Interleukin-3 (IL-3)-induced c-fos Activation Is Modulated by Gab2-Calcineurin Interaction

Interleukin-3 (IL-3) regulates cell growth by affecting various processes such as cell death, survival, and proliferation. Cues from the external environment are sensed by surface receptors, and complex signaling mechanisms arise within the cells, leading to specific functional outcomes. In this study, we demonstrate that the cytokine IL-3 induces the activation of the Ca2+-dependent phosphatase, calcineurin (Cn). Furthermore Cn dephosphorylates Gab2, resulting in c-fos activation and cell proliferation. We also report that there is a direct interaction between Cn and Gab2 upon IL-3 stimulation, and Akt can regulate this interaction. Interleukin-3 (IL-3) regulates cell growth by affecting various processes such as cell death, survival, and proliferation. Cues from the external environment are sensed by surface receptors, and complex signaling mechanisms arise within the cells, leading to specific functional outcomes. In this study, we demonstrate that the cytokine IL-3 induces the activation of the Ca2+-dependent phosphatase, calcineurin (Cn). Furthermore Cn dephosphorylates Gab2, resulting in c-fos activation and cell proliferation. We also report that there is a direct interaction between Cn and Gab2 upon IL-3 stimulation, and Akt can regulate this interaction. The tight control of survival, proliferation, and apoptosis is essential for normal cellular growth and development. A variety of extracellular signals including growth factors and cytokines regulate viability, proliferation, differentiation, and function in bone marrow progenitor cells to maintain normal hematopoiesis. Interleukin-3 (IL-3) 3The abbreviations used are:ILinterleukinCncalcineurinCaMcalmodulinHAhemagglutininIEGimmediate early geneWTwild typeBAPTA1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidPI3phosphatidylinositol 3. is a multipotent hematopoietic growth factor, which acts on progenitor, myeloid, and mast cells to promote cell survival, induce proliferation, and facilitate differentiation. interleukin calcineurin calmodulin hemagglutinin immediate early gene wild type 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid phosphatidylinositol 3. Upon IL-3 treatment, there is rapid phosphorylation of tyrosine residues in the β-subunit of the IL-3 receptor followed by tyrosine phosphorylation of a variety of signaling molecules including Gab2 (Grb2-associated binder-2) (1Gu 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). Gab2 is a member of a family of pleckstrin homology domain-containing adaptor proteins that includes mammalian Gab-1, Gab-3, and Drosophila DOS (daughter of sevenless). Gab2 has been implicated in a variety of cellular functions including the negative regulation of T-cell receptor signaling (2Pratt J.C. Igras V.E. Maeda H. Baksh S. Gelfand E.W. Burakoff S.J. Neel B.G. Gu H. J. Immunol. 2000; 165: 4158-4163Crossref PubMed Scopus (58) Google Scholar) and the positive regulation of growth factor, cytokine, and antigen receptor signaling (3Gu H. Griffin J.D. Neel B.G. J. Biol. Chem. 1997; 272: 16421-16430Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 4Lynch D.K. Daly R.J. EMBO J. 2002; 21: 72-82Crossref PubMed Scopus (73) Google Scholar, 5Miyakawa Y. Rojnuckarin P. Habib T. Kaushansky K. J. Biol. Chem. 2001; 276: 2494-2502Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Gab2 knock-out mice show tissue-specific defects in mast cell signaling (6Gu H. Saito K. Klaman L.D. Shen J. Fleming T. Wang Y. Pratt J.C. Lin G. Lim B. Kinet J.P. Neel B.G. Nature. 2001; 412: 186-190Crossref PubMed Scopus (278) Google Scholar) and osteoclast differentiation (7Wada T. Nakashima T. Oliveira-dos-Santos A.J. Gasser J. Hara H. Schett G. Penninger J.M. Nat. Med. 2005; 11: 394-399Crossref PubMed Scopus (151) Google Scholar). Gab2 knock-out mice also have defective hematopoiesis (8Zhang Y. Diaz-Flores E. Li G. Wang Z. Kang Z. Haviernikova E. Rowe S. Qu C.K. Tse W. Shannon K.M. Bunting K.D. Blood. 2007; 110: 116-124Crossref PubMed Scopus (40) Google Scholar). It has been reported that Gab2 plays a pivotal role in BCR/ABL-induced transformation (9Sattler M. Mohi M.G. Pride Y.B. Quinnan L.R. Malouf N.A. Podar K. Gesbert F. Iwasaki H. Li S. Van Etten R.A. Gu H. Griffin J.D. Neel B.G. Cancer Cell. 2002; 1: 479-492Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) and breast cancer (10Daly R.J. Gu H. Parmar J. Malaney S. Lyons R.J. Kairouz R. Head D.R. Henshall S.M. Neel B.G. Sutherland R.L. Oncogene. 2002; 21: 5175-5181Crossref PubMed Scopus (85) Google Scholar). Gab2 is one of several genes in a highly amplified locus in some breast cancers (11Bentires-Alj M. Gil S.G. Chan R. Wang Z.C. Wang Y. Imanaka N. Harris L.N. Richardson A. Neel B.G. Gu H. Nat. Med. 2006; 12: 114-121Crossref PubMed Scopus (173) Google Scholar). Gab2 has also been shown to be a direct target of the transcription factor E2F and an essential effector of E2F-dependent Akt activation during cell cycle progression (12Chaussepied M. Ginsberg D. Mol. Cell. 2004; 16: 831-837Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Calcium (Ca2+) is a universal second messenger, and the temporal and spatial regulation of intracellular Ca2+ enables cells to respond to various stimuli and control cellular processes including proliferation, development, contraction, secretion, and motility (13Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar). Ca2+ can act directly on target proteins, or its effect can be mediated via intracellular Ca2+-binding proteins. A major intracellular modulator for Ca2+ is calmodulin (CaM), which is a highly conserved four EF-hand-containing Ca2+-binding protein. One of the Ca2+/CaM-dependent targets is the serine/threonine phosphatase, calcineurin (Cn) (reviewed in Refs. 14Baksh S. Burakoff S.J. Semin. Immunol. 2000; 12: 405-415Crossref PubMed Scopus (52) Google Scholar and 15Kahl C.R. Means A.R. Endocr. Rev. 2003; 24: 719-736Crossref PubMed Scopus (399) Google Scholar). Although Ca2+/CaM has been shown to be required for proliferation in both unicellular and multicellular eukaryotes, the nature and regulation of the Ca2+-dependent pathway, including the role of Cn in regulating cell proliferation, is not clear. Here we report a novel, cytokine (IL-3)-induced, calcium- and Cn-dependent activation of c-fos, modulated by the scaffolding adaptor protein Gab2. We demonstrate that Gab2 directly associates with Cn and regulates c-fos, and this association modulates IL-3-mediated cell growth and proliferation. These results reveal a novel link between cytokine signals and cell growth and proliferation signals mediated via Gab2 and Cn. We also show that the Cn-Gab2 interaction can be regulated by the Akt Kinase. Cell Lines—Baf3 cells were cultured in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% fetal bovine serum, 2 μm l-glutamine, 1 μm HEPES, 0.05 μm 2-β-mercaptoethanol) supplemented with 10% WEHI conditioned medium. For IL-3 deprivation experiments, Baf3 cells were starved in RPMI with 0.8% bovine serum albumin or 0.5% serum for 16–18 h. For IL-3 stimulation, recombinant mouse IL-3 from R&D systems (Minneapolis, MN) was added to the deprivation medium at 10 ng/ml. Antibodies and Reagents—Antibodies were purchased from the following vendors: anti-Gab2 from Upstate Biotechnology (Lake Placid, NY) and AbCam (Cambridge, MA); anti-phospho-Gab2 (Ser-159), anti-Akt, anti-phospho (Ser/Thr) Akt substrate, anti-phospho Akt (Ser-473), all from Cell Signaling (Beverly, MA); anti-Cn A from BD Transduction Laboratories; and anti-Cn B from Affinity Bioreagents (Golden, CO). Antihemagglutinin (HA) antibody (12CA5) was purified from hybridoma supernatants in our laboratory. Propidium iodide and verapamil were purchased from Sigma-Aldrich, and BAPTA-1 AM was from Invitrogen – Molecular Probes. Trypan Blue 0.4% was from Invitrogen. The Dual-Luciferase assay kit was purchased from Promega (Madison, WI). Plasmid Constructs—The Gab2 constructs used in these studies have been described previously (1Gu 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) or were generated by PCR-assisted mutation strategies. Each mutated construct was sequenced to verify the existence of the desired mutation and for the absence of PCR-generated mutations. All the Cn constructs used have been described previously (16Baksh S. DeCaprio J.A. Burakoff S.J. Oncogene. 2000; 19: 2820-2827Crossref PubMed Scopus (49) Google Scholar). Cn A subunit or mutants were always co-transfected with Cn B subunit to obtain the optimal Cn function in vivo. The Akt and Akt (kinase dead) constructs were a kind gift from Dr. Moses Chao (NYU School of Medicine, NY). Luciferase Assay—Baf3 cells (107) were transfected with 10 μg of the indicated plasmids at 800 microfarads/250 V using an Invitrogen electroporator. All cells were cotransfected with 30 ng of a Renilla luciferase construct (Promega) to normalize for transfection efficiency. Once the cells were recovered in complete RPMI supplemented with WEHI conditioned medium for 2–3 h, they were spun down, washed, and resuspended in IL-3 deprivation medium lacking IL-3 for 16–18 h. An aliquot (106 cells) was set aside for immunoblot analysis to confirm expression of the transfected proteins. For IL-3 stimulation, the transfected cells were resuspended in 1 ml of deprivation medium and divided into aliquots to be left unstimulated or stimulated with IL-3 (10 ng/ml) for 6 h. Cells were then washed once with phosphate-buffered saline and lysed. Luciferase activities were determined with a Dual-Luciferase kit (Promega) and Berthold Detection Systems Sirius single tube luminometer (Oakridge, TN). Immunoprecipitation and Immunoblotting—Cell lysis, immunoprecipitation, immunoblotting, and detection by enhanced chemiluminescence were performed as described previously (17Timms J.F. Carlberg K. Gu H. Chen H. Kamatkar S. Nadler M.J. Rohrschneider L.R. Neel B.G. Mol. Cell Biol. 1998; 18: 3838-3850Crossref PubMed Scopus (175) Google Scholar). Anti-Cn B or anti-HA immunoprecipitations utilized 1 μg of antibody/107 Baf3 cell equivalents. Briefly, unstimulated or stimulated transfected cells were washed and lysed in buffer containing 1% Triton X-100, 150 mm NaCl, 1 mm CaCl2, and a mixture of protease and phosphatase inhibitors. Lysates were spun at 13,000 rpm for 10 min and subjected to immunoprecipitation overnight with the appropriate antibody. Protein A-Sepharose beads and rabbit anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA), when required, were added the next day. The beads were washed extensively with immunoprecipitation wash buffer (20 mm HEPES, 150 mm NaCl, 10% glycerol, 1 mm CaCl2, and a mixture of protease and phosphatase inhibitors). Bound proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the appropriate antibodies. The enhanced chemiluminescence detection system (Amersham Biosciences) was used to visualize proteins. Cell Cycle Analysis—Plasmid transfections into Baf3 cells were carried out by Amaxa-based nucleofection following the manufacturer's instructions (Amaxa, Gaithersburg, MD). 10 ng of enhanced green fluorescent protein plasmid (eGFP) (Invitrogen) was also co-transfected with each transfection and used to sort the transfected cells using a MoFlo high speed cell sorter (Cytomation, Fort Collins, CO). The sorted cells were labeled with propidium iodide and analyzed on FACScan (BD Biosciences). Cell cycle analysis was done by FlowJo software package (Treestar, Ashland, OR). IL-3 has long been recognized as a key cytokine required for hematopoietic progenitor cell survival and proliferation. The process of induction of cell death upon withdrawal of IL-3 has been extensively investigated (18Maurer U. Charvet C. Wagman A.S. Dejardin E. Green D.R. Mol. Cell. 2006; 21: 749-760Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). IL-3-induced protection against cell death is thought to be mediated by the activation of the PI3-kinase/Akt signaling pathway. In other reports, calcium has been shown to protect against death and promote cell survival (19Yano S. Tokumitsu H. Soderling T.R. Nature. 1998; 396: 584-587Crossref PubMed Scopus (536) Google Scholar). Upon withdrawal of IL-3, [Ca2+]i and Cn activity were required for cell survival in an IL-3-dependent cell line (20Palaga T. Kataoka T. Nagai K. Int. Immunopharmacol. 2004; 4: 953-961Crossref PubMed Scopus (8) Google Scholar). There are numerous reports of calcium regulating the immediate early genes (IEGs), including c-fos (21Li S.L. Cougnon N. Bresson-Bepoldin L. Zhao S.J. Schlegel W. J. Mol. Endocrinol. 1996; 16: 229-238Crossref PubMed Scopus (14) Google Scholar), that are involved in cell survival and proliferation in a cell-specific manner. However, the details of proximal signaling mechanisms initiated by IL-3 resulting in cell survival and proliferation are not well understood. We, therefore, investigated the proximal signals induced by IL-3 in activating c-fos and leading to cell survival and proliferation. We utilized the IL-3-dependent Pro-B cell line Baf3 to investigate the IL-3-dependent activation of c-fos. We investigated: 1) whether verapamil, an ion channel blocker, could block IL-3 signals; 2) whether a constitutively of Cn that not activation by Ca2+ can the effect of and whether IL-3 signaling leading to the activation of c-fos is on intracellular c-fos is induced early upon activation and is to a role in cell growth and proliferation. We have shown previously that c-fos is one of the upon IL-3 of Baf3 cells (1Gu 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). We the of the IL-3 signals on a c-fos luciferase gene The Cn A subunit was always co-transfected with the Cn B subunit for optimal Cn Baf3 cells transfected with wild type Cn or a constitutively of with a c-fos luciferase construct were for 16–18 and then left unstimulated or stimulated with IL-3 for 6 h. reported there is a activation of the c-fos upon IL-3 of Baf3 cells IL-3-induced luciferase gene activity was upon with verapamil, an ion channel blocker, and this was by the expression of Cn not by Cn Baf3 cells in complete of [Ca2+]i that could be by not Upon and with IL-3, the Baf3 cells a activation of the c-fos of intracellular Ca2+ by to a complete of c-fos activity in the of there was effect of when Cn was Gab2, a scaffolding adaptor is upon IL-3 (1Gu 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). investigate the role for both Cn and Gab2 in IL-3-mediated c-fos we to the nature of the complex that is upon IL-3 whether there is a direct interaction between Cn and Gab2 upon IL-3 stimulation, we We the or Cn in the of Gab2 in Baf3 A association could be by with an anti-Cn B antibody and for Gab2. Although IL-3 to an of Gab2 to the constitutively on IL-3 for Cn has been extensively as an phosphatase regulating the transcription factor factor of the activation of c-fos via Ca2+ and Cn has not been We, therefore, whether the IL-3-mediated interaction could to a activation of c-fos. Gab2 and Cn constructs were or with a c-fos as described Although expression of Gab2 or Cn c-fos activity and there was when the proteins were leading to a in c-fos activity as with the unstimulated control The expression of the constitutively of Cn in c-fos activity of IL-3 stimulation, this was not when Cn was these show that Cn and Gab2 can directly and of Cn with Gab2 c-fos We also the phosphatase Cn in Baf3 cells with the c-fos luciferase shown in the expression of the phosphatase Cn in complete of IL-3-induced c-fos activity in the of Gab2 of Gab2 by also c-fos activity to not These results an role for both Gab2 and Cn in IL-3-mediated c-fos It has been previously that the serine/threonine Akt could be a of Gab2 activity by at D.K. Daly R.J. EMBO J. 2002; 21: 72-82Crossref PubMed Scopus (73) Google Scholar). We an analysis to the of Gab2 that with Cn not analysis a direct and the to the pleckstrin homology of Gab2 was to be for the interaction. It was that the that we as for Cn the We, therefore, investigated the IL-3-dependent regulation of Gab2 by Akt has been previously implicated in cell survival and is upon IL-3 Baf3 cells were stimulated with IL-3, we could phosphorylation of Gab2 as indicated by the antibody We then determined whether the Gab2 could be immunoblotted with an antibody. For this Gab2 was with wild type Akt or We that Gab2 could be by the antibody when the Akt was and not when the Akt was We then determined whether Akt effect on IL-3-induced activation of c-fos mediated by we Akt with Gab2 and c-fos activation was negative regulation of the complex These results that Akt could Gab2 and regulate the complex and c-fos activation by whether Gab2 could be a for we transfected in Gab2 and Akt with Cn or Cn shown in Gab2 could be by antibody in the of phosphatase Cn and not Cn. These results that Gab2 can be a for Cn. We and have previously shown that and Gab2 could all be involved in cell growth and proliferation (3Gu H. Griffin J.D. Neel B.G. J. Biol. Chem. 1997; 272: 16421-16430Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, PubMed Scopus Google Scholar, T. D. Daly R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. W. Wang Y. M. Y. P. Neel B.G. Gu H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we show that upon IL-3 stimulation, Cn and Gab2 to c-fos. We, therefore, whether IL-3-induced activation of c-fos could regulate cell growth and proliferation. We performed cell cycle analysis of Baf3 cells Gab2 and Cn or were sorted by cell sorter the enhanced green fluorescent protein plasmid that was co-transfected at were analyzed for by propidium iodide on cell The of cells in the of cell cycle for each transfection are shown in shown in of Baf3 cells transfected with control were in the of cell when for IL-3 as with with of Gab2 or Cn not the of cells in the IL-3 or IL-3 Gab2 were a negative of proliferation, be that the expression of Gab2 the of cells in the of cell such is and this could be to the of Gab2 to be by Akt to be a negative this phosphorylation could be a that regulates the cell survival and proliferation. Upon of Cn with Gab2, a of cells were and the of cells in the of the cell cycle from to of IL-3 There was also a in the of cells in with IL-3 from to when Cn was with Gab2, of cells from apoptosis upon IL-3 withdrawal was with Gab2, the phosphatase activity of Cn is required as indicated by the in the of cells from to of IL-3 withdrawal and from to IL-3 Although this is this could be to the phosphatase Cn as a negative and Gab2 other proteins from the the phosphatase activity of Cn could be required for and Gab2 to to the as for S. J. S. DeCaprio J.A. J.F. Burakoff S.J. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google cycle analysis of cells in the of cell in a signaling that regulate cellular functions including survival, and proliferation. IL-3 is to an essential role in survival, and proliferation of hematopoietic cells both in and vivo. IL-3 that to cellular outcomes. Although IL-3 signals via the PI3-kinase/Akt for cell survival, also the protein for cell growth and the for cell differentiation. The by which activation of a cytokine receptor to signals that are within the cell to a that is to be The scaffolding adaptor Gab2 is one of the proteins that is upon IL-3 signaling (1Gu 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, H. Griffin J.D. Neel B.G. J. Biol. Chem. 1997; 272: 16421-16430Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). reports Gab2 in numerous proliferation signaling including receptor for factor B and (7Wada T. Nakashima T. Oliveira-dos-Santos A.J. Gasser J. Hara H. Schett G. Penninger J.M. Nat. Med. 2005; 11: 394-399Crossref PubMed Scopus (151) Google Scholar, M. J. W. Wang Y. M. Y. P. Neel B.G. Gu H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, J.F. J.M. S.M. J. Blood. 2002; PubMed Scopus Google Scholar). We that Gab2 modulates IL-3 signaling by or with other proliferation resulting in cell growth and proliferation. We show that IL-3-induced Cn activation and that its association with Gab2 modulates the regulation of c-fos. Although calcium and Cn have been previously implicated in cell growth and proliferation, the of its function by association with adaptor proteins the cellular or for the signals to be regulated in an manner. It could be that these can then cells cell cycle on the from both external and A report of E2F regulation of Gab2 transcription and Akt activation to this (12Chaussepied M. Ginsberg D. Mol. Cell. 2004; 16: 831-837Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Cell activation of Akt by Gab2 the regulation of and proliferation via the in the of cell survival signals mediated via Cn is a protein that also plays an essential role in cellular functions such as cell proliferation, differentiation, and death 2003; PubMed Scopus Google Scholar). The role of Cn in factor of signaling is there is a of proteins that a role in regulating or signaling Cn for with adaptor proteins such as Cn with protein and protein J. Cell Biol. 2003; PubMed Scopus (107) Google Scholar) within cellular such as which Cn to the complex J.P. A.J. S.M. Cell. Full Text PDF PubMed Scopus Google Scholar) and that Cn to in N. Richardson J.A. U. S. A. 2000; PubMed Scopus Google Scholar). All of these adaptor proteins to Cn a for its Gab2, an adaptor also the cellular for calcium and Cn function in cell growth and differentiation. is that Gab2 and Cn is to the in a of function in activating c-fos, which in is of the cell growth and proliferation In this we for Gab2 and Cn directly and the and of cell survival signals via Akt and cell proliferation signals of c-fos. The of Gab2 phosphorylation the for signaling in a these signals of cell survival and proliferation are regulated and amplified in cellular to be We the Burakoff for and We also Dr. for with

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