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• W2021904192 abstract "A subset of chromosomal translocations that participate in leukemia involve activated tyrosine kinases. Theets transcription factor, TEL, undergoes translocations with several distinct tyrosine kinases including JAK2. TEL-JAK2 transforms cell lines to factor independence, and constitutive tyrosine kinase activity results in the phosphorylation of several substrates including STAT1, STAT3, and STAT5. In this study we have shown that TEL-JAK2 can constitutively activate the phosphatidylinositol 3′-kinase (PI 3′-kinase) signaling pathway. The regulatory subunit of PI 3′-kinase, p85, associates with TEL-JAK2 in immunoprecipitations, and this was shown to be mediated by the amino-terminal SH2 domain of p85 but independent of a putative p85-binding motif within TEL-JAK2. The scaffolding protein Gab2 can also mediate the association of p85. TEL-JAK2 constitutively phosphorylates the downstream substrate protein kinase B/AKT. Importantly, the pharmacologic PI 3′-kinase inhibitor, LY294002, blocked TEL-JAK2 factor-independent growth and phosphorylation of protein kinase B. However, LY294002 did not alter STAT5 tyrosine phosphorylation, indicating that STAT5 and protein kinase B activation mediated by TEL-JAK2 are independent signaling pathways. Therefore, activation of the PI 3′-kinase signaling pathway is an important event mediated by TEL-JAK2 chromosomal translocations. A subset of chromosomal translocations that participate in leukemia involve activated tyrosine kinases. Theets transcription factor, TEL, undergoes translocations with several distinct tyrosine kinases including JAK2. TEL-JAK2 transforms cell lines to factor independence, and constitutive tyrosine kinase activity results in the phosphorylation of several substrates including STAT1, STAT3, and STAT5. In this study we have shown that TEL-JAK2 can constitutively activate the phosphatidylinositol 3′-kinase (PI 3′-kinase) signaling pathway. The regulatory subunit of PI 3′-kinase, p85, associates with TEL-JAK2 in immunoprecipitations, and this was shown to be mediated by the amino-terminal SH2 domain of p85 but independent of a putative p85-binding motif within TEL-JAK2. The scaffolding protein Gab2 can also mediate the association of p85. TEL-JAK2 constitutively phosphorylates the downstream substrate protein kinase B/AKT. Importantly, the pharmacologic PI 3′-kinase inhibitor, LY294002, blocked TEL-JAK2 factor-independent growth and phosphorylation of protein kinase B. However, LY294002 did not alter STAT5 tyrosine phosphorylation, indicating that STAT5 and protein kinase B activation mediated by TEL-JAK2 are independent signaling pathways. Therefore, activation of the PI 3′-kinase signaling pathway is an important event mediated by TEL-JAK2 chromosomal translocations. interleukin-3 phosphatidylinositol 3′-kinase protein kinase B nucleotide sodium 3,3′-{1-[(phenylamino)carbonyl]-3,4-tetrazolium)bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate} glutathione S-transferase horseradish peroxidase polyvinylidene difluoride bovine serum albumin polyacrylamide gel electrophoresis LY249002 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one phosphatidylinositol 7-aminoactinomycin D nucleotide Chromosomal translocations play a central role in the development of leukemia. The participating genes generally fall into three groups involving tyrosine kinases, transcription factors, or factors that modify transcriptional activation (1Gilliland D.G. Leukemia (Baltimore). 1998; 12 Suppl. 1: 7-12Google Scholar). The prototypical tyrosine kinase is the BCR-ABL translocation that is the causative agent in chronic myelogenous leukemia (2Sawyers C.L. N. Engl. J. Med. 1999; 340: 1330-1340Crossref PubMed Scopus (1283) Google Scholar). The ets transcription factor, TEL, is a frequent participant in chromosomal translocations, and a subset of these fusions involve tyrosine kinases including PDGFβR (3Golub T.R. Barker G.F. Lovett M. Gilliland D.G. Cell. 1994; 77: 307-316Abstract Full Text PDF PubMed Scopus (1088) Google Scholar), ABL (4Janssen J.W. Ridge S.A. Papadopoulos P. Cotter F. Ludwig W.D. Fonatsch C. Rieder H. Ostertag W. Bartram C.R. Wiedemann L.M. Br. J. Haematol. 1995; 90: 222-224Crossref PubMed Scopus (49) Google Scholar, 5Golub T.R. Goga A. Barker G.F. Afar D.E. McLaughlin J. Bohlander S.K. Rowley J.D. Witte O.N. Gilliland D.G. Mol. Cell. Biol. 1996; 16: 4107-4116Crossref PubMed Scopus (304) Google Scholar), ARG (6Cazzaniga G. Tosi S. Aloisi A. Giudici G. Daniotti M. Pioltelli P. Kearney L. Biondi A. Blood. 1999; 94: 4370-4373Crossref PubMed Google Scholar), TRKC (7Knezevich S.R. McFadden D.E. Tao W. Lim J.F. Sorensen P.H. Nat. Genet. 1998; 18: 184-187Crossref PubMed Scopus (655) Google Scholar, 8Eguchi M. Eguchi-Ishimae M. Tojo A. Morishita K. Suzuki K. Sato Y. Kudoh S. Tanaka K. Setoyama M. Nagamura F. Asano S. Kamada N. Blood. 1999; 93: 1355-1363Crossref PubMed Google Scholar), and JAK2 (9Peeters P. Raynaud S.D. Cools J. Wlodarska I. Grosgeorge J. Philip P. Monpoux F. Van Rompaey L. Baens M. Van den Berghe H. Marynen P. Blood. 1997; 90: 2535-2540Crossref PubMed Google Scholar, 10Lacronique V. Boureux A. Valle V.D. Poirel H. Quang C.T. Mauchauffe M. Berthou C. Lessard M. Berger R. Ghysdael J. Bernard O.A. Science. 1997; 278: 1309-1312Crossref PubMed Scopus (682) Google Scholar). We are particularly interested in characterizing the properties of the TEL-JAK2 translocation that mediate leukemogenesis. TEL-JAK2 translocations have been described in three patients to date. Two patients, each harboring primary translocations, were diagnosed with acute lymphoblastic leukemia. One patient expressed a fusion of TEL exon 4 to JAK2 exon 17 (t(9;12)(p24;p13); TEL-JAK2-(4–17)) (9Peeters P. Raynaud S.D. Cools J. Wlodarska I. Grosgeorge J. Philip P. Monpoux F. Van Rompaey L. Baens M. Van den Berghe H. Marynen P. Blood. 1997; 90: 2535-2540Crossref PubMed Google Scholar), whereas the other had a TEL exon 5 to JAK2 exon 19 translocation (t(9;12)(p24;p13); TEL-JAK2-(5–19)) (10Lacronique V. Boureux A. Valle V.D. Poirel H. Quang C.T. Mauchauffe M. Berthou C. Lessard M. Berger R. Ghysdael J. Bernard O.A. Science. 1997; 278: 1309-1312Crossref PubMed Scopus (682) Google Scholar). The third isolated TEL-JAK2 product arose from a compound t(9;12;15)(p24;q15;p13) translocation in which one allele of TEL was fused to JAK2 (TEL-JAK2-(5–12)) (9Peeters P. Raynaud S.D. Cools J. Wlodarska I. Grosgeorge J. Philip P. Monpoux F. Van Rompaey L. Baens M. Van den Berghe H. Marynen P. Blood. 1997; 90: 2535-2540Crossref PubMed Google Scholar) and the other TEL allele was fused to EVI1 (11Peeters P. Wlodarska I. Baens M. Criel A. Selleslag D. Hagemeijer A. Van den Berghe H. Marynen P. Cancer Res. 1997; 57: 564-569PubMed Google Scholar). All three fusions have been shown to convert IL-31-dependent hematopoietic cells to factor independence (10Lacronique V. Boureux A. Valle V.D. Poirel H. Quang C.T. Mauchauffe M. Berthou C. Lessard M. Berger R. Ghysdael J. Bernard O.A. Science. 1997; 278: 1309-1312Crossref PubMed Scopus (682) Google Scholar, 12Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar). Many studies have focused on the mechanism of constitutive activation mediated by BCR-ABL. Substrates that are activated downstream of BCR-ABL include STAT1 (13Carlesso N. Frank D.A. Griffin J.D. J. Exp. Med. 1996; 183: 811-820Crossref PubMed Scopus (434) Google Scholar, 14Frank D.A. Varticovski L. Leukemia (Baltimore). 1996; 10: 1724-1730PubMed Google Scholar, 15Ilaria Jr., R.L. Van Etten R.A. J. Biol. Chem. 1996; 271: 31704-31710Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar), STAT3 (15Ilaria Jr., R.L. Van Etten R.A. J. Biol. Chem. 1996; 271: 31704-31710Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar), STAT5 (13Carlesso N. Frank D.A. Griffin J.D. J. Exp. Med. 1996; 183: 811-820Crossref PubMed Scopus (434) Google Scholar, 14Frank D.A. Varticovski L. Leukemia (Baltimore). 1996; 10: 1724-1730PubMed Google Scholar, 15Ilaria Jr., R.L. Van Etten R.A. J. Biol. Chem. 1996; 271: 31704-31710Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 16Shuai K. Halpern J. Hoeve J.T. Rao X. Sawyers C.L. Oncogene. 1996; 13: 247-254PubMed Google Scholar), and STAT6 (15Ilaria Jr., R.L. Van Etten R.A. J. Biol. Chem. 1996; 271: 31704-31710Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Grb2-Sos can be recruited to BCR-ABL either directly or indirectly through other adaptor proteins including Ship1 (17Odai H. Sasaki K. Iwamatsu A. Nakamoto T. Ueno H. Yamagata T. Mitani K. Yazaki Y. Hirai H. Blood. 1997; 89: 2745-2756Crossref PubMed Google Scholar), Shp2 (18Tauchi T. Feng G.S. Shen R. Song H.Y. Donner D. Pawson T. Broxmeyer H.E. J. Biol. Chem. 1994; 269: 15381-15387Abstract Full Text PDF PubMed Google Scholar), Shc (19Goga A. McLaughlin J. Afar D.E. Saffran D.C. Witte O.N. Cell. 1995; 82: 981-988Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 20Harrison-Findik D. Susa M. Varticovski L. Oncogene. 1995; 10: 1385-1391PubMed Google Scholar, 21Matsuguchi T. Salgia R. Hallek M. Eder M. Druker B. Ernst T.J. Griffin J.D. J. Biol. Chem. 1994; 269: 5016-5021Abstract Full Text PDF PubMed Google Scholar, 22Puil L. Liu J. Gish G. Mbamalu G. Bowtell D. Pelicci P.G. Arlinghaus R. Pawson T. EMBO J. 1994; 13: 764-773Crossref PubMed Scopus (401) Google Scholar, 23Tauchi T. Boswell H.S. Leibowitz D. Broxmeyer H.E. J. Exp. Med. 1994; 179: 167-175Crossref PubMed Scopus (139) Google Scholar), and Cbl (24Andoniou C.E. Thien C.B. Langdon W.Y. EMBO J. 1994; 13: 4515-4523Crossref PubMed Scopus (217) Google Scholar, 25de Jong R. ten Hoeve J. Heisterkamp N. Groffen J. J. Biol. Chem. 1995; 270: 21468-21471Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 26Jain S.K. Langdon W.Y. Varticovski L. Oncogene. 1997; 14: 2217-2228Crossref PubMed Scopus (54) Google Scholar, 27Salgia R. Sattler M. Pisick E. Li J.L. Griffin J.D. Exp. Hematol. 1996; 24: 310-313PubMed Google Scholar, 28Senechal K. Heaney C. Druker B. Sawyers C.L. Mol. Cell. Biol. 1998; 18: 5082-5090Crossref PubMed Scopus (63) Google Scholar). BCR-ABL has also been shown to stimulate activation of Ras (29Sawyers C.L. McLaughlin J. Witte O.N. J. Exp. Med. 1995; 181: 307-313Crossref PubMed Scopus (248) Google Scholar) and the related family member Rac (30Skorski T. Wlodarski P. Daheron L. Salomoni P. Nieborowska-Skorska M. Majewski M. Wasik M. Calabretta B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11858-11862Crossref PubMed Scopus (84) Google Scholar). BCR-ABL also has been shown to participate in pathways that are involved in the prevention of apoptosis. For example, BCR-ABL activates the PI 3′-kinase signaling pathway (31Skorski T. Kanakaraj P. Nieborowska-Skorska M. Ratajczak M.Z. Wen S.C. Zon G. Gewirtz A.M. Perussia B. Calabretta B. Blood. 1995; 86: 726-736Crossref PubMed Google Scholar, 32Neshat M.S. Raitano A.B. Wang H.G. Reed J.C. Sawyers C.L. Mol. Cell. Biol. 2000; 20: 1179-1186Crossref PubMed Scopus (165) Google Scholar). Recent studies have shown that the PI 3′-kinase inhibitor, LY294002, blocks growth of BCR-ABL-expressing hematopoietic cells (32Neshat M.S. Raitano A.B. Wang H.G. Reed J.C. Sawyers C.L. Mol. Cell. Biol. 2000; 20: 1179-1186Crossref PubMed Scopus (165) Google Scholar). PI 3′-kinases are important modulators of cell survival, mitogenesis, cytoskeletal remodeling, metabolic control, and vesicular trafficking (reviewed in Ref. 33Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1323) Google Scholar). There are three classes of these enzymes. Class I PI 3′-kinases are heterodimers consisting of a 110-kDa catalytic subunit and a 85-kDa regulatory subunit. Binding of the p85 subunit to phosphotyrosines stimulates activity of the associated p110 subunit (34Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (822) Google Scholar, 35Fruman D.A. Cantley L.C. Carpenter C.L. Genomics. 1996; 37: 113-121Crossref PubMed Scopus (99) Google Scholar, 36Rordorf-Nikolic T. Horn D.J.V. Chen D. White M.F. Backer J.M. J. Biol. Chem. 1995; 270: 3662-3666Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). The two SH2 domains of p85 can interact with phosphorylated tyrosines on activated receptor tyrosine kinases or on adaptor proteins such as Gab2 (37Gu H. Pratt J.C. Burakoff S.J. Neel B.G. Mol. Cell. 1998; 2: 729-740Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) and IRS-2 (38Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (767) Google Scholar). The activation of PI 3′-kinase catalyzes the phosphorylation of phosphatidylinositol (PtdIns) lipids on the D3-hydroxy group generating products such as PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These lipids can modulate the subcellular localization and activation of a number of proteins. The serine/threonine kinase, Akt/PKB, is one well studied target of PI 3′-kinase activation implicated in mediating signals for cell survival and growth (reviewed in Ref. 39Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar). TEL-JAK2 has been shown to transform cell lines to factor independence through constitutive tyrosine kinase activity (10Lacronique V. Boureux A. Valle V.D. Poirel H. Quang C.T. Mauchauffe M. Berthou C. Lessard M. Berger R. Ghysdael J. Bernard O.A. Science. 1997; 278: 1309-1312Crossref PubMed Scopus (682) Google Scholar, 12Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar, 40Ho J.M. Beattie B.K. Squire J.A. Frank D.A. Barber D.L. Blood. 1999; 93: 4354-4364Crossref PubMed Google Scholar). Importantly, TEL-JAK2 does not activate endogenous JAK kinases but does result in constitutive tyrosine phosphorylation and DNA binding of STAT1 (12Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar, 40Ho J.M. Beattie B.K. Squire J.A. Frank D.A. Barber D.L. Blood. 1999; 93: 4354-4364Crossref PubMed Google Scholar), STAT3 (40Ho J.M. Beattie B.K. Squire J.A. Frank D.A. Barber D.L. Blood. 1999; 93: 4354-4364Crossref PubMed Google Scholar), and STAT5 (12Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar, 40Ho J.M. Beattie B.K. Squire J.A. Frank D.A. Barber D.L. Blood. 1999; 93: 4354-4364Crossref PubMed Google Scholar). Bone marrow transplant studies demonstrate that TEL-JAK2-(5–19) gives rise to a biphenotypic disease with elements of myelo- and lymphoproliferation (12Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar). TEL-JAK2-(5–19) transgenic mice develop a fatal T cell leukemia (41Carron C. Cormier F. Janin A. Lacronique V. Giovannini M. Daniel M.T. Bernard O. Ghysdael J. Blood. 2000; 95: 3891-3899Crossref PubMed Google Scholar). The importance of STAT5 in TEL-JAK2-mediated leukemogenesis was recently demonstrated as TEL-JAK2-transduced bone marrow cells failed to induce neoplasia when introduced into a genetic background devoid of STAT5a/b (42Schwaller J. Parganas E. Wang D. Cain D. Aster J.C. Williams I.R. Lee C.K. Gerthner R. Kitamura T. Frantsve J. Anastasiadou E. Loh M.L. Levy D.E. Ihle J.N. Gilliland D.G. Mol. Cell. 2000; 6: 693-704Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). However, a constitutively activated form of STAT5 resulted in only a myeloproliferative disease (42Schwaller J. Parganas E. Wang D. Cain D. Aster J.C. Williams I.R. Lee C.K. Gerthner R. Kitamura T. Frantsve J. Anastasiadou E. Loh M.L. Levy D.E. Ihle J.N. Gilliland D.G. Mol. Cell. 2000; 6: 693-704Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). In summation, these elegant studies have shown that signaling pathways distinct from STAT5a/b activation play a role in leukemogenesis mediated by TEL-JAK2. The goal of this study is to characterize PI 3′-kinase-dependent signaling mitigated by TEL-JAK2. Constructs were generated as described. 2J. M.-Y. Ho, M. H.-H. Nguyen, B. K. Beattie, and D. L. Barber, submitted for publication. The Quick-Change site-directed mutagenesis kit (Stratagene) was used to introduce the Y624F mutation into TEL-JAK2-(5–19) with the following primers: 5′-GCCCAGATGAGATCTTTATG-3′ and 5′-GCATTCTGTCATGATCATAAAGATCTCATCTGGGC-3′. Murine Ba/F3 cells were maintained in complete media (RPMI 1640 medium with antibiotics, 10% (v/v) fetal bovine serum (Sigma), 50 µm β-mercaptoethanol (Fisher)) containing 100 pg/ml of recombinant murine IL-3 (IL-3) (R & D Systems) in a 5% CO2 incubator at 37 °C. The same conditions using G418 selection media (complete media containing 100 pg/ml IL-3 with 1 mg/ml Geneticin (Life Technologies, Inc.)) maintained subclones of Ba/F3 cells expressing TEL-JAK2-(4–17), TEL-JAK2-(5–19), TEL-JAK2-(5–19) Y624F, TEL-JAK2-(5–12), BCR-ABL p210, or pcDNA3 vector alone. Electroporations were performed as described (40Ho J.M. Beattie B.K. Squire J.A. Frank D.A. Barber D.L. Blood. 1999; 93: 4354-4364Crossref PubMed Google Scholar, 44Barber D.L. DeMartino J.C. Showers M.O. D'Andrea A.D. Mol. Cell. Biol. 1994; 14: 2257-2265Crossref PubMed Scopus (51) Google Scholar), using 20 µg of DNA for the various constructs, vector alone, or no vector (350 mV and 950 microfarads) into Ba/F3 cells (GenePulser, Bio-Rad). G418-resistant populations were selected, and subclones were isolated by limiting dilution. The expression of TEL-JAK2 and BCR-ABL was confirmed by immunoblotting, and the IL-3-dependent growth characteristics of each subclone was confirmed by performing an XTT assay. XTT assays were performed as described (40Ho J.M. Beattie B.K. Squire J.A. Frank D.A. Barber D.L. Blood. 1999; 93: 4354-4364Crossref PubMed Google Scholar, 44Barber D.L. DeMartino J.C. Showers M.O. D'Andrea A.D. Mol. Cell. Biol. 1994; 14: 2257-2265Crossref PubMed Scopus (51) Google Scholar). Cytokine-depleted cells (2000/well) were added to a 96-well plate in a final volume of 100 µl containing complete media with varying concentrations of LY294002 and a constant concentration of IL-3. Plates were incubated at 37 °C for 48 h prior to addition of sodium 3,3′-{1-[(phenylamino)carbonyl]-3,4-tetrazolium)bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate} (XTT) (2 mg/ml) (Diagnostic Chemicals) and phenazine methosulfate (3 µm) (Sigma) (final volume of 125 µl). Cells were incubated for an additional 4 h at 37 °C prior to measuring the absorption of the soluble formazan reduction product at 450 nm. Cells were depleted of cytokine by washing three times with Hanks' balanced salt solution containing 10 mm Hepes (pH 7.4) and incubating at 37 °C for 18 h in complete media. Cells were then stimulated in the presence or absence of 10 ng/ml IL-3 in complete media for 10 min at 37 °C. Cells were washed once in cold Hanks' balanced salt solution containing 10 mm sodium pyrophosphate, 10 mmsodium fluoride, 10 mm EDTA, and 1 mm sodium orthovanadate. Lysates were prepared in ice-cold lysis buffer, containing 50 mm Tris-HCl (pH 8.0), 150 mmNaCl, 1% Triton X-100, 10 mmNa4P2O7, 10 mm NaF, 10 mm EDTA, 1 mm Na3VO4, 1 µm phenylmethylsulfonyl fluoride, 1 µmaprotinin, 1 µm leupeptin, and 2 µmpepstatin A, incubated for 10 min on ice, and centrifuged at 10,000 × g for 5 min at 4 °C. Lysate concentrations were quantified by the Bradford colorimetric method (Bio-Rad). For immunoblot analyses of lysates, 100 µg of lysate was boiled for 5 min in Laemmli sample buffer with 100 µm dithiothreitol (DTT). Samples were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF transfer membrane (PerkinElmer Life Sciences). 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY249002) (Calbiochem) was resuspended in dimethyl sulfoxide (Me2SO) (Fisher) to a final concentration of 65.06 mm. Preparation of cellular protein lysates for inhibitor studies is exactly as described for the preparation of cellular protein lysates with an additional incubation step; prior to murine IL-3 stimulation, cells were incubated with 10 or 20 µmLY294002 or carrier alone for 30 min. The anti-phosphotyrosine antibody, 4G10, was generously provided by Dr. Brian Druker, Oregon Health Sciences University, Portland, OR. Rabbit anti-Myc was purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Mouse anti-p85 was obtained from Transduction Laboratories, Lexington, KY. Rabbit anti-IRS-2, rabbit anti-Gab2, and rabbit anti-p85 were purchased from Upstate Biotechnology, Inc., Lake Placid, NY. Anti-phospho-PKB (Ser-473) and anti-PKB antibodies were purchased from New England Biolabs, Beverly, MA. Phospho-STAT5 antibody was purchased from Zymed Laboratories Inc., South San Francisco, CA, and anti-STAT5 antibody was generously provided by Dr. James Ihle, St. Jude's Childrens Hospital, Memphis, TN. A peptide-specific anti-TEL antibody was generated using a keyhole limpet hemocyanin-coupled peptide corresponding to amino acids 138–154 of TEL. Immunoblotting secondary reagents used were horseradish peroxidase (HRP)-conjugated protein A or HRP-conjugated sheep anti-mouse immunoglobulin obtained from Amersham Pharmacia Biotech. Immunoprecipitations were performed with 1.5 mg of protein lysates. Primary antibody was added for 1 h, followed by 1-h incubation with protein A-Sepharose (Amersham Pharmacia Biotech). Alternatively, primary antibody and protein A-Sepharose was added together, and incubations were performed overnight. Bead-bound immune complexes were washed 3 times with ice-cold lysis buffer, eluted by boiling for 5 min in Laemmli sample buffer containing 100 µm DTT, and separated by SDS-PAGE and transferred to PVDF transfer membrane for immunoblotting. GST fusion proteins (2.5 µg) expressing the amino, carboxyl, or both amino- and carboxyl-terminal SH2 domains of p85 (generously provided by Dr. Ben Margolis, University of Michigan, Ann Arbor, MI) or GST alone immobilized to glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) were incubated with 1.5 mg of protein lysates. After a 1-h incubation at 4 °C, the precipitate was washed three times with ice-cold lysis buffer. Samples were boiled for 5 min in Laemmli sample buffer with 100 µm DTT to elute proteins before separation on SDS-PAGE gels and transfer to PVDF transfer membrane. For most immunoblotting experiments, membranes were blocked at room temperature with 2.5% BSA in Tris-buffered saline (TBS; 50 mm Tris (pH 8.0), 150 mm NaCl) for 1 h. Following two washes in TBST (TBS, 0.1% Tween 20), membranes were incubated with the appropriate dilution of primary antibody solution for 1 h at room temperature. Membranes were then washed four times in TBST and incubated with the relevant HRP-conjugated secondary antibody (1:5000 dilution in TBST) for 30 min. Following four washes in TBST, reactive proteins were visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech) with autoradiographic film (Amersham Pharmacia Biotech). PVDF membranes for phospho-PKB and PKB immunoblots were blocked in 5% skim milk in TBST for 1 h at room temperature, washed once in primary antibody dilution buffer, and incubated with primary antibody (1:1000 dilution in 1% BSA in TBST) overnight at 4 °C. After six washes in TBST, the membrane was incubated with HRP-protein A (1:2000 dilution in 2.5% skim milk in TBST) for 1 h at room temperature. The membrane was washed 6 times in TBST and visualized by ECL. Membranes for phospho-STAT5 immunoblots were blocked in 5% milk in TBST for 1 h at room temperature, washed 2 times in TBST, and incubated with primary antibody (1:1000 dilution in 3% BSA in TBST) for 3 h at room temperature. After 4 washes in TBST, the membrane was incubated with HRP-protein A (1:5000 dilution in 2.5% BSA in TBST) for 1 h at room temperature. The membrane was washed 4 times in TBST prior to visualization by ECL. For reprobing, membranes were stripped in 62.5 mm Tris-HCl (pH 6.8), 2% SDS, and 0.1m β-mercaptoethanol for 30 min at 50 °C and rinsed twice in TBST. Annexin V, 7AAD, and 10× binding buffer were purchased from PharMingen. Briefly, at distinct time points, untreated and treated cells were washed in 1× binding buffer (10 mm Hepes (pH 7.4), 140 mm NaCl, 2.5 mm CaCl2). 1 × 106 cells were then resuspended in 50 µl of 1× binding buffer and incubated with 2 µl of annexin V antibody conjugated to PE for 10 min at room temperature. Samples were adjusted to a final phycoerythrin volume of 1 ml prior to fluorescence-activated cell sorter analysis (Becton Dickinson). Acquisition and analysis were performed using the CellQuest software. TEL-JAK2 isoforms have been constructed with breakpoints as described from patient samples (9Peeters P. Raynaud S.D. Cools J. Wlodarska I. Grosgeorge J. Philip P. Monpoux F. Van Rompaey L. Baens M. Van den Berghe H. Marynen P. Blood. 1997; 90: 2535-2540Crossref PubMed Google Scholar, 10Lacronique V. Boureux A. Valle V.D. Poirel H. Quang C.T. Mauchauffe M. Berthou C. Lessard M. Berger R. Ghysdael J. Bernard O.A. Science. 1997; 278: 1309-1312Crossref PubMed Scopus (682) Google Scholar) (Fig.1). These constructs were introduced into the murine IL-3-dependent myeloid cell line Ba/F3 via electroporation. TEL-JAK2-(4–17), TEL-JAK2-(5–19), and TEL-JAK2-(5–12) subclones were isolated by limiting dilution, and those displaying similar expression were selected for further characterization. Expression of TEL-JAK2 in Ba/F3 cells resulted in factor-independent proliferation and constitutive tyrosine phosphorylation of each fusion protein in all subclones, consistent with previous reports (10Lacronique V. Boureux A. Valle V.D. Poirel H. Quang C.T. Mauchauffe M. Berthou C. Lessard M. Berger R. Ghysdael J. Bernard O.A. Science. 1997; 278: 1309-1312Crossref PubMed Scopus (682) Google Scholar, 12Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar). Growth factors and cytokines, including IL-3, induce the activity of PI 3′-kinases. In addition, oncogenic tyrosine kinase fusions have been shown to activate PI 3′-kinase. The transforming ability of BCR-ABL has been shown to require the PI 3′-kinase signaling pathway and activation of the serine-threonine kinase PKB (45Skorski 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 (558) Google Scholar). PKB is one downstream component of the PI 3′-kinase signaling pathway important in influencing cell survival. We were interested in determining whether TEL-JAK2 mediated PKB activation (Fig.2). Since the phosphorylation of PKB is associated with its activation (46Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar), activation-specific antibodies have been developed that detect PKB phosphorylated at Ser-473. IL-3 stimulation of Ba/F3 cells led to a strong activation of PKB phosphorylation (lane 2). TEL-JAK2-(4–17) stimulated a level of PKB phosphorylation in the absence of IL-3 stimulation (lane 3) that was higher than that in unstimulated Ba/F3 cells (lane 1). Expression of TEL-JAK2-(5–12) and TEL-JAK2-(5–19) in Ba/F3 cells resulted in higher levels of constitutive PKB phosphorylation (lanes 5 and 7, respectively). Ba/F3 cells expressing BCR-ABL also stimulated PKB phosphorylation in the absence of IL-3 (lane 9). Upon IL-3 stimulation, all cell lines exhibited comparable levels of PKB phosphorylation (even lanes). Equal loading was confirmed by reprobing the blot with a total PKB antibody (lower panel). This experiment demonstrated that TEL-JAK2 expression constitutively activates PKB phosphorylation. To determine whether TEL-JAK2-mediated factor-independent growth was dependent on PI 3′-kinase, we performed XTT assays in the absence or presence of IL-3 (100 pg/ml) and increasing concentrations of the PI 3′-kinase inhibitor, LY294002 (47Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar) (Fig. 3). A reduction in the number of Ba/F3 and all TEL-JAK2-expressing Ba/F3 cells was observed with increasing LY294002 concentrations (upper panel), even in the presence of IL-3 (lower panel). Subclones of Ba/F3 cells expressing vector alone had identical kinetics as untransfected Ba/F3 cells (data not shown). Moreover, this decrease in cell number was not seen in the presence of the carrier, Me2SO (data not shown). These results suggest that TEL-JAK2, and IL-3 (48Craddock B.L. Orchiston E.A. Hinton H.J. Welham M.J. J. Biol. Chem. 1999; 274: 10633-10640Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 49Gold M.R. Duronio V. Saxena S.P. Schrader J.W. Aebersold R. J. Biol. Chem. 1994; 269: 5403-5412Abstract Full Text PDF PubMed Google Scholar, 50Scheid M.P. Lauener R.W. Duronio V. Biochem. J. 1995; 312: 159-162Crossref PubMed Sco" @default.
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• W2021904192 date "2001-08-01" @default.
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• W2021904192 title "TEL-JAK2 Mediates Constitutive Activation of the Phosphatidylinositol 3′-Kinase/Protein Kinase B Signaling Pathway" @default.
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• W2021904192 doi "https://doi.org/10.1074/jbc.m103100200" @default.
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