CML CD34+ cells, STI571 inhibition of Bcr-Abl tyrosine kinase activity results in a G1 cell cycle arrest mediated by the PI3K pathway. A study that used inhibitors of both Bcr-Abl and PI3K found that the decrease in the p27Kip1 protein levels in Bcr-Abl+ cells is due to regulation at the levels of transcription and degradation by activating the PI3K pathway (31Klejman A. Rushen L. Morrione A. Slupianek A. Skorski T. Oncogene. 2002; 21: 5868-5876Crossref PubMed Scopus (138) Google Scholar).The PI3K signaling pathway is deregulated in many human cancers and is considered an attractive target for the development of novel chemotherapeutic agents. The PI3K pathway contributes to transformation by Bcr-Abl, and PI3K inhibitors synergize with Abl kinase inhibitors (imatinib) to greatly increase apoptosis of CML cells from chronic phase and blast crisis patients (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). The PI3K effecter most closely associated with cell transformation is Akt, and the substrates of activated Akt regulate the cell cycle, growth, metabolism, and survival. Our study may indicate that Akt isoforms phosphorylated by activated PI3K are maintained by the Bcr-Abl-mediated suppression of PHLPP1 and PHLPP2 in CML cells. These mechanisms were clarified by our study. We found that the expression of PHLPP1 and PHLPP2 mRNA and protein was increased by Abl kinase inhibitors or depletion of Bcr-Abl expression. Moreover in K562 and Meg01 cells, the three Akt isoforms were constitutively phosphorylated on Ser-473, and the Abl kinase inhibitors reduced the phosphorylation of Ser-473 on Akt1, -2, and -3. We also showed that the depletion of PHLPP1 inhibited the phosphorylation of Ser-473 on AKt2 and Akt3, and the depletion of PHLPP2 inhibited the phosphorylation of Ser-473 on Akt1 and Akt3.We investigated the effects of depletion of PHLPP1 and PHLPP2 in CML cells. In both K562 and Meg01 cells, the cell proliferation was remarkably inhibited by treatment with STI571, AMN107, and BMS354825, whereas cell proliferation was moderately inhibited when these cells transfected with PHLPP1 and/or PHLPP2 siRNA were treated with STI571, AMN107, and BMS354825. These results reveal that PHLPP1 and PHLPP2 have an important role in the inhibition of CML cell proliferation by the Abl kinase inhibitors.The CFU-GEMM, CFU-GM, and BFU-E derived from normal progenitor cells were moderately reduced when they were treated with STI571, AMN107, or BNM354825 (data not shown). In contrast, the CFU-GEMM, CFU-GM, and BFU-E derived from CML progenitor cells were significantly reduced when they were treated with STI571, AMN107, and BMS354825 or transfected with Bcr-Abl siRNA. Moreover the relative expression of PHLPP1 and PHLPP2 mRNA increased in CFU-GEMM, CFU-GM, and BFU-E treated with the Abl kinase inhibitors or transfected with Bcr-Abl siRNA, respectively. In CFU-GEMM, CFU-GM, and BFU-E transfected with PHLPP1 and/or PHLPP2 siRNA, the inhibition effects of CFU by the Abl kinase inhibitors were weakened. These results indicate that the increased expression of PHLPP1 and PHLPP2 induced by the Abl kinase reduced the committed colony-forming cells derived from CML progenitor cells. These findings indicate that Bcr-Abl inhibited the expression of PHLPP1 and PHLPP2, resulting in continuously phosphorylated Ser-473 on Akt1, -2, and -3 and the promotion of Bcr-Abl+ progenitor cell proliferation.In conclusion, this study shows for the first time that Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and that the Abl kinase inhibitors induced both PHLPPs. These induced PHLPPs play an important role in cell growth inhibition in CML cells in vitro. We investigated the role of PHLPP expression on the induction of apoptosis or growth inhibition of CML cells treated with Abl kinase inhibitors. We demonstrated that Bcr-Abl inhibited the expression of PHLPP1 and PHLPP2 and promoted the CML cell progression through continuous phospho-Akt activation. The PI3K/Akt pathway is a central node in determining cellular fate. Hyperactivation or continuous activation of this pathway promotes cell survival, growth, and proliferation (24Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5073) Google Scholar). This pathway is activated in cancer by mechanisms including amplification or gain-of-function mutations in upstream receptor protein-tyrosine kinases (25Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3097) Google Scholar, 26Marx J.L. Science. 1989; 244: 654-655Crossref PubMed Scopus (4) Google Scholar), activating mutations in PI3K and Akt (27Carpten J.D. Faber A.L. Horn C. Donoho G.P. Briggs S.L. Robbins C.M. Hostetter G. Boguslawski S. Moses T.Y. Savage S. Uhlik M. Lin A. Du J. Qian Y.W. Zeckner D.J. Tucker-Kellogg G. Touchman J. Patel K. Mousses S. Bittner M. Schevitz R. Lai M.H. Blanchard K.L. Thomas J.E. Nature. 2007; 448: 439-444Crossref PubMed Scopus (990) Google Scholar), and loss-of-function mutations in the regulatory phosphatase PTEN (28Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4232) Google Scholar). In addition, PP2A or PHLPPs play key roles in tumorigenesis. Growth factors and cytokines activate PI3K to initiate Akt signaling. PI3K phosphorylates phosphatidylinositol to generate the lipid phosphatidylinositol 3,4,5-trisphosphate, which binds the pleckstrin homology domains of the three Akt isoforms (Akt1, -2, and -3) and phosphoinositide-dependent kinase 1, recruiting them to the membrane. The Akt isoforms are activated by phosphoinositide-dependent kinase 1 phosphorylation of their activation loop (Thr-308) and phosphoinositide-dependent kinase 2 phosphorylation of their hydrophobic motif (Ser-473). Akt1 is ubiquitously expressed; controls organism size, adipogenesis, and skeletal muscle differentiation; and inhibits cell motility. The Akt2 expression is elevated in insulin-responsive tissues, such as muscle and liver. Akt2 is required for glucose homeostasis and promotes cell motility. Akt3 expression is more specific to neural tissue and is required for the development to the normal neuronal cell size (29Stambolic V. Woodgett J.R. Trends. Cell Biol. 2006; 16: 461-466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Akt activity is directly repressed by PHLPP1 and PHLPP2 (14Gao T. Furnari F. Newton A.C. Mol. Cell. 2005; 18: 13-24Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar), which specifically dephosphorylate the hydrophobic motif (Ser-473) of Akt isoforms. This dephosphorylation decreases the activity of Akt isoforms, increases apoptosis, and inhibits cell proliferation. Coimmunoprecipitation studies have shown that an interaction between PHLPP1 and Akt2 or Akt3 influences their phosphorylation state, whereas PHLPP2 affects the phosphorylation of Akt1 and Akt3 (13Brognard J. Newton A.C. Trends Endocrinol. Metab. 2008; 19: 223-230Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Depletion of PHLPP1 not only increases the phosphorylation state of many Akt substrates (glycogen synthase kinase-3β, tuberin (TSC2), and forkhead box O) but also increases the phosphorylation state of a unique set of Akt substrates including the E3 ligase, HDM2 (human homologue to murine double minute 2), and glycogen synthase kinase-3α (15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Moreover depletion of PHLPP2 results in increased phosphorylation of glycogen synthase kinase-3β, TSC2, forkhead box O, and p27Kip1 (15Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Thus, PHLPP1 and PHLPP2 inactivate multiple cancer-related signaling pathways. In the present study, we found that treatment with Abl kinase inhibitors or depletion of Bcr-Abl induced the expression of both PHLPP1 and PHLPP2 in CML cells and that this expression of PHLPP1 and PHLPP2 in CML cells inhibited their proliferation. The development of Abl kinase inhibitors, such as STI571, AMN107, and BMS354825, has provided a beneficial treatment for CML patients as well as a new tool for studying the effect of inhibition of the Abl kinase activity in cells harboring the endogenous Bcr-Abl gene (30Andreu E.J. Lledo E. Poch E. Ivorra C. Albero M.P. Martinez-Climent J.A. Montiel-Duarte C. Rifon J. Perez-Calvo J. Arbona C. Prosper F. Perez-Roger I. Cancer Res. 2005; 65: 3264-3272Crossref PubMed Scopus (104) Google Scholar). In both Bcr-Abl+ cells and primary CML CD34+ cells, STI571 inhibition of Bcr-Abl tyrosine kinase activity results in a G1 cell cycle arrest mediated by the PI3K pathway. A study that used inhibitors of both Bcr-Abl and PI3K found that the decrease in the p27Kip1 protein levels in Bcr-Abl+ cells is due to regulation at the levels of transcription and degradation by activating the PI3K pathway (31Klejman A. Rushen L. Morrione A. Slupianek A. Skorski T. Oncogene. 2002; 21: 5868-5876Crossref PubMed Scopus (138) Google Scholar). The PI3K signaling pathway is deregulated in many human cancers and is considered an attractive target for the development of novel chemotherapeutic agents. The PI3K pathway contributes to transformation by Bcr-Abl, and PI3K inhibitors synergize with Abl kinase inhibitors (imatinib) to greatly increase apoptosis of CML cells from chronic phase and blast crisis patients (22Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). The PI3K effecter most closely associated with cell transformation is Akt, and the substrates of activated Akt regulate the cell cycle, growth, metabolism, and survival. Our study may indicate that Akt isoforms phosphorylated by activated PI3K are maintained by the Bcr-Abl-mediated suppression of PHLPP1 and PHLPP2 in CML cells. These mechanisms were clarified by our study. We found that the expression of PHLPP1 and PHLPP2 mRNA and protein was increased by Abl kinase inhibitors or depletion of Bcr-Abl expression. Moreover in K562 and Meg01 cells, the three Akt isoforms were constitutively phosphorylated on Ser-473, and the Abl kinase inhibitors reduced the phosphorylation of Ser-473 on Akt1, -2, and -3. We also showed that the depletion of PHLPP1 inhibited the phosphorylation of Ser-473 on AKt2 and Akt3, and the depletion of PHLPP2 inhibited the phosphorylation of Ser-473 on Akt1 and Akt3. We investigated the effects of depletion of PHLPP1 and PHLPP2 in CML cells. In both K562 and Meg01 cells, the cell proliferation was remarkably inhibited by treatment with STI571, AMN107, and BMS354825, whereas cell proliferation was moderately inhibited when these cells transfected with PHLPP1 and/or PHLPP2 siRNA were treated with STI571, AMN107, and BMS354825. These results reveal that PHLPP1 and PHLPP2 have an important role in the inhibition of CML cell proliferation by the Abl kinase inhibitors. The CFU-GEMM, CFU-GM, and BFU-E derived from normal progenitor cells were moderately reduced when they were treated with STI571, AMN107, or BNM354825 (data not shown). In contrast, the CFU-GEMM, CFU-GM, and BFU-E derived from CML progenitor cells were significantly reduced when they were treated with STI571, AMN107, and BMS354825 or transfected with Bcr-Abl siRNA. Moreover the relative expression of PHLPP1 and PHLPP2 mRNA increased in CFU-GEMM, CFU-GM, and BFU-E treated with the Abl kinase inhibitors or transfected with Bcr-Abl siRNA, respectively. In CFU-GEMM, CFU-GM, and BFU-E transfected with PHLPP1 and/or PHLPP2 siRNA, the inhibition effects of CFU by the Abl kinase inhibitors were weakened. These results indicate that the increased expression of PHLPP1 and PHLPP2 induced by the Abl kinase reduced the committed colony-forming cells derived from CML progenitor cells. These findings indicate that Bcr-Abl inhibited the expression of PHLPP1 and PHLPP2, resulting in continuously phosphorylated Ser-473 on Akt1, -2, and -3 and the promotion of Bcr-Abl+ progenitor cell proliferation. In conclusion, this study shows for the first time that Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and that the Abl kinase inhibitors induced both PHLPPs. These induced PHLPPs play an important role in cell growth inhibition in CML cells in vitro. Retraction: Depletion of pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 by Bcr-Abl promotes chronic myelogenous leukemia cell proliferation through continuous phosphorylation of Akt isoforms.Journal of Biological ChemistryVol. 285Issue 20PreviewVOLUME 284 (2009) PAGES 22155–22165 Full-Text PDF Open Access
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