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

Kaori Shinjo,Kazunori Ohnishi,Isao Hirano,Kazuyuki Shigeno,Daisuke Yokota,Satoki Nakamura,Shinya Fujisawa,Takaaki Ono

doi:10.1074/jbc.m808182200

Abstract

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.

Highlights

PP2A is inactivated in blast crisis Chronic myelogenous leukemia (CML) through Bcr-Ablmediated transcriptional up-regulation of the PP2A inhibitor SET
MRNA Expression in CML Cell Lines—PHLPP1 and PHLPP2 mRNAs were constitutively expressed in CML cell lines (K562, Meg01, and SHG3 cells) and acute myeloblastic leukemia (AML) cell lines (U937, HL60, and YRK2 cells) (Fig. 1A)
We found that the mRNA expression of PHLPP1 and PHLPP2 increased in the three CML cell lines treated with Abl kinase inhibitors (STI571, AMN107, or BMS354825) for 24 h as compared with untreated cells

Summary

EXPERIMENTAL PROCEDURES

Reagents—Imatinib mesylate (STI571) and AMN107 were kindly provided by Novartis Pharmaceuticals (Basel, Switzerland). To measure isoform-specific Ser-473 phosphorylations, Akt, Akt, or Akt was immunoprecipitated from 400 ␮g of protein lysates prepared from K562 and Meg cells treated with STI571 (10 ␮M), AMN107 (10 ␮M), or BMS354825 (10 nM) for 30 min or transfected with PHLPP1 and PHLPP2 siRNA. Immunofluorescence Microscopy—ALDHhi cells from CML bone marrow cells were transfected with control siRNA and Bcr-Abl siRNA. C, expression of PHLPP1, PHLPP2, and Bcr-Abl mRNA in ALDHhi cells isolated from the bone marrow of a CML patient. The effects of the Abl kinase inhibitors on PHLPP1 (upper right) and PHLPP2 (lower right) expression were assessed by RT-PCR in ALDHhi cells from CML patients (n ϭ 4). The means were compared by using the Student’s t test. p values less than 0.05 were considered statistically significant

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS