Effects of a Leukemia-associated Gain-of-Function Mutation of SHP-2 Phosphatase on Interleukin-3 Signaling
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m507622200
ISSN1083-351X
AutoresWen-Mei Yu, Hanako Daino, Jing Chen, Kevin D. Bunting, Cheng‐Kui Qu,
Tópico(s)Galectins and Cancer Biology
ResumoMutations in SHP-2 phosphatase that cause hyperactivation of its catalytic activity have been identified in human leukemias, particularly juvenile myelomonocytic leukemia, which is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce hematopoietic malignancies are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in both catalytic-dependent and -independent manners and that overexpression (5–6-fold) of wild type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through accelerated dephosphorylation of STAT5. These results raised the possibility that SHP-2-associated leukemias are not solely attributed to the increased catalytic activity of GOF mutant SHP-2. GOF mutant SHP-2 must have gained additional capacities. To test this possibility, we investigated effects of a GOF mutation of SHP-2 (SHP-2 E76K) on hematopoietic cell function and IL-3 signal transduction by comparing with those of overexpressed WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. The E76K mutation in the N-terminal Src homology 2 domain increased interactions of mutant SHP-2 with Grb2, Gab2, and p85, leading to hyperactivation of IL-3-induced Erk and phosphatidylinositol 3-kinase (PI3K) pathways. In addition, despite the substantial increase in the catalytic activity, dephosphorylation of STAT5 by SHP-2 E76K was dampened. Furthermore, catalytically inactive SHP-2 E76K with an additional C459S mutation retained the capability to increase the interaction with Gab2 and to enhance the activation of the PI3K pathway. Taken together, these studies suggest that in addition to the elevated catalytic activity, fundamental changes in physical and functional interactions between GOF mutant SHP-2 and signaling partners also play an important role in SHP-2-related leukemigenesis. Mutations in SHP-2 phosphatase that cause hyperactivation of its catalytic activity have been identified in human leukemias, particularly juvenile myelomonocytic leukemia, which is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce hematopoietic malignancies are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in both catalytic-dependent and -independent manners and that overexpression (5–6-fold) of wild type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through accelerated dephosphorylation of STAT5. These results raised the possibility that SHP-2-associated leukemias are not solely attributed to the increased catalytic activity of GOF mutant SHP-2. GOF mutant SHP-2 must have gained additional capacities. To test this possibility, we investigated effects of a GOF mutation of SHP-2 (SHP-2 E76K) on hematopoietic cell function and IL-3 signal transduction by comparing with those of overexpressed WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. The E76K mutation in the N-terminal Src homology 2 domain increased interactions of mutant SHP-2 with Grb2, Gab2, and p85, leading to hyperactivation of IL-3-induced Erk and phosphatidylinositol 3-kinase (PI3K) pathways. In addition, despite the substantial increase in the catalytic activity, dephosphorylation of STAT5 by SHP-2 E76K was dampened. Furthermore, catalytically inactive SHP-2 E76K with an additional C459S mutation retained the capability to increase the interaction with Gab2 and to enhance the activation of the PI3K pathway. Taken together, these studies suggest that in addition to the elevated catalytic activity, fundamental changes in physical and functional interactions between GOF mutant SHP-2 and signaling partners also play an important role in SHP-2-related leukemigenesis. Hematopoietic cell fate is tightly controlled by environmental cues, such as growth factors, cytokines, and extracellular matrix, which exert their functions by activation of intracellular signaling mechanisms. Therefore, intracellular signaling processes play an important role in the determination of hematopoietic cell function. Dysregulation of signal transduction in hematopoietic cells by mutations in cell surface receptors or intracellular signaling components results in malfunction of hematopoietic cells, leading to blood disorders including leukemias. For instance, juvenile myelomonocytic leukemia (JMML), 2The abbreviations used are: JMML, juvenile myelomonocytic leukemia; NF1, neurofibromatosis type 1; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; PI3K, phosphatidylinositol 3-kinase; SH, Src homology; WT, wild type; GOF, gain-of-function; FBS, fetal bovine serum; Ab, antibody; BrdUrd, bromodeoxyuridine; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; PI, propidium iodide. 2The abbreviations used are: JMML, juvenile myelomonocytic leukemia; NF1, neurofibromatosis type 1; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; PI3K, phosphatidylinositol 3-kinase; SH, Src homology; WT, wild type; GOF, gain-of-function; FBS, fetal bovine serum; Ab, antibody; BrdUrd, bromodeoxyuridine; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; PI, propidium iodide. a clonal myeloproliferative disease characterized by overproduction of myeloid lineage cells, is thought to result from increased Ras signaling (1Arico M. Biondi A. Pui C.H. Blood. 1997; 90: 479-488Crossref PubMed Google Scholar, 2Emanuel P.D. Shannon K.M. Castleberry R.P. Mol. Med. Today. 1996; 2: 468-475Abstract Full Text PDF PubMed Scopus (72) Google Scholar). Activating mutations in the Ras gene or homozygous inactivation of the neurofibromatosis type 1 (NF1) gene, whose product, neurofibromin 1, is a Ras-GTPase-activating protein (Ras-GAP), have been identified in ∼50% of JMML (3Cichowski K. Santiago S. Jardim M. Johnson B.W. Jacks T. Genes Dev. 2003; 17: 449-454Crossref PubMed Scopus (108) Google Scholar). Due to activating Ras mutations or inactivation of NF1 mutations, hematopoietic progenitor cells in JMML are hypersensitive to granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 (4Birnbaum R.A. O'Marcaigh A. Wardak Z. Zhang Y.Y. Dranoff G. Jacks T. Clapp D.W. Shannon K.M. Mol. Cell. 2000; 5: 189-195Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5Zhang Y.Y. Vik T.A. Ryder J.W. Srour E.F. Jacks T. Shannon K. Clapp D.W. J. Exp. Med. 1998; 187: 1893-1902Crossref PubMed Scopus (127) Google Scholar). GM-CSF, IL-3, and IL-5 are a small family of hematopoietic growth factors that induce intracellular signal transduction mainly through the common receptor β chains (6Okuda K. Foster R. Griffin J.D. Ann. N. Y. Acad. Sci. 1999; 872: 305-313Crossref PubMed Scopus (30) Google Scholar, 7Reddy E.P. Korapati A. Chaturvedi P. Rane S. Oncogene. 2000; 19: 2532-2547Crossref PubMed Scopus (189) Google Scholar). They regulate hematopoietic cell survival, proliferation, and differentiation via shared intracellular signaling pathways. Although GM-CSF-, IL-3-, and IL-5-triggered signal transduction has been extensively investigated, the precise regulation of their signaling mechanisms is still not well understood. IL-3 exerts its function through binding to two distinct high affinity receptors composed of a ligand-specific α subunit and one of two β subunits: a ligand-specific β chain (Aic2A) or a common β chain (Aic2B). Upon binding by ligands, IL-3 receptors heterodimerize and become quickly tyrosyl phosphorylated. Activated receptors, in particular, the common receptor β chains, then trigger a signal relay to the targets in the nucleus to induce cellular responses through shared Jak-STAT, Ras-Raf-MAP, PI3K, and Src kinase pathways (6Okuda K. Foster R. Griffin J.D. Ann. N. Y. Acad. Sci. 1999; 872: 305-313Crossref PubMed Scopus (30) Google Scholar, 7Reddy E.P. Korapati A. Chaturvedi P. Rane S. Oncogene. 2000; 19: 2532-2547Crossref PubMed Scopus (189) Google Scholar). Upon receptor engagement by IL-3, non-receptor tyrosine kinases such as Jak2 and Src family members are activated. Activated Jak2 and/or Src family kinases in turn phosphorylate the receptor β chains on multiple tyrosine residues, which thereafter serve as docking sites for other Src homology (SH) 2 or phosphotyrosine binding domain-containing signaling molecules, such as the Shc adaptor protein or SHP-2 phosphatase, to couple downstream signaling pathways (8Pratt J.C. Weiss M. Sieff C.A. Shoelson S.E. Burakoff S.J. Ravichandran K.S. J. Biol. Chem. 1996; 271: 12137-12140Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 9Bone H. Welham M.J. Cell. Signal. 2000; 12: 183-194Crossref PubMed Scopus (23) Google Scholar, 10Gu H. Maeda H. Moon J.J. Lord J.D. Yoakim M. Nelson B.H. Neel B.G. Mol. Cell Biol. 2000; 20: 7109-7120Crossref PubMed Scopus (222) Google Scholar). Jak2 has been demonstrated to play a central role in receptor β chain signaling. Deficiency of Jak2 abrogates physiological and biochemical responses to IL-3 or GM-CSF in hematopoietic cells (11Neubauer H. Cumano A. Muller M. Wu H. Huffstadt U. Pfeffer K. Cell. 1998; 93: 397-409Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 12Parganas E. Wang D. Stravopodis D. Topham D.J. Marine J.C. Teglund S. Vanin E.F. Bodner S. Colamonici O.R. van Deursen J.M. Grosveld G. Ihle J.N. Cell. 1998; 93: 385-395Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar). However, detailed signaling mechanisms downstream of the common receptor β chain and exact components involved in the signaling processes are still not fully characterized. SHP-2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase, has been implicated in diverse signaling pathways induced by a number of stimuli including growth factors, cytokines, extracellular matrix, and even cellular stress (13Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar, 14Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (459) Google Scholar, 15Qu C.K. Biochim. Biophys. Acta. 2002; 1592: 297-301Crossref PubMed Scopus (108) Google Scholar). In many cases, especially in receptor tyrosine kinase-initiated intracellular signaling, SHP-2 enhances signal transmission. SHP-2 is highly expressed in hematopoietic cells. Our previous studies have shown that SHP-2 plays a critical role in hematopoietic cell development and function (16Qu C.K. Shi Z.Q. Shen R. Tsai F.Y. Orkin S.H. Feng G.S. Mol. Cell. Biol. 1997; 17: 5499-5507Crossref PubMed Scopus (148) Google Scholar, 17Qu C.K. Yu W.M. Azzarelli B. Cooper S. Broxmeyer H.E. Feng G.S. Mol. Cell. Biol. 1998; 18: 6075-6082Crossref PubMed Scopus (106) Google Scholar, 18Qu C.K. Nguyen S. Chen J. Feng G.S. Blood. 2001; 97: 911-914Crossref PubMed Scopus (98) Google Scholar) and that SHP-2 is indispensable in IL-3-mediated hematopoietic cell activities (19Yu W.M. Hawley T.S. Hawley R.G. Qu C.K. Oncogene. 2003; 22: 5995-6004Crossref PubMed Scopus (61) Google Scholar, 20Chen J. Yu W.M. Bunting K.D. Qu C.K. Oncogene. 2004; 23: 3659-3669Crossref PubMed Scopus (36) Google Scholar). SHP-2 appears to act at multiple sites in IL-3 signaling pathways, functioning in both catalytic-dependent and -independent manners. While it promotes Erk and PI3K pathways and enhances Jak2 activation (19Yu W.M. Hawley T.S. Hawley R.G. Qu C.K. Oncogene. 2003; 22: 5995-6004Crossref PubMed Scopus (61) Google Scholar, 20Chen J. Yu W.M. Bunting K.D. Qu C.K. Oncogene. 2004; 23: 3659-3669Crossref PubMed Scopus (36) Google Scholar), SHP-2 does have a negative role in hematopoietic cell survival (20Chen J. Yu W.M. Bunting K.D. Qu C.K. Oncogene. 2004; 23: 3659-3669Crossref PubMed Scopus (36) Google Scholar). Overexpression (5–6-fold increase) of wild type (WT) SHP-2 enhances growth factor deprivation-induced apoptosis and compromises hematopoietic cell function in vitro, and this is achieved by direct dephosphorylation of STAT5 (20Chen J. Yu W.M. Bunting K.D. Qu C.K. Oncogene. 2004; 23: 3659-3669Crossref PubMed Scopus (36) Google Scholar). Recently, genetic lesions in SHP-2 that cause hyperactivation of its catalytic activity have emerged as major genetic events underlying the developmental disorder Noonan syndrome and JMML. Fifty percent of Noonan syndrome patients and 35% of JMML cases carry gain-of-function (GOF) mutations in SHP-2 (21Tartaglia M. Mehler E.L. Goldberg R. Zampino G. Brunner H.G. Kremer H. van der Burgt I. Crosby A.H. Ion A. Jeffery S. Kalidas K. Patton M.A. Kucherlapati R.S. Gelb B.D. Nat. Genet. 2001; 29: 465-468Crossref PubMed Scopus (1287) Google Scholar, 22Tartaglia M. Niemeyer C.M. Fragale A. Song X. Buechner J. Jung A. Hahlen K. Hasle H. Licht J.D. Gelb B.D. Nat. Genet. 2003; 34: 148-150Crossref PubMed Scopus (795) Google Scholar, 23Loh M.L. Vattikuti S. Schubbert S. Reynolds M.G. Carlson E. Lieuw K.H. Cheng J.W. Lee C.M. Stokoe D. Bonifas J.M. Curtiss N.P. Gotlib J. Meshinchi S. Le Beau M.M. Emanuel P.D. Shannon K.M. Blood. 2004; 103: 2325-2331Crossref PubMed Scopus (340) Google Scholar, 24Bentires-Alj M. Paez J.G. David F.S. Keilhack H. Halmos B. Naoki K. Maris J.M. Richardson A. Bardelli A. Sugarbaker D.J. Richards W.G. Du J. Girard L. Minna J.D. Loh M.L. Fisher D.E. Velculescu V.E. Vogelstein B. Meyerson M. Sellers W.R. Neel B.G. Cancer Res. 2004; 64: 8816-8820Crossref PubMed Scopus (404) Google Scholar). The SHP-2 mutations appear to play a causal role in the development of these diseases. SHP-2 mutations and other JMML-associated Ras or NF1 mutations are mutually exclusive in leukemic patients. Moreover, a single SHP-2 GOF mutation (D61G) does induce myeloproliferative disease and Noonan syndrome in mice (25Araki T. Mohi M.G. Ismat F.A. Bronson R.T. Williams I.R. Kutok J.L. Yang W. Pao L.I. Gilliland D.G. Epstein J.A. Neel B.G. Nat. Med. 2004; 10: 849-857Crossref PubMed Scopus (334) Google Scholar). These new findings underscore the importance of the role of SHP-2 in cellular processes, particularly hematopoietic cell development. However, the molecular mechanisms of JMML induced by SHP-2 GOF mutations and detailed signaling activities of SHP-2 GOF mutants in hematopoietic cells are not well characterized. The hallmark of JMML is hypersensitivity of myeloid progenitor cells to GM-CSF and IL-3 (4Birnbaum R.A. O'Marcaigh A. Wardak Z. Zhang Y.Y. Dranoff G. Jacks T. Clapp D.W. Shannon K.M. Mol. Cell. 2000; 5: 189-195Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5Zhang Y.Y. Vik T.A. Ryder J.W. Srour E.F. Jacks T. Shannon K. Clapp D.W. J. Exp. Med. 1998; 187: 1893-1902Crossref PubMed Scopus (127) Google Scholar). Since our previous studies showed that excessive WT SHP-2 in hematopoietic cells attenuated IL-3-induced cellular responses (20Chen J. Yu W.M. Bunting K.D. Qu C.K. Oncogene. 2004; 23: 3659-3669Crossref PubMed Scopus (36) Google Scholar), we reasoned that SHP-2-associated JMML was not solely attributed to the increased phosphatase activity of mutant SHP-2. GOF mutant SHP-2 must have gained other capacities. To test this hypothesis and to gain more insight into the pathogenesis of SHP-2-associated leukemias, we investigated the consequences of SHP-2 E76K, the most frequent SHP-2 GOF mutation seen in JMML, on IL-3 signal transduction by comparing signaling activities of SHP-2 E76K to those of overexpressed WT SHP-2. Our results have suggested that profound changes in physical and functional interactions between GOF mutant SHP-2 and its signaling partners also play an important role in the pathogenesis of SHP-2-related JMML. Mice, Cell Line, and Reagents—WT C57BL/6 and congenic HW80 mice were purchased from the Jackson Laboratory (Bar Harbor, MN). All animal procedures complied with the National Institutes of Health Guideline for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Ba/F3, an IL-3-dependent murine hematopoietic cell line, was maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 10% conditioned medium containing mouse IL-3 (19Yu W.M. Hawley T.S. Hawley R.G. Qu C.K. Oncogene. 2003; 22: 5995-6004Crossref PubMed Scopus (61) Google Scholar). Anti-SHP-2, anti-STAT5a, anti-STAT5b, anti-Grb2, anti-Sos, anti-Erk, anti-phospho-Erk, antiphospho-Jnk1, and anti-Jnk1 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine (PY) (4G10), anti-p85, anti-Jak2 antiserum, anti-SHP-1, and anti-Gab2 Abs were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-phospho-p38, anti-phospho-Akt, and anti-Akt Abs were purchased from Cell Signaling Technology (Beverly, MA). Anti-Ly5.1 Ab was provided by BD Biosciences. AG490, PP2, LFM-A13, and piceatannol were supplied by Calbiochem. BrdUrd- and fluorescein isothiocyanate-conjugated anti-BrdUrd Ab were purchased from Roche Applied Science. Generation of SHP-2 E76K Retroviral Producer Cell Line and Transduction of Primary Bone Marrow Hematopoietic Stem/Progenitor Cells—SHP-2 E76K mutation was generated by PCR-based mutagenesis. The mutation was confirmed by cDNA sequencing. SHP-2 E76K cDNA was cloned into the MSCV-IRES-GFP retroviral vector (26Persons D.A. Allay J.A. Allay E.R. Ashmun R.A. Orlic D. Jane S.M. Cunningham J.M. Nienhuis A.W. Blood. 1999; 93: 488-499Crossref PubMed Google Scholar) containing an internal ribosomal entry sequence (IRES) driving expression of a downstream green fluorescence protein (GFP) gene to facilitate tracking of transduced cells. Ecotropic GP+E86-based retroviral producer cell lines were generated by transduction with retroviral supernatant produced by 293T cells that were transiently co-transfected with pQEPAM3 (Minus E) packaging plasmid, pSrαG (vesicular stomatitis virus glycoprotein) envelope plasmid, and the recombinant SHP-2 E76K retroviral plasmid. To transduce primary hematopoietic stem/progenitor cells with SHP-2 E76K, nucleated bone marrow cells harvested from femurs of 4-week-old mice were prestimulated in RPMI 1640 medium containing 10% FBS, SCF (50 ng/ml), IL-3 (20 ng/ml), and IL-6 (50 ng/ml) for 2 days and then co-cultured with irradiated (1500 rads) retroviral producer cells in the presence of polybrene (6 μg/ml) for 48 h. Hematopoietic Progenitor Assay—Following retroviral-mediated gene transfer, transduction efficiencies of bone marrow cells were examined by fluorescence-activated cell sorting (FACS) based on expression of GFP. Transduced bone marrow cells (5 × 104 cells/ml) were then assayed for colony forming units in 0.9% methylcellulose Iscove's modified Dulbecco's medium containing 30% FBS, glutamine (10–4m), β-mercaptoethanol (3.3 × 10–5m), and GM-CSF or IL-3 at the indicated concentrations. After 7 days of culture at 37 °C in a humidified 5% CO2 incubator, GFP-positive hematopoietic cell colonies were counted under an inverted fluorescence microscope. Colony-forming efficiencies were determined based on numbers of GFP-positive colonies and transduction efficiencies of the starting cells. Cell Cycle Analysis—Cell cycle analysis was performed as previously described (27Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar) with minor modifications. Exponentially growing cells (2 × 106) were synchronized by serum starvation for 24 h and then cultured in serum-free IL-3-containing (1 ng/ml) RPMI medium for 24 h. Cells were pulsed with BrdUrd (10 μm) for 45 min, washed twice in cold phosphate-buffered saline (PBS), and fixed in 75% ethanol at –20 °C overnight. After cells were suspended in 2 n HCl containing 0.5% Tween 20 at room temperature for 30 min, cells were washed twice in PBS (pH 7.4) to restore physiological pH and then resuspended with PBS containing 1% bovine serum albumin and 0.5% Tween 20. Fluorescein isothiocyanate-conjugated anti-BrdUrd monoclonal Ab (1 μg) was added and incubated at room temperature for 30 min. Cells were washed twice in PBS, stained with propidium iodide (PI) (50 μg/ml) dissolved in PBS in the presence of 100 μg/ml of RNase A at room temperature for 30 min, and then analyzed by FACS using BD-LSR flow cytometry (BD Biosciences). Immunoprecipitation and Immunoblotting—Cells were lysed in RIPA buffer (50 mm Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm NaF, 2 mm Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotin, and 1 mm phenylmethylsulfonyl fluoride). Whole cell lysates (500 μg) were immunoprecipitated with 1–2 μg of purified Abs or 2 μl of antiserum Abs. Immunoprecipitates were washed three times with HNTG buffer (20 mm Hepes (pH 7.5), 150 mm NaCl, 1% glycerol, 0.1% Triton X-100, and 1 mm Na3VO4) and resolved by SDS-PAGE followed by immunoblotting with the indicated Abs. Hematopoietic Cell Transplantation—Bone marrow cells freshly harvested from C57BL/6 mice were transduced with SHP-2 E76K, WT SHP-2, or catalytically inactive SHP-2 C459S by using the retroviral producer cell lines as described above. Transduced cells were harvested and examined for transduction efficiencies by FACS based on GFP expression. Transduced cells (1–2 × 106 cells) were injected through lateral tail veins into congenic HW80 or isogenic C57BL/6 recipient mice that had been irradiated with γ-radiation (1100 rads). Animals were bled 3 months after transplantation for hematology analyses. Our previous studies have shown that although SHP-2 phosphatase plays an overall positive role in hematopoietic cell development and function (13Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar, 14Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (459) Google Scholar, 15Qu C.K. Biochim. Biophys. Acta. 2002; 1592: 297-301Crossref PubMed Scopus (108) Google Scholar), overexpression (5–6-fold) of WT SHP-2 enhances growth factor deprivation-induced apoptosis in hematopoietic cells and decreases cellular responses to IL-3-induced proliferation and differentiation in bone marrow progenitor cells (20Chen J. Yu W.M. Bunting K.D. Qu C.K. Oncogene. 2004; 23: 3659-3669Crossref PubMed Scopus (36) Google Scholar). To confirm that overexpression of SHP-2 attenuates hematopoietic activity, we transduced bone marrow hematopoietic cells with WT SHP-2 as well as catalytically inactive SHP-2 C459S and then examined hematopoietic potential of the transduced cells in recipient animals. The bone marrow cell transduction efficiencies of WT SHP-2, SHP-2 C459S, and control vector were 28, 25, and 30%, respectively. Whole cell populations containing transduced and non-transduced cells were directly used for transplantation. As the catalytic activity of SHP-2 plays an important role in hematopoietic growth factor-induced activation of Erk kinase and Jak2 kinase (19Yu W.M. Hawley T.S. Hawley R.G. Qu C.K. Oncogene. 2003; 22: 5995-6004Crossref PubMed Scopus (61) Google Scholar), inhibition of the catalytic activity of endogenous SHP-2 by overexpression of catalytically inactive SHP-2 C459S decreased hematopoietic potential of the transduced progenitor cells. Three months after transplantation, compared with the vector control, contribution of SHP-2 C459S-transduced cells to nucleated cells in peripheral blood of the recipient mice was dramatically decreased (Fig. 1A), even though the hematopoietic system of the recipients was well reconstituted by donor cells (including both SHP-2 C459S-transduced and non-transduced cells), as evidenced by high percentages (>95%) of donor-derived progeny cells (Ly5.1-positive) in peripheral blood (Fig. 1B). Interestingly, overexpression of WT SHP-2 also decreased hematopoietic activities of the transduced progenitor cells (Fig. 1A). Three months after transplantation, the percentages of nucleated cells derived from WT SHP-2-transduced progenitor cells in peripheral blood of the recipient animals decreased to 1–7% (Fig. 1A). These data suggest that WT SHP-2-overexpressing progenitor cells lost growth advantage when competing with non-transduced donor cells in recipient mice. GOF mutations in SHP-2 causing hyperactivation of its catalytic activity have recently been identified in human leukemias, in particular JMML (22Tartaglia M. Niemeyer C.M. Fragale A. Song X. Buechner J. Jung A. Hahlen K. Hasle H. Licht J.D. Gelb B.D. Nat. Genet. 2003; 34: 148-150Crossref PubMed Scopus (795) Google Scholar, 23Loh M.L. Vattikuti S. Schubbert S. Reynolds M.G. Carlson E. Lieuw K.H. Cheng J.W. Lee C.M. Stokoe D. Bonifas J.M. Curtiss N.P. Gotlib J. Meshinchi S. Le Beau M.M. Emanuel P.D. Shannon K.M. Blood. 2004; 103: 2325-2331Crossref PubMed Scopus (340) Google Scholar, 24Bentires-Alj M. Paez J.G. David F.S. Keilhack H. Halmos B. Naoki K. Maris J.M. Richardson A. Bardelli A. Sugarbaker D.J. Richards W.G. Du J. Girard L. Minna J.D. Loh M.L. Fisher D.E. Velculescu V.E. Vogelstein B. Meyerson M. Sellers W.R. Neel B.G. Cancer Res. 2004; 64: 8816-8820Crossref PubMed Scopus (404) Google Scholar). Since the hallmark of JMML is hypersensitivity of myeloid progenitor cells to GM-CSF and IL-3 (4Birnbaum R.A. O'Marcaigh A. Wardak Z. Zhang Y.Y. Dranoff G. Jacks T. Clapp D.W. Shannon K.M. Mol. Cell. 2000; 5: 189-195Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5Zhang Y.Y. Vik T.A. Ryder J.W. Srour E.F. Jacks T. Shannon K. Clapp D.W. J. Exp. Med. 1998; 187: 1893-1902Crossref PubMed Scopus (127) Google Scholar), these findings prompted us to compare the effects of GOF mutant SHP-2 with those of WT SHP-2 on IL-3 induced cellular responses. We generated SHP-2 E76K, the most common SHP-2 GOF mutant seen in JMML (22Tartaglia M. Niemeyer C.M. Fragale A. Song X. Buechner J. Jung A. Hahlen K. Hasle H. Licht J.D. Gelb B.D. Nat. Genet. 2003; 34: 148-150Crossref PubMed Scopus (795) Google Scholar), and transduced this mutant into bone marrow hematopoietic progenitor cells via retroviral-mediated gene transfer. Transduced cells were assessed for their sensitivity to GM-CSF and IL-3 by hematopoietic colony assay. As shown in Fig. 2, A and B, SHP-2 E76K transduction increased cellular responses to GM-CSF and IL-3 in hematopoietic progenitor cells. Colony-forming capacities, in particular, at low concentrations of growth factors, were greatly enhanced by SHP-2 E76K transduction, consistent with recent reports (28Mohi M.G. Williams I.R. Dearolf C.R. Chan G. Kutok J.L. Cohen S. Morgan K. Boulton C. Shigematsu H. Keilhack H. Akashi K. Gilliland D.G. Neel B.G. Cancer Cell. 2005; 7: 179-191Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 29Chan R.J. Leedy M.B. Munugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (130) Google Scholar, 30Schubbert S. Lieuw K. Rowe S.L. Lee C.M. Li X. Loh M.L. Clapp D.W. Shannon K.M. Blood. 2005; 106: 311-317Crossref PubMed Scopus (125) Google Scholar). Furthermore, transduced cells were transplanted into recipient mice. Peripheral blood of these animals was examined three months after transplantation. In direct contrast to recipient mice transplanted with WT SHP-2-overexpressing cells (Fig. 1A), the contribution from SHP-2 E76K-transduced progenitors to the progeny cells in peripheral blood of all recipient mice was increased (Fig. 2C). These results suggest that SHP-2 E76K mutation confers hematopoietic progenitor cells growth advantages over non-transduced donor cells, consistent with the in vitro colony assay (Fig. 2A and 2B). Although E76K is an activating mutation and SHP-2 E76K phosphatase activity is about five times higher than that of WT SHP-2 (21Tartaglia M. Mehler E.L. Goldberg R. Zampino G. Brunner H.G. Kremer H. van der Burgt I. Crosby A.H. Ion A. Jeffery S. Kalidas K. Patton M.A. Kucherlapati R.S. Gelb B.D. Nat. Genet. 2001; 29: 465-468Crossref PubMed Scopus (1287) Google Scholar, 24Bentires-Alj M. Paez J.G. David F.S. Keilhack H. Halmos B. Naoki K. Maris J.M. Richardson A. Bardelli A. Sugarbaker D.J. Richards W.G. Du J. Girard L. Minna J.D. Loh M.L. Fisher D.E. Velculescu V.E. Vogelstein B. Meyerson M. Sellers W.R. Neel B.G. Cancer Res. 2004; 64: 8816-8820Crossref PubMed Scopus (404) Google Scholar), it does not seem that its enhanced catalytic activity is the sole contributing factor to blood disorders, since excessive (5–6-fold increase) WT SHP-2 did not cause myeloproliferative disease in recipient mice but decreased hematopoietic potential of the transduced cells (Fig. 1A). We next decided to dissect signaling activities of SHP-2 E76K by comparing to overexpressed WT SHP-2. As SHP-2 E76K results in hypersensitivity to GM-CSF and IL-3 in myeloid progenitor cells (Fig. 2, A and B), we focused on IL-3 signal transduction. SHP-2 E76K was transduced into an IL-3-dependent hematopoietic cell line, Ba/F3 cells, and transduced cells were sorted based on GFP expression. Sorted cell pools and the WT SHP-2-overexpressing Ba/F3 cell pool that we previously generated (20Chen J. Yu W.M. Bunting K.D. Qu C.K. Oncogene. 2004; 23: 3659-3669Crossref PubMed Scopus (36) Google Scholar) were used for subsequent signaling studies. In agreement with the progenitor cell assay data shown in Fig. 2, A and B, transduction of SHP-2 E76K into Ba/F3 cells greatly inc
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