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• W1969793909 abstract "A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were ∼3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function. A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were ∼3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function. Sequence-specific transcription factors are modular proteins containing distinct domains that mediate their actions, including the ability to move among different subcellular compartments, to bind to DNA in chromatin in the nucleus, and to interact with transcriptional co-activators, co-repressors, and other regulatory molecules (1Ptashne M. Trends Biochem. Sci. 2005; 30: 275-279Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The Stat 2The abbreviations used are: Stat, signal transducers and activators of transcription; GH, growth hormone; IGF, insulin-like growth factor; ANS, 1-anilino-8-naphthalenesulfonic acid.2The abbreviations used are: Stat, signal transducers and activators of transcription; GH, growth hormone; IGF, insulin-like growth factor; ANS, 1-anilino-8-naphthalenesulfonic acid. family consists of seven related proteins (Stats 1, 2, 3, 4, 5a, 5b, and 6) that are responsible for many of the transcriptional effects of cytokines, growth factors, and hormones (2Darnell J.E.J. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3320) Google Scholar, 3Levy D.E. Darnell J.E.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 651-662Crossref PubMed Scopus (2421) Google Scholar). Stat proteins are found in latent form in the cytoplasm of unstimulated cells. They are activated upon ligand binding to its receptor by a series of steps consisting of receptor-initiated tyrosine phosphorylation, dimerization, transport into the nucleus, binding to DNA response elements on target genes, and recruitment of a complex of co-activator proteins that stimulate transcription (2Darnell J.E.J. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3320) Google Scholar, 3Levy D.E. Darnell J.E.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 651-662Crossref PubMed Scopus (2421) Google Scholar). Growth hormone (GH) plays a central role in regulating somatic growth and intermediary metabolism in many vertebrate species, including humans (4Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Upon binding to its transmembrane receptor, GH triggers the activation of the receptor-associated tyrosine-protein kinase Jak2, which in addition to recruiting a variety of other signaling molecules leads to the activation of Stats 1, 3, 5a, and 5b (4Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 5Horvath C.M. Trends Biochem. Sci. 2000; 25: 496-502Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Many of the growth promoting actions of GH are mediated by the peptide growth factor, IGF-I (6Schoenle E. Zapf J. Humbel R.E. Froesch E.R. Nature. 1982; 296: 252-253Crossref PubMed Scopus (473) Google Scholar, 7Guler H.P. Zapf J. Scheiwiller E. Froesch E.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4889-4893Crossref PubMed Scopus (403) Google Scholar, 8Le Roith D. Bondy C. Yakar S. Liu J.L. Butler A. Endocr. Rev. 2001; 22: 53-74Crossref PubMed Scopus (859) Google Scholar), and IGF gene expression is under the control of GH (9Daughaday W.H. Rotwein P. Endocr. Rev. 1989; 10: 68-91Crossref PubMed Scopus (1601) Google Scholar). Recent studies have demonstrated that GH stimulates IGF-I gene transcription through the mediation of Stat5b (10Woelfle J. Billiard J. Rotwein P. J. Biol. Chem. 2003; 278: 22696-22702Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and results in experimental animals have shown that the absence of Stat5b is associated with diminished post-natal growth (11Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (816) Google Scholar, 12Teglund S. McKay C. Schuetz E. van Deursen 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 (1063) Google Scholar). Decreased activity through the GH-IGF-I axis causes growth defects in humans that may lead to short stature (13Lopez-Bermejo A. Buckway C.K. Rosenfeld R.G. Trends Endocrinol. Metab. 2000; 11: 39-49Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Rosenbloom A.L. Rosenfeld R.G. Guevara-Aguirre J. Pediatr. Clin. N. Am. 1997; 44: 423-442Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Several genetic and acquired abnormalities have been shown to inhibit GH gene expression or biosynthesis (13Lopez-Bermejo A. Buckway C.K. Rosenfeld R.G. Trends Endocrinol. Metab. 2000; 11: 39-49Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), and mutations in the GH receptor have been described that impair its synthesis or biological actions (15Rosenfeld R.G. Rosenbloom A.L. Guevara-Aguirre J. Endocr. Rev. 1994; 15: 369-390Crossref PubMed Scopus (433) Google Scholar, 16Woods K.A. Dastot F. Preece M.A. Clark A.J. Postel-Vinay M.C. Chatelain P.G. Ranke M.B. Rosenfeld R.G. Amselem S. Savage M.O. J. Clin. Endocrinol. Metab. 1997; 82: 3529-3535Crossref PubMed Scopus (161) Google Scholar). Individuals also have been reported with growth deficiency and inactivating mutations in the genes for IGF-I and its receptor (17Woods K.A. Camacho-Hubner C. Savage M.O. Clark A.J. N. Engl. J. Med. 1996; 335: 1363-1367Crossref PubMed Scopus (933) Google Scholar, 18Bonapace G. Concolino D. Formicola S. Strisciuglio P. J. Med. Genet. 2003; 40: 913-917Crossref PubMed Scopus (130) Google Scholar, 19Abuzzahab M.J. Schneider A. Goddard A. Grigorescu F. Lautier C. Keller E. Kiess W. Klammt J. Kratzsch J. Osgood D. Pfaffle R. Raile K. Seidel B. Smith R.J. Chernausek S.D. N. Engl. J. Med. 2003; 349: 2211-2222Crossref PubMed Scopus (492) Google Scholar). The recent description of a young woman with markedly impaired growth and a homozygous point mutation in the Stat5b gene predicted to change amino acid alanine 630 to proline within the SH2 domain of the protein (20Kofoed E.M. Hwa V. Little B. Woods K.A. Buckway C.K. Tsubaki J. Pratt K.L. Bezrodnik L. Jasper H. Tepper A. Heinrich J.J. Rosenfeld R.G. N. Engl. J. Med. 2003; 349: 1139-1147Crossref PubMed Scopus (429) Google Scholar) prompted us to investigate the biochemical consequences of this inherited defect. Here we demonstrate that Stat5bA630P is an inactive transcription factor. In cells, including those derived from the patient, Stat5bA630P accumulates primarily as stable protein aggregates in cytoplasmic inclusion bodies. The A630P mutation also predisposes the isolated SH2 domain to aggregate in solution under conditions in which the wild-type domain is a monomer. Our observations demonstrate that this individual has a protein folding disorder where the aberrantly folded SH2 domain of the mutant Stat5b molecule triggers primary transcription factor deficiency and protein aggregation. Recombinant Plasmids and Adenoviruses—An expression plasmid in pcDNA3 and an adenovirus encoding NH2-terminal FLAG-tagged rat Stat5bWT have been described previously (10Woelfle J. Billiard J. Rotwein P. J. Biol. Chem. 2003; 278: 22696-22702Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), as have an expression plasmid for the mouse GH receptor and for FLAG-tagged rat Stat5bY699F and reporter plasmids HS7-TK-Luc and HS7-IGF P1-Luc (21Woelfle J. Chia D.J. Rotwein P. J. Biol. Chem. 2003; 278: 51261-51266Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Codon 630 of FLAG-Stat5bWT was modified by site-directed mutagenesis (Stratagene, La Jolla, CA) to produce FLAG-Stat5bA630P in pcDNA3, and an adenovirus encoding this protein under the control of a tetracycline-repressible promoter was prepared and purified as described (10Woelfle J. Billiard J. Rotwein P. J. Biol. Chem. 2003; 278: 22696-22702Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). DNA encoding the SH2 domains of Stat5bWT and Stat5bA630P (from codon Asp591 to Glu689) was generated by PCR and cloned into pET-15b (Novagen, San Diego, CA) using NdeI and XhoI restriction sites so that a hexahistidine tag was added to the NH2 terminus. The coding regions of all recombinant plasmids were verified by DNA sequencing. Cell Culture and Transient Transfections—All of the cells were maintained in Dulbecco's modified Eagle's medium (Mediatech-Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in humidified air with 5% CO2. Preparation of primary dermal fibroblasts from the patient homozygous for the A630P mutation in Stat5b and from a 30-year old female of normal height has been described (20Kofoed E.M. Hwa V. Little B. Woods K.A. Buckway C.K. Tsubaki J. Pratt K.L. Bezrodnik L. Jasper H. Tepper A. Heinrich J.J. Rosenfeld R.G. N. Engl. J. Med. 2003; 349: 1139-1147Crossref PubMed Scopus (429) Google Scholar). All transient transfections were performed with TransIT-LT1 (Mirus, Madison, WI) and a protocol from the supplier. Reporter Gene Assays—Cos-7 cells (ATCC CRL-1651) were transfected in 6-well tissue culture dishes with expression plasmids for mouse GH receptor (100 ng), Stat5bWT or Stat5bA630P (100 ng), and the reporter plasmids HS7-TK-Luc or HS7-IGF P1-Luc (250 ng). After 24 h, the cells were incubated in serum-free medium with 1% bovine serum albumin, and recombinant rat GH (final concentration, 40 nm) or vehicle was added. The cell lysates were harvested 18 h later for luciferase assays (Promega, Madison, WI). The results were normalized for total cellular protein. Immunoblotting—Whole cell protein lysates were prepared after washing the cells with phosphate-buffered saline, and incubating on ice for 15 min in radioimmune precipitation assay buffer (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 1.25 mm EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Igepal CA-630, 1 mm dithiothreitol, 1× protease inhibitor mixture (Roche Applied Science), 0.25 mm phenylmethylsulfonyl fluoride) followed by passage through a 22-gauge needle and centrifugation at 15,000 rpm at 4 °C. The protein concentrations were determined using the BCA protein assay kit (Pierce). The indicated insoluble material was suspended in an equal volume of electrophoresis sample buffer containing 2% SDS. Protein samples (30 μg each) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with antibodies as described (10Woelfle J. Billiard J. Rotwein P. J. Biol. Chem. 2003; 278: 22696-22702Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), followed by detection with a LiCoR Odyssey infrared imager and version 1.2 analysis software (LiCoR Biosciences, Lincoln, NB). The antibodies were used at the following dilutions: FLAG M2 (Sigma) 1:2000, Stat5b (Zymed Laboratories Inc., South San Francisco, CA) 1:5000, phospho-Stat5 (Upstate Cell Signaling, Lake Placid, NY) 1:2000, tubulin (Sigma) 1:2000, mouse IR680 (LiCoR) 1:5000, and rabbit IR800 (LiCor) 1:5000. GH-stimulated Tyrosine Phosphorylation—Cos-7 cells were co-transfected with expression plasmids for the mouse GH receptor (100 ng) and Stat5bWT, Stat5bA630P, or Stat5bY699F (100 ng). After 24 h, serum-free medium with 1% bovine serum albumin was added for 18 h, followed by the addition of recombinant rat GH (40 nm) or vehicle for 30 or 60 min. The cell extracts (500 μg) were immunoprecipitated with FLAG M2 antibody coupled to agarose beads by incubation overnight at 4 °C with rocking. Pilot immunoblots were performed to equalize concentrations of Stat5b in all lanes. Studies of Stat5b Biosynthesis—Cos-7 cells were infected with a recombinant adenovirus expressing a tetracycline-repressible transcriptional activator. After 24 h, the cells were infected with adenoviruses encoding Stat5bWT or Stat5bA630P under control of a tetracycline-repressible transcriptional activator-activated promoter. Whole cell protein extracts were prepared starting 1 h after infection with the Stat5b adenovirus and assessed by immunoblotting with primary antibodies to FLAG M2 and tubulin. Studies of Protein Stability—Cos-7 cells in 60-mm dishes were transiently transfected with expression plasmids encoding either Stat5bWT or Stat5bA630P (500 ng). After 24 h, the cells were incubated in serum-free, methionine-free medium for 30 min, followed by a 15-min pulse in methionine-free medium supplemented with 8 μCi of [35S]methionine and then returned to fresh complete medium (chase). At various chase intervals, cell extracts were immunoprecipitated with FLAG M2 antibody coupled to agarose beads by incubation overnight at 4 °C with rocking. The immunoprecipitates were separated by SDS-PAGE, and the dried gels were analyzed by fluorography with En3Hance (PerkinElmer Life Sciences). A representative result of four independent experiments is shown in Fig. 2C. Immunocytochemistry—Cos-7 cells in 6-well plates were transiently transfected with expression plasmids for Stat5bWT or Stat5bA630P (500 ng). After 48 h, the cells were fixed in 4% paraformaldehyde for 15 min at 20 °C and permeabilized with a 50:50 mixture of methanol and acetone for 2 min before blocking in 0.25% normal goat serum for >1 h at 20 °C. After the addition of the FLAG M2 monoclonal antibody at 1:2000 dilution in blocking buffer overnight followed by a washing step and incubation in goat anti-mouse IgG1-Alexa 488 at 1:1000 dilution in blocking buffer for 2 h, the images were captured with a Roper Scientific Cool Snap FX CCD camera attached to a Nikon Eclipse T300 fluorescent microscope using IP Labs 3.5 software (Scanalytics, Rockville, MD). Purification of SH2 Domain—Escherichia coli strain BL21 cells transformed with pET15b-Stat5bWT, or pET15b-Stat5bA630P SH2 domains were grown with shaking at 37 °C in LB medium containing ampicillin (200 μg/ml) to an A600 of 0.6-1.0 and induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 4 h. Purification of the His-tagged SH2 domains from inclusion bodies was as previously described (22Haan S. Hemmann U. Hassiepen U. Schaper F. Schneider-Mergener J. Wollmer A. Heinrich P.C. Grotzinger J. J. Biol. Chem. 1999; 274: 1342-1348Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Upon elution from the nickel-nitrilotriacetic acid-agarose column, the fractions were dialyzed against 100 volumes of refolding buffer (20 mm Na2HPO4/KH2PO4, pH 7.5, 150 mm NaCl, 0.5 mm EDTA, 1 mm β-mercaptoethanol) with two buffer changes. Purification and yield were monitored by SDS-PAGE and staining with GelCode blue reagent (Pierce). For gel filtration chromatography and binding studies with 1-anilino-8-naphthalenesulfonic acid (ANS), purified samples were used immediately after refolding. Circular Dichroism Spectroscopy—CD measurements were performed on an AVIV model 215 CD spectrometer maintained at 4 °C. The spectra were measured from 260 to 200 nm at 0.5-nm intervals. The protein concentration was 18.6 μm, and a path length of 0.1 cm was used. The measurements were averaged over a 3-s time interval at each wavelength. The profiles depicted in Fig. 4 are averages of three independent experiments. Equilibrium folding and unfolding were monitored by CD absorbance at 222 nm upon increasing guanidine HCl concentrations from 0 to 6 m at 0.25 m intervals to obtain free energy differences for each transition as described previously (23Subbian E. Yabuta Y. Shinde U. Biochemistry. 2004; 43: 14348-14360Crossref PubMed Scopus (27) Google Scholar). The unfolding transitions best represented two states, native (N) and unfolded (U), as follows,N⇄KNUU The observed ellipticity, Aobs(c), at any concentration of the denaturant is given by the sum of the contributions from the two states as follows,Aobs(c)=AN+AUexp[-(ΔGNUH2O-mNUc)/RT]1+exp[-(ΔGNUH2O-mNUc)/RT] where AN and AU are the ellipticity values of the pure N and U states, respectively, ΔGNUH2O is ΔGNU at 0 m guanidine HCl, and mNUc represents the dependence of the respective free energy changes on c. The data were fitted using Prism Graphpad. Folding Kinetics—The change in fluorescence upon folding-unfolding was used to monitor kinetics. Stopped flow kinetics were measured on a Hi-Tech SF61-DX2 fluorometer using λexcitation of a 295- and 310-nm band pass filter. For these analyses, unfolded proteins (1-4 μm) in 6 m guanidine HCl were rapidly diluted (1:10 ratio) into renaturation buffer. The data were fitted to single and double exponential equations (GraphPad-Prism) to determine folding rate constants. All of the experiments were performed at 23 °C, and the results shown represent the averages of three to six independent replicates. Fluorescence Spectroscopy—The fluorescence measurements were performed on a Photon Technology International spectrometer with a model 810 photomultiplier detection system at 4 °C. The protein samples (2.5 μm) were immediately incubated with ANS (50 μm) for 30 min upon completion of refolding. The proteins were excited at 390 nm, and the emission scan was recorded from 400 to 600 nm. The excitation and emission bandwidths were maintained at 10 nm throughout the experiments. For the intrinsic fluorescence experiments, the protein samples (5 μm) were excited at 295 nm, and the emission scans were recorded from 300 to 400 nm. Gel Filtration Chromatography—Soluble protein (250 ml at 1 mg/ml) was applied to a Superdex S200 HR10/30 column (Amersham Biosciences) previously equilibrated with 20 mm Na2HPO4/KH2PO4, pH 7.5, 150 mm NaCl, 0.5 mm EDTA, 1 mm β-mercaptoethanol. The proteins were eluted at 20 °C using a flow rate of 0.5 ml/min. The elution was monitored at 280 nm using a Beckman Gold 166 UV-visible detector. Molecular Modeling—Modeling was performed using Swiss Model, a program that predicts structures reliably (root mean square deviation < 2 Å) for sequences with at least 50-60% identity, using coordinates from the crystal structure of human Stat1 (Swiss Protein Database 1YVL) as the template (24Chen X. Vinkemeier U. Zhao Y. Jeruzalmi D. Darnell J.E.J. Kuriyan J. Cell. 1998; 93: 827-839Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). Absent Transcriptional Activity and Diminished Tyrosine Phosphorylation of Stat5bA630P after GH Treatment—The overall domain structure of Stat5b is pictured in Fig. 1A. To study the consequences of the A630P substitution within the SH2 domain of the protein, we engineered the mutation into FLAG epitope-tagged rat Stat5b, which is 96% identical to the human protein (25Lin J.X. Mietz J. Modi W.S. John S. Leonard W.J. J. Biol. Chem. 1996; 271: 10738-10744Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), and generated recombinant expression plasmids and adenoviruses. In initial experiments we addressed the potential transcriptional properties of the mutant protein and the effects of acute GH treatment on its tyrosine phosphorylation after transient transfection with the GH receptor into Cos-7 cells. As seen in Fig. 1B, in cells expressing Stat5bA630P, GH had no stimulatory effect on the activity of reporter genes containing a GH-responsive element from the rat IGF-I gene (21Woelfle J. Chia D.J. Rotwein P. J. Biol. Chem. 2003; 278: 51261-51266Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In contrast, hormone treatment led to a 4- or 7-fold rise in reporter gene expression in cells transfected with Stat5bWT. The earliest event in GH-stimulated activation of Stat5b is recruitment of the protein to the tyrosine-phosphorylated cytoplasmic tail of the GH receptor, followed by phosphorylation of tyrosine 699 of Stat5b by receptor-associated Jak2 (4Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 5Horvath C.M. Trends Biochem. Sci. 2000; 25: 496-502Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). As seen in Fig. 1C, GH caused rapid and robust tyrosine phosphorylation of Stat5bWT but had a minimal effect on Stat5bA630P, because levels of its tyrosine phosphorylation were barely above those of the nonphosphorylated Stat5bY699F mutant. Thus, based on these results, Stat5bA630P is transcriptionally inert secondary to its inability to be activated by GH. Diminished Expression of Stat5bA630P Is Secondary to Its Decreased Stability—Analysis of steady state protein levels in transfected Cos-7 cells revealed that the amount of Stat5bA630P was only ∼30-35% of Stat5bWT (Fig. 2A). Treatment with GH had no effect on the accumulation of either protein (data not shown). Assessment of the biosynthetic rates of each transcription factor after infection of Cos-7 cells with recombinant adenoviruses showed that each protein could be detected within 2 h of infection, but that Stat5bA630P reached constant levels sooner than Stat5bWT (8 versus <24 h), and that its apparent concentration at steady state was reduced by ∼70% compared with Stat5bWT (Fig. 2B). Evaluation of protein half-life by pulse-chase assays demonstrated that Stat5bWT was a fairly long-lived protein, with a T1/2 > 24 h, whereas Stat5bA630P appeared to be short-lived, with a T1/2 of ∼3.5 h (Fig. 2C and data not shown). Thus, at least in transfected cells, in addition to being transcriptionally silent secondary to reduced activation by GH, Stat5bA630P appears to turn over at least seven times faster than Stat5bWT. Stat5bA630P Accumulates in Cells in Insoluble Protein Aggregates That Interfere with Proteasomal Function—We next addressed the mechanism of apparently increased turnover of Stat5bA630P by assessing the potential role of lysosomal or proteasomal degradation. Neither steady state protein levels nor the T1/2 of either Stat5bWT or Stat5bA630P were altered by incubation of cells with a variety of lysosomal and proteasomal inhibitors including chloroquine, MG132, and N-acetyl-Leu-Leu-Nle-CHO (where Nle is norleucine; data not shown). Some disease-associated proteins when mutated, including the cystic fibrosis transmembrane conductance regulator (26Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1745) Google Scholar), rhodopsin (27Saliba R.S. Munro P.M. Luthert P.J. Cheetham M.E. J. Cell Sci. 2002; 115: 2907-2918Crossref PubMed Google Scholar, 28Illing M.E. Rajan R.S. Bence N.F. Kopito R.R. J. Biol. Chem. 2002; 277: 34150-34160Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), and huntingtin (29Ross C.A. Neuron. 2002; 35: 819-822Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar), have been shown to be misfolded, and at least when overexpressed, form insoluble protein aggregates in cells (26Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1745) Google Scholar). To determine whether Stat5bA630P is prone to aggregation, we looked for accumulation of the protein in the “insoluble” fraction after extraction of transfected cells with buffer containing primarily nonionic detergents. As seen in Fig. 3A and in contrast to Stat5bWT and tubulin, which were found exclusively in the soluble fraction, over 80% of Stat5bA630P was located in the insoluble portion and could be solubilized fully only with high concentrations of ionic detergents. Analogous results were observed when transfected Cos-7 cells were evaluated by immunocyto-chemistry. In the absence of GH, Stat5bWT was found throughout the cytoplasm in a diffuse and uniform pattern, whereas Stat5bA630P was concentrated in multiple discrete cytoplasmic inclusions (Fig. 3B), which have been termed “aggresomes” when detected previously in cells expressing proteins prone to misfolding (26Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1745) Google Scholar). To determine whether aggregates of Stat5bA630P occurred in an endogenous setting, we examined skin fibroblasts derived from the index patient. As pictured in Fig. 3C, unlike cells from an individual expressing Stat5bWT, where the protein was found only in the soluble fraction, in the fibroblasts of the patient ∼90% of Stat5bA630P was located in the insoluble portion. By contrast, in cells from both individuals highly related Stat5a (91% identical to Stat5b (25Lin J.X. Mietz J. Modi W.S. John S. Leonard W.J. J. Biol. Chem. 1996; 271: 10738-10744Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar)) partitioned to the soluble fraction. We were unable to observe inclusion bodies in the cells of the patient by immunocytochemistry (data not shown), a finding that we interpret as secondary to low levels of expression of Stat5b in fibroblasts. Based on our results, the accumulation of Stat5bA630P in an insoluble protein complex is an inherent property of the mutant protein and is not just induced by its overexpression in Cos-7 cells. Several studies have suggested that proteins that aggregate may disrupt proteasome function, leading to alterations in metabolism of short-lived cellular proteins (30Bence N.F. Sampat R.M. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1781) Google Scholar, 31Bennett E.J. Bence N.F. Jayakumar R. Kopito R.R. Mol. Cell. 2005; 17: 351-365Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). To determine whether Stat5bA630P could cause gain-of-function abnormalities secondary to diminished proteasomal activity, we examined in Cos-7 cells steady state levels of co-expressed MyoD, a muscle-specific transcription factor with a T1/2 of <30 min (32Abu Hatoum O. Gross-Mesilaty S. Breitschopf K. Hoffman A. Gonen H. Ciechanover A. Bengal E. Mol. Cell. Biol. 1998; 18: 5670-5677Crossref PubMed Google Scholar). As pictured in Fig. 3D, Stat5bWT had no effect on levels of MyoD, whereas co-expression with Stat5bA630P led to a >10-fold increase in its abundance, similar to that seen after incubation with the proteasome inhibitor N-acetyl-Leu-Leu-Nle-CHO. Based on these results, it is possible that some of the pathological changes observed in the individual with the homozygous Stat5bA630P mutation are secondary to alterations in levels of selected proteins because of diminished proteasome function. Misfolding of the Isolated SH2 Domain of Stat5bA630P—To understand how the A630P mutation might perturb the structure and function of the SH2 domain, we cloned, expressed, and characterized the isolated SH2 domains of wild-type and mutant human Stat5b. Both proteins were purified from an E. coli expression system to >95% purity (data not shown) and refolded as described under “Experimental Procedures.” We first an" @default.
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• W1969793909 date "2006-03-01" @default.
• W1969793909 modified "2023-10-11" @default.
• W1969793909 title "Aberrant Folding of a Mutant Stat5b Causes Growth Hormone Insensitivity and Proteasomal Dysfunction" @default.
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