FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2016207813> ?p ?o ?g. }

• W2016207813 endingPage "26775" @default.
• W2016207813 startingPage "26767" @default.
• W2016207813 abstract "Immature myeloid cells have been shown to transduce signals through a carboxyl-terminally truncated isoform of Stat5. This functionally distinct signal transducer and activator of transcription isoform is generated through a unique protein-processing event. Evaluation of numerous cell lines has determined that there is a direct correlation between the expression of truncated Stat5 and protease activity. Moreover, protease activity is found only in the myeloid and not in lymphoid progenitors. To further characterize the protease small quantities have been purified to near homogeneity. Studies on this purified material indicate that the protease has an apparent molecular mass of ∼25 kDa and is active over a wide range of pH values. The protease will also cleave both activated (i.e. tyrosine-phosphorylated) and inactivate Stat5. Although this activity is sensitive to phenylmethylsulfonyl fluoride, it is notably not sensitive to several other serine protease inhibitors. Additional studies have led to the identification of the unique site where the protease cleaves Stat5. Mutagenesis of this site renders Stat5 resistant to cleavage. Consistent with the model that Stat5 cleavage is important for early myeloid development, introduction of a “non-cleavable” isoform of Stat5 into FDC-P1 cells (a myeloid progenitor line) leads to significant phenotypic changes. Immature myeloid cells have been shown to transduce signals through a carboxyl-terminally truncated isoform of Stat5. This functionally distinct signal transducer and activator of transcription isoform is generated through a unique protein-processing event. Evaluation of numerous cell lines has determined that there is a direct correlation between the expression of truncated Stat5 and protease activity. Moreover, protease activity is found only in the myeloid and not in lymphoid progenitors. To further characterize the protease small quantities have been purified to near homogeneity. Studies on this purified material indicate that the protease has an apparent molecular mass of ∼25 kDa and is active over a wide range of pH values. The protease will also cleave both activated (i.e. tyrosine-phosphorylated) and inactivate Stat5. Although this activity is sensitive to phenylmethylsulfonyl fluoride, it is notably not sensitive to several other serine protease inhibitors. Additional studies have led to the identification of the unique site where the protease cleaves Stat5. Mutagenesis of this site renders Stat5 resistant to cleavage. Consistent with the model that Stat5 cleavage is important for early myeloid development, introduction of a “non-cleavable” isoform of Stat5 into FDC-P1 cells (a myeloid progenitor line) leads to significant phenotypic changes. interferon polyacrylamide gel electrophoresis electrophoretic mobility shift assay 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid interleukin phenylmethylsulfonyl fluoride signal transducers and activators of transcription granulocyte-monocyte-colony stimulating factor matrix-assisted laser desorption ionization granulocyte-colony stimulating factor Characterization of the ability of IFNs1 to induce genes rapidly has led to the identification of the JAK-STAT pathway (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 2Mui A. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (545) Google Scholar, 3Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (464) Google Scholar). In this signaling paradigm, JAKs are receptor-associated tyrosine kinases, and STATs (signal transducers and activators of transcription) are the cytoplasmic transcription factors they activate. Once activated, STATs dimerize, translocate to the nucleus, and bind to enhancer elements, culminating in gene induction. Subsequent studies have determined that all members of the cytokine family transduce signals through one or more of the seven members of the STAT family (4Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1269) Google Scholar, 5Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3421) Google Scholar). These STATs share several functionally conserved domains including an amino-terminal coiled-coil domain, a DNA binding domain, a linker domain, an SH2 domain, a tyrosine activation domain, and a divergent carboxyl-terminal transcriptional activation domain (6Chen X. Winkemeier U. Zhao Y. Jeruzalmi D. Darnell J.E. Kuriyan J. Cell. 1998; 93: 827-839Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 7Becker S. Groner B. Müller C.W. Nature. 1998; 394: 145-151Crossref PubMed Scopus (675) Google Scholar). Interleukin (IL)-3 is a member of a subfamily of functionally related cytokines (i.e. IL-3, IL-5, and GM-CSF) that all signal through a common receptor chain (8Goodall G.J. Bagley C.J. Vadas M.A. Lopez A.F. Growth Factors. 1993; 8: 87-97Crossref PubMed Scopus (65) Google Scholar, 9Miyajima A. Mui A.L. Ogorochi T. Sakamaki K. Blood. 1993; 82: 1960-1974Crossref PubMed Google Scholar). Consistent with this, all three ligands play an important role in the maturation, proliferation, and activation of myeloid lineages (10Sachs L. Blood Cells. 1993; 19: 709-726PubMed Google Scholar). Moreover, a number of studies have determined that these ligands transduce signals through several isoforms of Stat5. In most cell types, full-length Stat5a (96 kDa) and Stat5b (94 kDa) are activated in response to stimulation with IL-3 or other members of this family (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 2Mui A. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (545) Google Scholar, 3Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (464) Google Scholar). This leads to the induction of several known Stat5 target genes (11Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (719) Google Scholar, 12Sliva D. Wood T.J. Schindler C. Lobie P.E. Norstedt G. J. Biol. Chem. 1994; 269: 26208-26214Abstract Full Text PDF PubMed Google Scholar, 13Wood T.J. Sliva D. Lobie P.E. Goullieux F. Mui A.L. Groner B. Norstedt G. Haldosen L.A. Mol. Cell. Endocrinol. 1997; 130: 69-81Crossref PubMed Scopus (46) Google Scholar, 14Lecine P. Algarte M. Rameil P. Beadling C. Bucher P. Nabholz M. Imbert J. Mol. Cell. Biol. 1996; 16: 6829-6840Crossref PubMed Google Scholar, 15John S. Robbins C. Leonard W. EMBO J. 1996; 15: 5627-5635Crossref PubMed Scopus (131) Google Scholar, 16Yoshimura A. Ichihara M. Kinjyo I. Moriyama M. Copeland N.G. Gilbert D.J. Jenkins N.A. Hara T. Miyajima A. EMBO J. 1996; 15: 1055-1063Crossref PubMed Scopus (197) Google Scholar, 17Verdier F. Rabionet R. Gouilleux F. Beisenherz-Huss C. Varlet P. Muller O. Mayeux P. Lacombe C. Gisselbrecht G. Chretien S. Mol. Cell. Biol. 1998; 18: 5852-5860Crossref PubMed Scopus (131) Google Scholar, 18Yip-Schneider M.T. Horie M. Broxmeyer H.E. Blood. 1995; 85: 3494-3502Crossref PubMed Google Scholar, 19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In the absence of both Stat5a and Stat5b, there are significant defects in the development of CFU-Mix, CFU-Eos, and CFU-GM colonies, as well as defects in the induction of target genes (20Teglund S. McKay C. Schuetz E. VanDeursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar). In myeloid progenitors, IL-3 stimulates the induction of carboxyl-terminally truncated isoforms of both Stat5a (i.e. 77 kDa) and Stat5b (i.e. 80 kDa). These isoforms, which are missing their transcriptional activation domain, are functionally distinct and fail to promote the induction of Stat5 target genes (19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar,21Mui A.L.-F. Wakao H. Kinoshita T. Kitamura T. Miyajima A. EMBO J. 1996; 15: 2425-2433Crossref PubMed Scopus (378) Google Scholar, 22Wang D. Stravopodis D. Teglund S. Kitazawa J. Ihle J.N. Mol. Cell. Biol. 1996; 16: 6141-6148Crossref PubMed Scopus (226) Google Scholar). In contrast to other STATs (23Müller M. Laxton C. Briscoe J. Schindler C. Improta T. Darnell J.E. Stark G.R. Kerr I.M. EMBO J. 1993; 12: 4221-4228Crossref PubMed Scopus (373) Google Scholar, 24Sasse J. Hemmann U. Schwartz C. Schiertshauer U. Heesel B. Landgraf C. Scheider-Mergener J. Heinrich P.C. Horn F. Mol. Cell. Biol. 1997; 17: 4677-4686Crossref PubMed Scopus (112) Google Scholar, 25Schaefer T.S. Sanders L.K. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9097-9101Crossref PubMed Scopus (328) Google Scholar), the truncated isoforms of Stat5 are generated through a unique protein-processing event. Previous studies have indicated that this protease is specific for Stat5 and can only be found in several immature cell lines (19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In this work, we extend these studies and demonstrate that there is a direct correlation between the expression of truncated Stat5 isoforms and protease activity. Moreover, this activity is only found in myeloid progenitors, supporting our hypothesis that the Stat5 protease plays an important regulatory role during myelopoiesis. We find no evidence for this during lymphoid development. Biochemical characterization of this protease has revealed that it has an apparent molecular mass of ∼25 kDa and exhibits a unique pattern of sensitivity to serine protease inhibitors. Functional evidence is provided for a role of this protease in myeloid development. Cell culture reagents were purchased from Life Technologies, Inc. DA-3, 32Dc1, WEHI3b, Ba/F3, FdTrk, and HSC15 (a generous gift from J. Pierce) cells were grown as described previously in RPMI supplemented with 10% heat-inactivated fetal calf serum, penicillin/streptomycin, and conditioned media as required (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 26Gupta S. Yan H. Wong L.H. Ralph S. Krolewski J. Schindler C. EMBO J. 1996; 15: 1075-1084Crossref PubMed Scopus (135) Google Scholar, 27Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 28Pernis A. Gupta S. Yopp J. Garfein E. Kashleva H. Schindler C. Rothman P. J. Biol. Chem. 1995; 270: 14517-14522Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). 3T3, 293, and HeLa cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium supplemented either with 10% fetal calf serum (3T3 and 293 cells) or calf serum (HeLa cells). Thymocytes were prepared by mechanical disruption of harvested thymuses as previously reported (28Pernis A. Gupta S. Yopp J. Garfein E. Kashleva H. Schindler C. Rothman P. J. Biol. Chem. 1995; 270: 14517-14522Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Prior to treatment with cytokines, cells were starved of growth factors and/or serum for 4–6 h. DA-3, 32Dc1, WEHI3b, Ba/F3, cl.19 (an FDC-P1 derivative (29Matsuguchi T. Zhao Y. Lilly M.B. Kraft A.S. J. Biol. Chem. 1997; 272: 17450-17459Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 30Bourette R.P. Myles G.M. Carlberg K. Chen A.R. Rohrschneider L.R. Cell Growth Differ. 1995; 6: 631-645PubMed Google Scholar)), and FdTrk (an FDC-P1 derivative (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar)) cells were either stimulated with 10% WEHI3b conditioned media or IL-3 (10 units/ml; Peprotech). FDC-P1 (cl.19) cells, a generous gift of L. Rohrschneider, were differentiated by stimulation with murine GM-CSF (10 units/ml; Peprotech) for 3 days, as previously reported (29Matsuguchi T. Zhao Y. Lilly M.B. Kraft A.S. J. Biol. Chem. 1997; 272: 17450-17459Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 30Bourette R.P. Myles G.M. Carlberg K. Chen A.R. Rohrschneider L.R. Cell Growth Differ. 1995; 6: 631-645PubMed Google Scholar). HSC15 cells and thymocytes were stimulated with IL-7 (10 units/ml; Genzyme). The R10 chimeric receptor represents a modest variation of G/aRα, where the ecto- and transmembrane domains of the human G-CSF receptor are fused to a truncated endodomain from the human IFN-α receptor α-chain (IFNAR1) (31Strehlow I. Schindler C. J. Biol. Chem. 1998; 273: 28049-28056Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In R10, the Stat1 recruitment motif has been substituted with the Stat5 recruitment cassette from the human IL-2 receptor β-chain (NTDAYLSLQELQ (32Lin J.-X. Migone T.S. Tsang M. Friedmann M. Weartherbee J.A. Zhou L. Yamauchi A. Bloom E.T. Mietz J. John S. Leonard W.J. Immunity. 1995; 2: 331-339Abstract Full Text PDF PubMed Scopus (678) Google Scholar)). Stat5b point mutants were generated through site-directed mutagenesis (QuickChange; Stratagene) of a Stat5b RcCMV (Invitrogen)-driven expression construct (19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), with the following pairs of oligonucleotides (Operon): S5Cmtl, ggAgTggCgCCACCTCAgCagATCAggCTCCTTCC, and S5Cmtu, ggAAggAgcCTgATCTgCTgAggTggCgCCACTCC; S5CSm1u, ACAgATgCTgggAgTggCgCCACCgCCATggATCAggCTCCTTCC, and S5CSm1l, ggAAggAgCCTgATCCATggCggTggCgCCACTCCCAgCATCTgT; S5CSm2u, gATgCTgggAgTggCgCCACCTACCTTgATCAggCTCCTTCCCCA, and S5CSm2l, TggggAAggAgCCTgATCAAggTAggTggCgCCACTCCCAgCATC; S5CSm3u, ACAgATgCTgggAgTggCgCCgTATACAtggATCAggCTCCTTCC, and S5CSm3l, ggAAggAgCCTgATCCATgTATACggCgCCACTCCCAgCATCTgT; S5CSm4u, TCCACAgATgCTgggAgTggCAgTACTTACATggATCAggCTCCT, and S5CSm4l, AggAgCCTgATCCATgTAAgTACTgCCACTCCCAgCATCTgTggA. The Stat5bm/m mutant (i.e. Y724S↓M725A) was also cloned into MSCV (a generous gift of I. Dusanter-Fourt (33Hawley R.G. Fong A.Z. Ngan B.Y. Hawley T.S. Oncogene. 1995; 6: 1113-1123Google Scholar)) for stable expression in FDC-P1 cells. Prior to transfection by the calcium phosphate method (34Pear E.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2307) Google Scholar), 293 cells were treated with 25 mmchloroquine (Sigma) for 15 min. They were then transfected with 10 μg of the appropriate STAT construct and 5 μg of R10 (31Strehlow I. Schindler C. J. Biol. Chem. 1998; 273: 28049-28056Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), per 10-ml culture plate. After 24 h, transfectants were stimulated with human G-CSF (250 ng/ml for 30 min; Amgen). Extracts were then prepared by lysis in CHAPS (Sigma) buffer (0.1% CHAPS, 10 mm Hepes (pH 7.4), 2 mm EDTA, 1 mm dithiothreitol, and 10% glycerol (19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 35Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T., Yu, V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3816) Google Scholar)) or Nonidet P-40 buffer (0.5% Nonidet P-40, 20 mm Tris (pH 7.5), 150 mm NaCl, 10% glycerol (27Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar)) as indicated. FDC-P1 (cl.19) cells were transfected by electroporation (Gentronics model ECM600; 260 V, 1050 farads with infinite resistance) and neomycin-resistant clones (G418 at 0.5 mg/ml) selected by limiting dilution (36Pernis A. Gupta S. Gollob K. Garfein E. Coffman B. Schindler C. Rothman P. Science. 1995; 269: 245-247Crossref PubMed Scopus (284) Google Scholar). Stat5 cleaving activity was evaluated either through immunoblotting or electrophoretic mobility shift assay (EMSA). Briefly, 1 μl of recombinant Stat5b (or Stat5a) substrate (prepared by transient transfection; see above) was incubated with 1–5 μl of crude or partially purified protease for 30–60 min at 37 °C. The reactants were then evaluated either by SDS-PAGE or EMSA with an IRF1-GAS probe (gatcGATTTCCCCGAAAT; Oligos Etc.), as described previously (37Bonni A. Frank D.A. Schindler C. Greenberg M.E. Science. 1993; 262: 1575-1579Crossref PubMed Scopus (165) Google Scholar, 38Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (340) Google Scholar). For pH optima studies, either 1 mHepes or 1 m Tris, at the appropriate pH, was added to each cleavage assay to achieve a final concentration of ∼330 mm (i.e. 1 μl of buffer + 1 μl of protease + 1 μl of rStat5b*). Proteins were fractionated by SDS-PAGE as described previously (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). For immunoblotting, proteins were transferred to nitrocellulose membranes (Schleicher & Schuell) and probed with antibodies at a 1:2000-fold dilution or as recommended by the manufacturer (38Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (340) Google Scholar). Pan-Stat5 and antiphosphotyrosine (4G10) monoclonal antibodies were purchased from Transduction Laboratories and Upstate Biotechnology Inc., respectively. The mass of peptides was determined by MALDI mass spectrometry (Voyager-DE RP with a 337-nm pulsed nitrogen laser; Perspective Biosystems) at the Columbia University Protein Core facility. Briefly, 200–300 pmol of biotinylated peptides, biotin-GSGATYMDQAPS and biotin-GASATYMDQAPS (prepared at Amgen, Boulder, CO), were incubated with 5–8 μl of crude or partially purified protease in CHAPS buffer in a final volume of 10 μl at 37 °C for 60 min. The reactants were dried and redissolved in 10 μl of matrix solution (10 mg/ml 4-hydroxy-α-cyanocinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid + bradykinin as an internal standard). Crude protease was prepared by gentle resuspension of a pellet of washed cells (∼2 × 106cells) in 2 volumes of cold (4 °C) CHAPS buffer adjusted to 150 mm NaCl (CHAPS, 150 mm). This material was then homogenized by 30 strokes in a 7-ml glass Dounce with a “type A” pestle (Kontes). After a 1-h incubation at 4 °C, the lysate was cleared by centrifugation (12000 × g for 20 min at 4 °C). For purification, 10 ml of extracts were prepared from 500 ml of cultured FdTrk cells at one time and then snap-frozen. 20 ml of accumulated extract was fractionated on a DEAE-Sephacel (Amersham Pharmacia Biotech) column (2.5 × 10 cm), equilibrated in 1× CHAPS, 150 mm. Proteins were eluted with a linear 0.15–1.2m NaCl gradient (in 1× CHAPS buffer), and protease activity was determined by EMSA. Peak fractions, eluting between 500 and 650 mm NaCl (radiometer conductivity meter), were pooled, diluted to 150 mm NaCl, and applied to a heparin-agarose (Sigma) column (1.5 × 12 cm), also equilibrated in 1× CHAPS, 150 mm. Bound proteins were again eluted with a linear 0.15–1.2 m NaCl gradient (in 1× CHAPS buffer). Peak fractions, which eluted between 600 and 800 mm NaCl, were diluted to 150 mm NaCl and applied to a Econo-Pac CM Cartridge (2 tandem 5-ml columns; Bio-Rad), equilibrated as described above. Peak activity eluted between 280 and 380 mm NaCl. Next, 1–2 ml of this peak activity was either applied to a 50-ml size exclusion column (2 × 27 cm; SE100/40, Bio-Rad) or a second 3-ml DEAE-MacroPrep column (Bio-Rad), equilibrated to pH 8.4. A concentrated sample (∼4-fold; Centricon-3, Millipore) was applied to the exclusion column both before and after calibration with Sigma molecular weight standards. These included blue dextran (200,000 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), lysozyme (14.3 kDa), and cytochrome c (12.4 kDa). Conditions for the second DEAE fractionation were similar to the first, except binding and elution were carried out at pH 8.5. In both of these final chromatographic steps, fractions with peak activity were concentrated 3–5-fold by Centricon 10 membranes (Millipore) or 50–60-fold by lyophilization and then fractionated on a 12% SDS-PAGE. Stimulation of cells with members of the IL-3 family of cytokines leads to the induction of two distinct types of Stat5 DNA binding complexes (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 27Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar). The faster migrating complex, consisting of two carboxyl-terminally truncated isoforms of Stat5a (i.e. p77) and Stat5b (i.e. 80), is found in nuclear extracts prepared from immature cell types (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 22Wang D. Stravopodis D. Teglund S. Kitazawa J. Ihle J.N. Mol. Cell. Biol. 1996; 16: 6141-6148Crossref PubMed Scopus (226) Google Scholar). A slower migrating complex, consisting of the full-length isoforms of Stat5a (i.e. p96) and Stat5b (i.e. p94), is found in most other cells (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 2Mui A. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (545) Google Scholar). The faster migrating complex has been well characterized in DA-3 and FDC-P1 cells, which represent myeloid progenitors (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 39Bovolenta C. Testolin L. Benussi L. Lievens P.M.-J. Liboi E. J. Biol. Chem. 1998; 273: 20779-20784Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 40Lokuta M.A. McDowell M.A. Paulnock D.M. J. Immunol. 1998; 161: 1594-1597PubMed Google Scholar, 41Meyer J. Jücker M. Ostertag W. Stocking C. Blood. 1998; 91: 1901-1908Crossref PubMed Google Scholar). This complex has also been reported in other early myeloid cell lines, including 32Dc1 (27Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar). But a more differentiated subline of 32Dc1 cells, which exhibits early erythroid features (42Migliaccio G. Migliaccio A.R. Kreider B.L. Rovera G. Adamson J.W. J. Cell Biol. 1989; 109: 833-841Crossref PubMed Scopus (67) Google Scholar), yields only the full-length complex after stimulation with IL-3 or erythropoietin (27Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar). Recent studies have determined that a protease is responsible for the generation of the truncated isoforms of Stat5 in DA-3 and FDC-P1 cells (19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 41Meyer J. Jücker M. Ostertag W. Stocking C. Blood. 1998; 91: 1901-1908Crossref PubMed Google Scholar). To extend these initial observations, extracts were prepared from several additional cell lines and evaluated for Stat5 protease activity (Fig. 1). In the absence of protease activity, the full-length DNA binding activity (i.e. an “Intact” p94 homodimer) was recovered. If protease activity was present, then the full-length recombinant activated Stat5b (i.e. rStat5b) was cleaved into the faster migrating “truncated” DNA binding activity (i.e. the “Cleaved” p80 homodimer). Extracts prepared from FdTrk cells served as the positive control for protease activity ((19) see Fig. 1, lane 2). In several cases, an additional intermediate complex (i.e. a p94:p80 heterodimer; data not shown), which correlated with less potent protease activity (e.g. see Fig.3 A), was recovered (see Fig. 3 A, lanes 3–5). Similar results were obtained with the slower migrating (19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), recombinant, activated Stat5a (rStat5a; see Fig. 1, lanes 10 and 11).Figure 3Purification of the Stat5 protease. A, protease activity recovered from the final SE100/40 size exclusion chromatography step was evaluated as outlined in Fig. 1. Recombinant activated Stat5b (rSt5b*) was either loaded directly (lane 1), incubated with buffer alone (lane 2), incubated with the column load (lane 3), or incubated with the indicated column fractions (Frn.) (lanes 4–16). The mobilities of the DNA binding complex representing Intact (i.e. p94:p94) and Cleaved (i.e. p80:p80) are indicated in the margins. p80:p94 represents an intermediate complex. B, fractions from subsequent chromatographic steps were run on an SDS-PAGE and evaluated by silver staining to evaluate the progress in purification. Included in the analysis were the peak fractions from the DEAE-Sephacel (lane 1), CM Econo-Pac (lane 2), and DEAE-MacroPrep (lanes 6 and 7) chromatographic steps. The peak fraction (#31) from the SE100/40 column (see A) was evaluated both before (lane 4) and after (lane 5) an ∼10-fold concentration. An inactive fraction (#24, lane 3) from this column was included for comparison. Likewise, both an inactive (I, lane 6) and active (A, lane 7) fraction from the DEAE-MacroPrep eluate have been included. Molecular mass markers and the mobility of a faint ∼25-kDa band are indicated in the left and right margins, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Stat5 protease activity was recovered from each of four cell lines where truncated Stat5 had previously been identified (i.e. FdTrk, WEHI-3b, DA-3, and 32Dc1 (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 27Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar)). Yet, in four cell lines, where only full-length Stat5 had previously been identified (i.e. 293, BaF/3, HeLa, and 3T3 cells (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 27Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 43Meinke A. Barahmand-Pour F. Woehrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (156) Google Scholar)), rStat5b was not cleaved (see Fig. 1, lanes 6–9). As noted previously (1Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (303) Google Scholar, 19Azam M. Lee C. Strehlow I. Schindler C. Immunity. 1997; 6: 1-20Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 22Wang D. Stravopodis D. Teglund S. Kitazawa J. Ihle J.N. Mol. Cell. Biol. 1996; 16: 6141-6148Crossref PubMed Scopus (226) Google Scholar, 27Rothman P. Kreider B. Azam M. Levy D. Wegenka U. Eilers A. Decker T. Horn F. Kashleva H. Ihle J. Schindler C. Immunity. 1994; 1: 457-468Abstract Full Text PDF PubMed Scopus (76) Google Scholar), the truncated species of Stat5 exhibits a more robust level of DNA binding activity than the full-length isoform. An important control for these studies, evaluation of the intrinsic DNA binding activity recovered from each of the CHAPs extracts (Fig. 1, lanes 12–19), demonstrated that there was an insufficient level of endogenous Stat5 DNA binding activity to confound the results. These studies demonstrate that there is a direct correlation between the activation of the carboxyl-terminally truncated" @default.
• W2016207813 created "2016-06-24" @default.
• W2016207813 creator A5009287624 @default.
• W2016207813 creator A5020896338 @default.
• W2016207813 creator A5022503287 @default.
• W2016207813 creator A5043889830 @default.
• W2016207813 creator A5058623923 @default.
• W2016207813 creator A5075407171 @default.
• W2016207813 creator A5083917358 @default.
• W2016207813 creator A5084813252 @default.
• W2016207813 date "1999-09-01" @default.
• W2016207813 modified "2023-10-18" @default.
• W2016207813 title "Characterization of the Stat5 Protease" @default.
• W2016207813 cites W1013290308 @default.
• W2016207813 cites W1510347472 @default.
• W2016207813 cites W1543392049 @default.
• W2016207813 cites W1543438262 @default.
• W2016207813 cites W1970342497 @default.
• W2016207813 cites W1972941866 @default.
• W2016207813 cites W1974659740 @default.
• W2016207813 cites W1974968368 @default.
• W2016207813 cites W1978300695 @default.
• W2016207813 cites W1981372022 @default.
• W2016207813 cites W1992168782 @default.
• W2016207813 cites W1995297089 @default.
• W2016207813 cites W1998051987 @default.
• W2016207813 cites W2003052785 @default.
• W2016207813 cites W2011030679 @default.
• W2016207813 cites W2025198871 @default.
• W2016207813 cites W2030584421 @default.
• W2016207813 cites W2035509101 @default.
• W2016207813 cites W2042952724 @default.
• W2016207813 cites W2047267173 @default.
• W2016207813 cites W2052982889 @default.
• W2016207813 cites W2053937978 @default.
• W2016207813 cites W2067804511 @default.
• W2016207813 cites W2075207336 @default.
• W2016207813 cites W2075507370 @default.
• W2016207813 cites W2079341971 @default.
• W2016207813 cites W2079518341 @default.
• W2016207813 cites W2081145901 @default.
• W2016207813 cites W2083802827 @default.
• W2016207813 cites W2084108041 @default.
• W2016207813 cites W2086715846 @default.
• W2016207813 cites W2087852931 @default.
• W2016207813 cites W2094096693 @default.
• W2016207813 cites W2097166235 @default.
• W2016207813 cites W2098279326 @default.
• W2016207813 cites W2099072360 @default.
• W2016207813 cites W2107690874 @default.
• W2016207813 cites W2108664888 @default.
• W2016207813 cites W2138009059 @default.
• W2016207813 cites W2139441497 @default.
• W2016207813 cites W2140489687 @default.
• W2016207813 cites W2143760520 @default.
• W2016207813 cites W2169649037 @default.
• W2016207813 cites W2171316918 @default.
• W2016207813 cites W218554609 @default.
• W2016207813 cites W2232689465 @default.
• W2016207813 cites W2411590716 @default.
• W2016207813 cites W2423833205 @default.
• W2016207813 cites W25119911 @default.
• W2016207813 cites W2799941631 @default.
• W2016207813 cites W318497777 @default.
• W2016207813 cites W334134150 @default.
• W2016207813 cites W4237567990 @default.
• W2016207813 cites W4241299256 @default.
• W2016207813 cites W44416003 @default.
• W2016207813 cites W4553897 @default.
• W2016207813 cites W77986286 @default.
• W2016207813 doi "https://doi.org/10.1074/jbc.274.38.26767" @default.
• W2016207813 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10480881" @default.
• W2016207813 hasPublicationYear "1999" @default.
• W2016207813 type Work @default.
• W2016207813 sameAs 2016207813 @default.
• W2016207813 citedByCount "44" @default.
• W2016207813 countsByYear W20162078132016 @default.
• W2016207813 countsByYear W20162078132019 @default.
• W2016207813 crossrefType "journal-article" @default.
• W2016207813 hasAuthorship W2016207813A5009287624 @default.
• W2016207813 hasAuthorship W2016207813A5020896338 @default.
• W2016207813 hasAuthorship W2016207813A5022503287 @default.
• W2016207813 hasAuthorship W2016207813A5043889830 @default.
• W2016207813 hasAuthorship W2016207813A5058623923 @default.
• W2016207813 hasAuthorship W2016207813A5075407171 @default.
• W2016207813 hasAuthorship W2016207813A5083917358 @default.
• W2016207813 hasAuthorship W2016207813A5084813252 @default.
• W2016207813 hasBestOaLocation W20162078131 @default.
• W2016207813 hasConcept C171250308 @default.
• W2016207813 hasConcept C181199279 @default.
• W2016207813 hasConcept C185592680 @default.
• W2016207813 hasConcept C192562407 @default.
• W2016207813 hasConcept C2776714187 @default.
• W2016207813 hasConcept C2780841128 @default.
• W2016207813 hasConcept C55493867 @default.
• W2016207813 hasConcept C70721500 @default.
• W2016207813 hasConcept C86803240 @default.
• W2016207813 hasConceptScore W2016207813C171250308 @default.

• next