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Abstract
Shp2 has been known to mediate growth factor-stimulated cell proliferation, but its role in cell survival is less clear. Gain-of-function Shp2 mutants such as Shp2E76K are associated with myeloid leukemias. We found that Shp2E76K could transform cytokine-dependent human TF-1 myeloid cells into cytokine independence and further characterized the Shp2E76K-induced cell survival mechanism in this study. Expression of Shp2E76K suppressed the cytokine withdrawal-induced intrinsic/mitochondrial apoptosis pathway, which is controlled by the Bcl-2 family proteins. Analysis of Bcl-2 family proteins showed that Bcl-XL and Mcl-1 were up-regulated in Shp2E76K-transformed TF-1 (TF-1/Shp2E76K) cells. Knockdown of Bcl-XL but not Mcl-1 with short hairpin RNAs prevented Shp2E76K-induced cytokine-independent survival. Roscovitine, which down-regulated Mcl-1, also did not prevent cytokine-independent survival of TF-1/Shp2E76K cells, whereas the Bcl-XL inhibitor HA14-1 did. Ras and mitogen-activated protein kinases Erk1 and Erk2 (Erk1/2) were constitutively activated in TF-1/Shp2E76K cells, whereas little active Akt was detected under cytokine-free conditions. Shp2E76K-induced Bcl-XL expression was suppressed by Mek inhibitors and by a dominant-negative Mek1 mutant but not by the phosphoinositide 3-phosphate inhibitor LY294002 and the Akt inhibitor API-2. Inhibition of Erk1/2 blocked cytokine-independent survival of TF-1/Shp2E76K cells, whereas inhibition of Akt had a minimal effect on cytokine-independent survival of TF-1/Shp2E76K cells. These results show that Shp2E76K can evoke constitutive Erk1/2 activation in TF-1 cells. Furthermore, Shp2E76K induces cytokine-independent survival of TF-1 cells by a novel mechanism involving up-regulation of Bcl-XL through the Erk1/2 pathway.
Highlights
Shp2 is a non-receptor protein-tyrosine phosphatase (PTP)2 encoded by the PTPN11 gene (1)
It was reported that murine bone marrow or fetal liver cells transduced with retroviruses encoding the leukemia-associated Shp2E76K, Shp2D61Y, or Shp2D61V mutant could evoke cytokine-independent myeloid colonies and display hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) in methylcellulose cultures (22–24), suggesting that these Shp2 mutants protein; 7-AAD, 7-aminoactinomycin D; PARP, poly(ADP-ribose) polymerase; FBS, fetal bovine serum; shRNA, short hairpin RNA; Mek, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RNAi, RNA-mediated interference; HA, hemagglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
Immunoprecipitation of FLAGtagged Shp2 and Shp2E76K from GM-CSF-starved cells followed by PTP assay indicated that Shp2E76K had constitutively elevated PTP activity, which was ϳ25 times that detected in the wild type Shp2 (Fig. 1C)
Summary
Antibodies and Reagents—Monoclonal (M2) and polyclonal anti-FLAG antibodies, antibody to active Bax (6A7), and ␣-tubulin were from Sigma. 0.5 ml TF-1/Shp2E76K cells (1 ϫ 106 cells/ml) in RPMI 1640, 10% FBS, 10 ng/ml GM-CSF, 6 g/ml Polybrene were incubated with 0.5 ml of retrovirus in a 12-well plate and centrifuged at 2500 rpm at 30 °C for 1.5 h. Cells were transferred to 6-well plates containing 1 ml of RPMI 1640, 10% FBS, 5 ng/ml GM-CSF medium in each well and incubated for 24 h. Cells were washed twice with GM-CSF-free medium and incubated in 10 ml of RPMI 1640, 10% FBS for 4 days. Cells were washed and diluted with a 8ϫ volume of RPMI 1640, 10% FBS without GM-CSF and incubated for 4 days and analyzed for Mcl-1 knockdown and apoptosis. On day 7 cell colonies were stained with thiazolyl blue tetrazolium bromide and enumerated
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