FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2090936301> ?p ?o ?g. }

• W2090936301 endingPage "13880" @default.
• W2090936301 startingPage "13869" @default.
• W2090936301 abstract "Ikaros encodes a zinc finger protein that is involved in gene regulation and chromatin remodeling. The majority of Ikaros localizes at pericentromeric heterochromatin (PC-HC) where it regulates expression of target genes. Ikaros function is controlled by posttranslational modification. Phosphorylation of Ikaros by CK2 kinase determines its ability to bind DNA and exert cell cycle control as well as its subcellular localization. We report that Ikaros interacts with protein phosphatase 1 (PP1) via a conserved PP1 binding motif, RVXF, in the C-terminal end of the Ikaros protein. Point mutations of the RVXF motif abolish Ikaros-PP1 interaction and result in decreased DNA binding, an inability to localize to PC-HC, and rapid degradation of the Ikaros protein. The introduction of alanine mutations at CK2-phosphorylated residues increases the half-life of the PP1-nonbinding Ikaros mutant. This suggests that dephosphorylation of these sites by PP1 stabilizes the Ikaros protein and prevents its degradation. In the nucleus, Ikaros forms complexes with ubiquitin, providing evidence that Ikaros degradation involves the ubiquitin/proteasome pathway. In vivo, Ikaros can target PP1 to the nucleus, and a fraction of PP1 colocalizes with Ikaros at PC-HC. These data suggest a novel function for the Ikaros protein; that is, the targeting of PP1 to PC-HC and other chromatin structures. We propose a model whereby the function of Ikaros is controlled by the CK2 and PP1 pathways and that a balance between these two signal transduction pathways is essential for normal cellular function and for the prevention of malignant transformation. Ikaros encodes a zinc finger protein that is involved in gene regulation and chromatin remodeling. The majority of Ikaros localizes at pericentromeric heterochromatin (PC-HC) where it regulates expression of target genes. Ikaros function is controlled by posttranslational modification. Phosphorylation of Ikaros by CK2 kinase determines its ability to bind DNA and exert cell cycle control as well as its subcellular localization. We report that Ikaros interacts with protein phosphatase 1 (PP1) via a conserved PP1 binding motif, RVXF, in the C-terminal end of the Ikaros protein. Point mutations of the RVXF motif abolish Ikaros-PP1 interaction and result in decreased DNA binding, an inability to localize to PC-HC, and rapid degradation of the Ikaros protein. The introduction of alanine mutations at CK2-phosphorylated residues increases the half-life of the PP1-nonbinding Ikaros mutant. This suggests that dephosphorylation of these sites by PP1 stabilizes the Ikaros protein and prevents its degradation. In the nucleus, Ikaros forms complexes with ubiquitin, providing evidence that Ikaros degradation involves the ubiquitin/proteasome pathway. In vivo, Ikaros can target PP1 to the nucleus, and a fraction of PP1 colocalizes with Ikaros at PC-HC. These data suggest a novel function for the Ikaros protein; that is, the targeting of PP1 to PC-HC and other chromatin structures. We propose a model whereby the function of Ikaros is controlled by the CK2 and PP1 pathways and that a balance between these two signal transduction pathways is essential for normal cellular function and for the prevention of malignant transformation. The Ikaros gene encodes a C2H2 zinc finger protein that is essential for normal hematopoiesis (1Nichogiannopoulou A. Trevisan M. Neben S. Friedrich C. Georgopoulos K. J. Exp. Med. 1999; 190: 1201-1214Crossref PubMed Scopus (183) Google Scholar). The absence of Ikaros expression leads to severely impaired lymphoid development as well as defects in myeloid and erythroid differentiation (2Wu L. Nichogiannopoulou A. Shortman K. Georgopoulos K. Immunity. 1997; 7: 483-492Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 3Avitahl N. Winandy S. Friedrich C. Jones B. Ge Y. Georgopoulos K. Immunity. 1999; 10: 333-343Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Winandy S. Wu L. Wang J.H. Georgopoulos K. J. Exp. Med. 1999; 190: 1039-1048Crossref PubMed Scopus (130) Google Scholar, 5Dumortier A. Kirstetter P. Kastner P. Chan S. Blood. 2003; 101: 2219-2226Crossref PubMed Scopus (65) Google Scholar). Studies of mice with a disruption in the Ikaros gene demonstrate that Ikaros acts as a tumor suppressor (6Winandy S. Wu P. Georgopoulos K. Cell. 1995; 83: 289-299Abstract Full Text PDF PubMed Scopus (355) Google Scholar). In humans, impaired Ikaros activity has been associated with the development of infant T-cell acute lymphoblastic leukemia (ALL) (7Sun L. Heerema N. Crotty L. Wu X. Navara C. Vassilev A. Sensel M. Reaman G.H. Uckun F.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 680-685Crossref PubMed Scopus (168) Google Scholar), adult B cell ALL (8Nakase K. Ishimaru F. Avitahl N. Dansako H. Matsuo K. Fujii K. Sezaki N. Nakayama H. Yano T. Fukuda S. Imajoh K. Takeuchi M. Miyata A. Hara M. Yasukawa M. Takahashi I. Taguchi H. Matsue K. Nakao S. Niho Y. Takenaka K. Shinagawa K. Ikeda K. Niiya K. Harada M. Cancer Res. 2000; 60: 4062-4065PubMed Google Scholar), myelodysplastic syndrome (9Crescenzi B. La Starza R. Romoli S. Beacci D. Matteucci C. Barba G. Aventin A. Marynen P. Ciolli S. Nozzoli C. Martelli M.F. Mecucci C. Haematologica. 2004; 89: 281-285PubMed Google Scholar), acute myeloid leukemia (10Yagi T. Hibi S. Takanashi M. Kano G. Tabata Y. Imamura T. Inaba T. Morimoto A. Todo S. Imashuku S. Blood. 2002; 99: 1350-1355Crossref PubMed Scopus (65) Google Scholar), and adult and juvenile chronic myelogenous leukemia (11Nakayama H. Ishimaru F. Avitahl N. Sezaki N. Fujii N. Nakase K. Ninomiya Y. Harashima A. Minowada J. Tsuchiyama J. Imajoh K. Tsubota T. Fukuda S. Sezaki T. Kojima K. Hara M. Takimoto H. Yorimitsu S. Takahashi I. Miyata A. Taniguchi S. Tokunaga Y. Gondo H. Niho Y. Nakao S. Kgo T. Dohy H. Kamada N. Harada M. Cancer Res. 1999; 59: 3931-3934PubMed Google Scholar). Recently, the deletion of Ikaros has been demonstrated in more than 30% of childhood leukemias, whereas a single copy or the complete absence of Ikaros was observed in more than 80% of patients with bcr-abl-positive acute lymphoblastic leukemia (12Mullighan C.G. Goorha S. Radtke I. Miller C.B. Coustan-Smith E. Dalton J.D. Girtman K. Mathew S. Ma J. Pounds S.B. Su X. Pui C.H. Relling M.V. Evans W.E. Shurtleff S.A. Downing J.R. Nature. 2007; 446: 758-764Crossref PubMed Scopus (1349) Google Scholar, 13Mullighan C.G. Miller C.B. Radtke I. Phillips L.A. Dalton J. Ma J. White D. Hughes T.P. Le Beau M.M. Pui C.H. Relling M.V. Shurtleff S.A. Downing J.R. Nature. 2008; 453: 110-114Crossref PubMed Scopus (792) Google Scholar). These studies established Ikaros as a key regulator of hematopoiesis and a major tumor suppressor whose loss of function is associated with the malignant transformation of hematopoietic cells. The mechanism by which Ikaros exerts its tumor suppressor activity is less clear. Ikaros exhibits a punctate staining pattern in normal and leukemia cells (14Klug C.A. Morrison S.J. Masek M. Hahm K. Smale S.T. Weissman I.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 657-662Crossref PubMed Scopus (139) Google Scholar). Co-staining for Ikaros and HP1 proteins showed that this punctate staining pattern is because of the localization of Ikaros to pericentromeric heterochromatin (PC-HC) 3The abbreviations used are: PC-HC, pericentromeric heterochromatin; PP1, protein phosphatase 1; DRB, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole; TBB, 4,5,6,7-tetrabromo-1H-benzotriazole; GST, glutathione S-transferase; RIPA, radioimmune precipitation assay buffer. 3The abbreviations used are: PC-HC, pericentromeric heterochromatin; PP1, protein phosphatase 1; DRB, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole; TBB, 4,5,6,7-tetrabromo-1H-benzotriazole; GST, glutathione S-transferase; RIPA, radioimmune precipitation assay buffer. (15Brown K.E. Guest S.S. Smale S.T. Hahm K. Merkenschlager M. Fisher A.G. Cell. 1997; 91: 845-854Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Ikaros binds in vitro and in vivo to the murine and human gamma satellite repeats that are located within the PC-HC, and Ikaros DNA binding ability is essential for its localization to PC-HC (16Cobb B.S. Morales-Alcelay S. Kleiger G. Brown K.E. Fisher A.G. Smale S.T. Genes Dev. 2000; 14: 2146-2160Crossref PubMed Scopus (209) Google Scholar). Ikaros binds to the regulatory elements of its target genes in a sequence-dependent manner. Ikaros binding leads to the activation or repression of target genes via chromatin remodeling (17Koipally J. Heller E.J. Seavitt J.R. Georgopoulos K. J. Biol. Chem. 2002; 277: 13007-13015Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 18Thompson E.C. Cobb B.S. Sabbattini P. Meixlsperger S. Parelho V. Liberg D. Taylor B. Dillon N. Georgopoulos K. Jumaa H. Smale S.T. Fisher A.G. Merkenschlager M. Immunity. 2007; 26: 335-344Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Thus, optimal Ikaros DNA binding is essential both for its subcellular localization and for its regulation of target genes. Ikaros associates with histone deacetylase (HDAC)-containing complexes (NuRD and Sin3) (19Koipally J. Renold A. Kim J. Georgopoulos K. EMBO J. 1999; 18: 3090-3100Crossref PubMed Scopus (256) Google Scholar), although HDAC-independent binding to the transcriptional corepressor C-terminal binding protein has been reported as well (20Koipally J. Georgopoulos K. J. Biol. Chem. 2000; 275: 19594-19602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Ikaros also associates with Brg-1, a catalytic subunit of the SWI/SNF nucleosome remodeling complex that acts as an activator of gene expression (21O'Neill D.W. Schoetz S.S. Lopez R.A. Castle M. Rabinowitz L. Shor E. Krawchuk D. Goll M.G. Renz M. Seelig H.P. Han S. Seong R.H. Park S.D. Agalioti T. Munshi N. Thanos D. Erdjument-Bromage H. Tempst P. Bank A. Mol. Cell. Biol. 2000; 20: 7572-7582Crossref PubMed Scopus (138) Google Scholar, 22Kim J. Sif S. Jones B. Jackson A. Koipally J. Heller E. Winandy S. Viel A. Sawyer A. Ikeda T. Kingston R. Georgopoulos K. Immunity. 1999; 10: 345-355Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). The current hypothesis is that Ikaros binds the upstream region of target genes and aids in their recruitment to PC-HC, resulting in repression or activation of the gene (15Brown K.E. Guest S.S. Smale S.T. Hahm K. Merkenschlager M. Fisher A.G. Cell. 1997; 91: 845-854Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 23Liberg D. Smale S.T. Merkenschlager M. Trends Immunol. 2003; 24: 567-570Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Thus, Ikaros can act both as an activator and a repressor, depending on whether it associates with the NuRD, the C-terminal binding protein, or the SWI/SNF complex. Ikaros activity is regulated at a posttranslational level by various mechanisms. The association of the full-length Ikaros protein with its dominant negative isoforms impairs its activity, whereas the formation of complexes with other active isoforms can modify its function (24Molnár A. Georgopoulos K. Mol. Cell. Biol. 1994; 14: 8292-8303Crossref PubMed Scopus (367) Google Scholar, 25Ronni T. Payne K.J. Ho S. Bradley M.N. Dorsam G. Dovat S. J. Biol. Chem. 2007; 282: 2538-2547Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Posttranslational modifications at specific residues regulate Ikaros activity. Sumoylation at two amino acids has been reported to regulate Ikaros repressor function (26Gomez-del Arco P. Koipally J. Georgopoulos K. Mol. Cell. Biol. 2005; 25: 2688-2697Crossref PubMed Scopus (82) Google Scholar). The cell cycle-specific phosphorylation of Ikaros at an evolutionarily conserved linker regulates its DNA binding ability and nuclear localization during mitosis (27Dovat S. Ronni T. Russell D. Ferrini R. Cobb B.S. Smale S.T. Genes Dev. 2002; 16: 2985-2990Crossref PubMed Scopus (103) Google Scholar). Phosphorylation of Ikaros by CK2 kinase at its C-terminal region regulates its ability to control G1/S cell cycle progression (28Gomez-del Arco P. Maki K. Georgopoulos K. Mol. Cell. Biol. 2004; 24: 2797-2807Crossref PubMed Scopus (75) Google Scholar). Our previous work identified additional N-terminal phosphorylation sites that are phosphorylated either directly by CK2 kinase or by another kinase in the CK2 signal transduction pathway (29Gurel Z. Ronni T. Ho S. Kuchar J. Payne K.J. Turk C.W. Dovat S. J. Biol. Chem. 2008; 283: 8291-8300Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Reversible phosphorylation of these amino acids regulates the subcellular localization of Ikaros to PC-HC as well as its ability to bind the upstream regulatory element of the Ikaros target gene, TdT (29Gurel Z. Ronni T. Ho S. Kuchar J. Payne K.J. Turk C.W. Dovat S. J. Biol. Chem. 2008; 283: 8291-8300Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These data identified CK2 kinase as a major regulator of Ikaros function. Because the overexpression of CK2 kinase leads to T-cell leukemia, it has been hypothesized that the impairment of Ikaros function is one of the mechanisms by which CK2 kinase promotes malignant transformation. In this report we demonstrate that Ikaros is dephosphorylated by protein phosphatase 1 (PP1) and identify the specific amino acids that are responsible for Ikaros-PP1 interactions. We show that the process of Ikaros dephosphorylation by PP1 is essential for normal Ikaros function and to prevent accelerated Ikaros degradation via the ubiquitin pathway. We also propose a novel function for Ikaros; that is, to target PP1 to PC-HC in hematopoietic cells. Cells—The murine VL3-3M2 thymocyte leukemia cell line has been described previously (30Groves T. Katis P. Madden Z. Manickam K. Ramsden D. Wu G. Guidos C.J. J. Immunol. 1995; 154: 5011-5022PubMed Google Scholar). The human HEK 293T (293T) endothelial kidney cell line, the human T cell leukemia cell lines MOLT-4 and CCRF-CEM (CEM), and the human Burkitt's B cell lymphoma cell line, Ramos, were obtained from American Type Culture Collection (ATCC), Manassas, VA. The murine B cell lines BAL17 and HAFTL have been described previously (31Alessandrini A. Pierce J.H. Baltimore D. Desiderio S.V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1799-1803Crossref PubMed Scopus (25) Google Scholar, 32Mizuguchi J. Tsang W. Morrison S.L. Beaven M.A. Paul W.E. J. Immunol. 1986; 137: 2162-2167PubMed Google Scholar). Antibodies—The antibodies used to detect the C terminus (Ikaros-CTS) of Ikaros (comprising amino acids (320-515 of the murine IK-VI isoform) have been described previously (33Hahm K. Cobb B.S. McCarty A.S. Brown K.E. Klug C.A. Lee R. Akashi K. Weissman I.L. Fisher A.G. Smale S.T. Genes Dev. 1998; 12: 782-796Crossref PubMed Scopus (208) Google Scholar). The antibodies used to detect PP1 were purchased from Santa Cruz Biotechnology, Santa Cruz, CA (sc-7482). The antibodies against ubiquitin were from Assay Designs/Stressgen Bioreagents, Ann Arbor, MI (SPA-200). Antibodies to detect the hemagglutinin tag (12CA5) were purchased from Covance (Princeton, NJ). In Vivo Labeling—For in vivo labeling, 293T cells were incubated with radioactive orthophosphate. Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were washed twice with phosphate-free RPMI 1640 medium and incubated for 4 h with 0.5 mCi/ml [32P]orthophosphate (PerkinElmer Life Sciences) in phosphate-free medium. Cells were collected by centrifugation, lysed on ice for 20 min in solubilizing buffer (50 mm Tris-HCL pH 7.2, 1% v/v Nonidet P-40, 150 mm NaCl, 5 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 5 μm leupeptin), and centrifuged at 15,000 rpm at 4 °C for 20 min. Lysates were incubated with anti-Ikaros-CTS antibodies for 1 h at 4 °C, and the resulting immune complexes were absorbed to protein G-Sepharose (Amersham Biosciences), washed 4 times with solubilizing buffer, separated by SDS-PAGE, transferred to a nylon membrane, and subjected to autoradiography. Expression and Purification of Recombinant Ikaros Proteins—cDNA for the murine full-length Ikaros isoform, IK-VI, was amplified and cloned into a pGEX-2T GST-containing vector (Amersham Biosciences) that was cleaved with BamH1 and EcoR1 as described previously (34Hahm K. Ernst P. Lo K. Kim G.S. Turck C. Smale S.T. Mol. Cell. Biol. 1994; 14: 7111-7123Crossref PubMed Scopus (196) Google Scholar). IK-VI-GST-containing pGEX-2T plasmid was transformed and expressed in the SCS-1 strain of Escherichia coli (Stratagene, La Jolla, CA). E. coli were induced with 1 mm iospropylthiogalactopyranoside for 2 h in the presence of 100 μm ZnCl2. The GST-IK-VI fusion protein was purified from E. coli according to the standard procedures (Amersham Biosciences) except that all of the buffers contained 10 μm ZnCl2 as described previously (34Hahm K. Ernst P. Lo K. Kim G.S. Turck C. Smale S.T. Mol. Cell. Biol. 1994; 14: 7111-7123Crossref PubMed Scopus (196) Google Scholar). Fractions that contained purified GST-IK-VI protein were pooled and stored at -80 °C. GST Pulldown Assay—The full-length Ikaros-GST fusion protein (10 μg) or GST protein, as a negative control, were incubated with glutathione-agarose beads and then mixed with cell lysate of VL3-3M2 cells (a total of 2 mg of protein in lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 120 mm NaCl, protease inhibitors (phenylmethylsulfonyl fluoride, pepstatin and leupeptin)) for 2 h. After incubations, samples were washed with lysis buffer, and proteins were eluted with SDS sample buffer. Proteins were separated by SDS-PAGE, and the presence of PP1 was detected by Western blot with anti-PP1 antibody. Plasmids and Transfection—Alanine substitution mutants for IK-VI were generated using the QuikChange method (Stratagene). The full-length Ikaros isoform IK-VI and its alanine mutants were cloned into the mammalian expression vector pcDNA3 (Invitrogen). For transfection, 1 μg of each construct was used to transfect 293T cells via the calcium phosphate method. Confocal Microscopy—293T cells were transfected with appropriate constructs and analyzed by confocal microscopy as described previously (16Cobb B.S. Morales-Alcelay S. Kleiger G. Brown K.E. Fisher A.G. Smale S.T. Genes Dev. 2000; 14: 2146-2160Crossref PubMed Scopus (209) Google Scholar). Images were acquired at room temperature by a Leica TCS-SP MP Confocal and Multiphoton Microscope with a Leica DM-LFS body (upright fixed-stage microscope) using a 100× Leica HX PLAPO (Planapo) oil immersion lens with numerical aperture of 1.4 (Heidelberg, Germany). Biochemical Experiments—Nuclear extractions, Western blots, and gel-shift experiments were performed as described previously (16Cobb B.S. Morales-Alcelay S. Kleiger G. Brown K.E. Fisher A.G. Smale S.T. Genes Dev. 2000; 14: 2146-2160Crossref PubMed Scopus (209) Google Scholar, 35Trinh L.A. Ferrini R. Cobb B.S. Weinmann A.S. Hahm K. Ernst P. Garraway I.P. Merkenschlager M. Smale S.T. Genes Dev. 2001; 15: 1817-1832Crossref PubMed Scopus (123) Google Scholar). The γ satellite A and γ satellite B gel shift probes have been described previously (16Cobb B.S. Morales-Alcelay S. Kleiger G. Brown K.E. Fisher A.G. Smale S.T. Genes Dev. 2000; 14: 2146-2160Crossref PubMed Scopus (209) Google Scholar). Degradation Assay—Pulse-chase analysis was performed in 293T cells as described previously (36Li Y. Suresh Kumar K.G. Tang W. Spiegelman V.S. Fuchs S.Y. Mol. Cell Biol. 2004; 24: 4038-4048Crossref PubMed Scopus (74) Google Scholar, 37Bhatia N. Thiyagarajan S. Elcheva I. Saleem M. Dlugosz A. Mukhtar H. Spiegelman V.S. J. Biol. Chem. 2006; 281: 19320-19326Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The 293T cells were grown in 100-mm tissue culture dishes and transfected with full-length Ikaros constructs or the indicated mutants. Cells were grown in Dulbecco's modified Eagle's medium for 24 h, and then medium was replaced with cysteine- and methionine-free Dulbecco's modified Eagle's medium. After starvation for 1 h, cells were labeled with a [35S]methionine/[35S]cysteine mixture (PerkinElmer Life Sciences). Chase was performed in Dulbecco's modified Eagle's medium with 10% fetal bovine serum supplemented with 2 mm unlabeled methionine and cysteine. Cells were harvested at indicated time points and lysed in RIPA buffer with proteinase and phosphatase inhibitors. Ikaros proteins were immunoprecipitated using anti-Ikaros-CTS antibodies, separated by SDS-PAGE, and visualized by autoradiography. In the degradation assay described in Fig. 4D, MOLT-4 cells were treated with cycloheximide (50 μg/ml) (Sigma-Aldrich catalogue #C1988) in the presence or absence of calyculin (10 nm) (Calbiochem catalogue #208851) and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB, 50 μm) (Calbiochem catalogue #287891) or 4,5,6,7-tetrabromo-1H-benzotriazole (TBB, 10 μm) (Sigma). Cells were harvested at the indicated times, nuclear extract was obtained, and proteins from 20 μg of nuclear extracts from indicated time points were separated by SDS-PAGE. The presence of Ikaros isoforms was detected by anti-Ikaros-CTS antibodies. In Vitro Phosphatase Assay—Recombinant PP1 and PP2A were purchased from New England Biolabs, Ipswich, MA. Ikaros protein and its mutants were expressed in 293T cells. Cells were labeled in vivo with radioactive orthophosphate as described above and harvested in RIPA, and proteins were immunoprecipitated and washed three times in NTN buffer (100 mm NaCl, 20 mm Tris, pH 8.0, 0.5% NP-40), then once in phosphatase reaction buffer. Immunoprecipitated Ikaros proteins bound to protein A beads were incubated with phosphatase buffer (50 mm Tris-HCl, pH 7.0, 0.1% 2-mercaptoethanol, 0.3 mg/ml bovine serum albumin, 0.01% Brij 35, 5 mm caffeine, and 0.1 mm EGTA) in the presence or absence of PP1 or PP2A (∼150 pm) as indicated. The reaction was performed at 30 °C for 30 min and was terminated by the addition of SDS sample buffer. Proteins were separated by SDS-PAGE and visualized by autoradiography. In Vivo Binding Assay—293T cells transfected with Ikaros or its mutant were lysed in RIPA lysis buffer. The Ikaros-PP1 interaction was tested by immunoprecipitation with anti-PP1 antibodies (Santa Cruz, sc 7482) followed by Western blot with anti-Ikaros CTS antibodies. The interaction between endogenous Ikaros and PP1 was tested in untransfected VL3-3M2, MOLT-4, CEM, HAFTL, and BAL17 cells. Cells were lysed in RIPA lysis buffer, the lysates were immunoprecipitated with anti-PP1 antibodies, and the presence of Ikaros was detected by Western blot using anti-Ikaros-CTS antibodies. For experiments described in Fig. 1C, 293T cells expressing Ikaros were labeled in vivo with radioactive orthophosphates (PerkinElmer Life Science) for 4 h and lysed in RIPA lysis buffer, and lysates were immunoprecipitated with anti-Ikaros-CTS antibodies on protein A beads. Immunoprecipitates were washed three times with NTN buffer and once in phosphatase reaction buffer (described above). After washing, immunoprecipitates were incubated with phosphatase reaction buffer supplemented with phosphatase inhibitors as indicated: a phosphatase inhibitor mixture, PPi (Sigma-Aldrich) in lane 1;no phosphatase inhibitor (lane 2); or PP1-specific inhibitor (inhibitor 2, lane 3). The reactions shown in all lanes were supplemented with the CK2 kinase inhibitors DRB and heparin. Proteins were separated by SDS-PAGE and visualized by autoradiography. Analysis of Ikaros Ubiquitination—MOLT-4 or VL3-3M2 cells were treated with Cbz-LLL (MG132) proteasome inhibitor (50 μm) (Calbiochem, catalogue #474790) for 2 h or left untreated (as indicated). Cells were harvested in ice-cold phosphate-buffed saline and lysed in RIPA buffer along with protease and phosphatase inhibitors. Cell lysates were immunoprecipitated with anti-Ikaros-CTS antibody or with anti-ubiquitin antibody (Assay Designs/Stressgen Bioreagents, SPA-200) on protein A beads. Immunoprecipitates were eluted in SDS sample buffer and separated on SDS-PAGE. Ikaros-ubiquitin conjugates were detected by Western blot using anti-Ikaros-CTS or anti-ubiquitin antibodies as indicated. PP1 Binds and Dephosphorylates Ikaros in Vitro and in Vivo—We tested the ability of different phosphatases to dephosphorylate Ikaros in vitro. Wild-type Ikaros was expressed in 293T cells, labeled in vivo by radioactive orthophosphate, and immunoprecipitated using anti-Ikaros-CTS antibody. The phosphatases PP1, PP2A, PP2B, and PP2C were used for in vitro reactions with immunoprecipitated Ikaros. Changes in Ikaros phosphorylation were visualized by autoradiography (Fig. 1A and data not shown). Results showed that after in vitro reaction with PP1, Ikaros lost its radioactive phosphate (Fig. 1A, lanes 3 versus 4), whereas PP2A (Fig. 1A, lanes 1 and 2) and PP2B and PP2C (data not shown) failed to dephosphorylate Ikaros. These data demonstrate that PP1 can dephosphorylate Ikaros. The ability of Ikaros to associate with PP1 was tested using a GST pulldown assay. The nuclear extract from VL3-3M2 cells was mixed with glutathione-agarose beads that were preincubated with GST-Ikaros fusion protein (Fig. 1B, lane 2) or GST protein alone (Fig. 1B, lane 1). Proteins that were bound to the beads were eluted and tested for the presence of PP1 by Western blot (Fig. 1B). These results show that Ikaros interacts with PP1 in a GST pulldown assay. Next, we tested whether Ikaros could be co-immunoprecipitated with PP1. First we examined the ability of Ikaros to be dephosphorylated by a co-immunoprecipitated protein and the ability of a PP1-specific inhibitor to abrogate this dephosphorylation. Ikaros-transfected 293T cells were labeled in vivo with radioactive orthophosphate, and whole cell lysates were immunoprecipitated with anti-Ikaros-CTS antibody. Immunoprecipitates were then incubated with phosphatase buffer. Incubations were performed in the presence of a phosphatase inhibitor mixture (PPi, Fig. 1C, lane 1), the absence of phosphatase inhibitor (Fig. 1C, lane 2), or in the presence of a PP1-specific inhibitor (Inhibit-2, Fig. 1C, lane 3). After incubation, samples were separated by SDS-PAGE and visualized by autoradiography. Results showed that the radioactive, in vivo phosphorylated Ikaros undergoes dephosphorylation when incubated with phosphatase buffer in the absence of phosphatase inhibitors (Fig. 1C lane 2), suggesting that a phosphatase was co-immunoprecipitated with Ikaros. The co-immunoprecipitated phosphatase activity is inhibited by the addition of a nonspecific phosphatase inhibitor mixture (PPi) or inhibitor-2, a PP1-specific inhibitor (Fig. 1C, lanes 1 and 3, respectively). These results show that Ikaros co-immunoprecipitates a phosphatase whose activity is inhibited by inhibitor-2, a PP1-specific inhibitor. These results suggest that Ikaros interacts in vivo with PP1. To confirm this, we determined whether Ikaros could be co-immunoprecipitated with PP1-specific antibodies (Fig. 1D). Immunoprecipitation with anti-PP1 antibodies followed by Western blot with anti-IK antibodies showed that PP1 co-immunoprecipitated transfected Ikaros in 293T cells as well as endogenous Ikaros in VL3-3M2 cells (Fig. 1D, lanes 3 and 6, respectively). Immunoprecipitations of untransfected 293T cells with anti-PP1 antibodies (Fig. 1D, lane 1) of Ikaros-transfected 293T and VL3-3M2 cells with anti-hemagglutinin antibodies (Fig. 1D, lanes 2 and 4) and of VL3-3M2 cells with anti-PP2A antibodies (Fig. 1D, lane 5) were used as negative controls. The association of endogenous Ikaros with endogenous PP1 in vivo in other human and murine hematopoietic cell lines was confirmed by co-immunoprecipitation using anti-PP1 antibodies. The co-immunoprecipitated Ikaros isoforms were detected by Western blot with anti-IK antibodies (Fig. 1D, lanes 7-11). These experiments demonstrate that Ikaros associates with PP1 in hematopoietic cells in vivo. PP1 Interacts with Ikaros via a Specific Recognition Motif—To identify the recognition site that is required for PP1-Ikaros interaction, we tested the ability of PP1 to dephosphorylate Ikaros deletion mutants (Fig. 2A). In vivo phosphorylated Ikaros deletion mutants were immunoprecipitated and then incubated with appropriate buffer in the absence (Fig. 2A, lanes 1, 3, 5, 7, and 9) or presence (Fig. 2A, lanes 2, 4, 6, 8, and 10) of PP1, and their phosphorylation status was visualized by radiography. Results showed that PP1 is able to dephosphorylate in vitro all Ikaros deletion mutants except the mutant that lacked the C-terminal part of the protein (N-458, Fig. 2A, lanes 9 and 10). These results suggest that residues that are essential for the dephosphorylation of Ikaros by PP1 are located at the C-terminal part of Ikaros (Fig. 2B). It has been demonstrated that PP1 interacts with its substrate via a specific recognition motif, (R/K)X0-1(V/I)X(F/W) (38Wakula P. Beullens M. Ceulemans H. Stalmans W. Bollen M. J. Biol. Chem. 2003; 278: 18817-18823Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 39Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar, 40Zhao S. Lee E.Y. J. Biol. Chem. 1997; 272: 28368-28372Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). A search for potential residues involved in PP1-Ikaros interaction at the C-terminal end of the Ikaros protein revealed the presence of an evolutionarily conserved PP1 recognition motif at amino acids 459-470. A sequence comparison of this motif in the Ikaros protein to the PP1 recognition site in proteins known to interact with PP1 is provided in Fig. 2C. The Ikaros PP1 recognition motif is located within one of the C-terminal zinc fingers involved in protein-protein interactions with other Ikaros family members (Fig. 2B). Because valine and phenylalanine have previously been sho" @default.
• W2090936301 created "2016-06-24" @default.
• W2090936301 creator A5006301881 @default.
• W2090936301 creator A5046890064 @default.
• W2090936301 creator A5068792925 @default.
• W2090936301 creator A5072518893 @default.
• W2090936301 creator A5075303062 @default.
• W2090936301 creator A5077299625 @default.
• W2090936301 creator A5087655461 @default.
• W2090936301 date "2009-05-01" @default.
• W2090936301 modified "2023-10-03" @default.
• W2090936301 title "Ikaros Stability and Pericentromeric Localization Are Regulated by Protein Phosphatase 1" @default.
• W2090936301 cites W1973814019 @default.
• W2090936301 cites W1974531826 @default.
• W2090936301 cites W1977795985 @default.
• W2090936301 cites W1982295124 @default.
• W2090936301 cites W1984065010 @default.
• W2090936301 cites W1985187876 @default.
• W2090936301 cites W1989695798 @default.
• W2090936301 cites W1989718163 @default.
• W2090936301 cites W1991783305 @default.
• W2090936301 cites W1998508073 @default.
• W2090936301 cites W2003138942 @default.
• W2090936301 cites W2005115869 @default.
• W2090936301 cites W2005750341 @default.
• W2090936301 cites W2008744347 @default.
• W2090936301 cites W2010601153 @default.
• W2090936301 cites W2012519319 @default.
• W2090936301 cites W2013036001 @default.
• W2090936301 cites W2018170238 @default.
• W2090936301 cites W2019881883 @default.
• W2090936301 cites W2028805224 @default.
• W2090936301 cites W2030163569 @default.
• W2090936301 cites W2036733966 @default.
• W2090936301 cites W2052453105 @default.
• W2090936301 cites W2053167235 @default.
• W2090936301 cites W2053632669 @default.
• W2090936301 cites W2056521686 @default.
• W2090936301 cites W2064667001 @default.
• W2090936301 cites W2070994243 @default.
• W2090936301 cites W2071628963 @default.
• W2090936301 cites W2072713101 @default.
• W2090936301 cites W2086780553 @default.
• W2090936301 cites W2098679175 @default.
• W2090936301 cites W2111822843 @default.
• W2090936301 cites W2114894459 @default.
• W2090936301 cites W2115482702 @default.
• W2090936301 cites W2119870415 @default.
• W2090936301 cites W2119967034 @default.
• W2090936301 cites W2120553816 @default.
• W2090936301 cites W2123227888 @default.
• W2090936301 cites W2125378951 @default.
• W2090936301 cites W2135702315 @default.
• W2090936301 cites W2146547823 @default.
• W2090936301 cites W2165959813 @default.
• W2090936301 cites W2169923455 @default.
• W2090936301 cites W2170927603 @default.
• W2090936301 cites W2305334404 @default.
• W2090936301 cites W4232011187 @default.
• W2090936301 doi "https://doi.org/10.1074/jbc.m900209200" @default.
• W2090936301 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2679487" @default.
• W2090936301 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19282287" @default.
• W2090936301 hasPublicationYear "2009" @default.
• W2090936301 type Work @default.
• W2090936301 sameAs 2090936301 @default.
• W2090936301 citedByCount "84" @default.
• W2090936301 countsByYear W20909363012012 @default.
• W2090936301 countsByYear W20909363012013 @default.
• W2090936301 countsByYear W20909363012014 @default.
• W2090936301 countsByYear W20909363012015 @default.
• W2090936301 countsByYear W20909363012016 @default.
• W2090936301 countsByYear W20909363012017 @default.
• W2090936301 countsByYear W20909363012018 @default.
• W2090936301 countsByYear W20909363012020 @default.
• W2090936301 countsByYear W20909363012021 @default.
• W2090936301 countsByYear W20909363012022 @default.
• W2090936301 countsByYear W20909363012023 @default.
• W2090936301 crossrefType "journal-article" @default.
• W2090936301 hasAuthorship W2090936301A5006301881 @default.
• W2090936301 hasAuthorship W2090936301A5046890064 @default.
• W2090936301 hasAuthorship W2090936301A5068792925 @default.
• W2090936301 hasAuthorship W2090936301A5072518893 @default.
• W2090936301 hasAuthorship W2090936301A5075303062 @default.
• W2090936301 hasAuthorship W2090936301A5077299625 @default.
• W2090936301 hasAuthorship W2090936301A5087655461 @default.
• W2090936301 hasBestOaLocation W20909363012 @default.
• W2090936301 hasConcept C112972136 @default.
• W2090936301 hasConcept C11960822 @default.
• W2090936301 hasConcept C119857082 @default.
• W2090936301 hasConcept C178666793 @default.
• W2090936301 hasConcept C185592680 @default.
• W2090936301 hasConcept C2779408958 @default.
• W2090936301 hasConcept C2988375501 @default.
• W2090936301 hasConcept C389152 @default.
• W2090936301 hasConcept C41008148 @default.
• W2090936301 hasConcept C55493867 @default.
• W2090936301 hasConcept C86803240 @default.
• W2090936301 hasConcept C95444343 @default.

• next