PTPN11 (Shp2) Mutations in LEOPARD Syndrome Have Dominant Negative, Not Activating, Effects
2005; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m513068200
ISSN1083-351X
AutoresMaria I. Kontaridis, Kenneth D. Swanson, Frank David, David Barford, Benjamin G. Neel,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoMultiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinasemediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation. Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinasemediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation. Mutations in the PTPN11 gene product, Shp2, are associated with several human diseases (for review, see Ref. 1Tartaglia M. Gelb B.D. Annu. Rev. Genomics Hum. Genet. 2005; 6: 45-68Crossref PubMed Scopus (279) Google Scholar), including multiple lentigines/LEOPARD syndrome (LS) 4The abbreviations used are: LS, LEOPARD syndrome; NS, Noonan syndrome WT, wild type Shp2; Erk, extracellular signal-regulated kinase; PTP, protein-tyrosine phosphatase; PDGF, platelet-derived growth factor; GST, glutathione S-transferase; pNPP, para-nitrophenyl phosphate; EGF, epidermal growth factor; HA, hemagglutinin; FGF, fibroblast growth factor. (2Digilio M.C. Conti E. Sarkozy A. Mingarelli R. Dottorini T. Marino B. Pizzuti A. Dallapiccola B. Am. J. Hum. Genet. 2002; 71: 389-394Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 3Legius E. Schrander-Stumpel C. Schollen E. Pulles-Heintzberger C. Gewillig M. Fryns J.-P. J. Med. Genet. 2002; 39: 571-574Crossref PubMed Scopus (205) Google Scholar), Noonan syndrome (NS) (4Tartaglia M. Mehler E.L. Goldberg R. Zampino G. Brunner H.G. Kremer H. van der Burgt I. Crosby A.H. Ion A. Jeffery S. Kalidas K. Patton M.A. Kucherlapati R.S. Gelb B.D. Nat. Genet. 2001; 29: 465-468Crossref PubMed Scopus (1341) Google Scholar), and various malignancies (5Tartaglia M. Niemeyer C.M. Fragale A. Song X. Buechner J. Jung A. Hahlen K. Hasle H. Licht J.D. Gelb B.D. Nat. Genet. 2003; 34: 148-150Crossref PubMed Scopus (830) Google Scholar, 6Loh M.L. Vattikuti S. Schubbert S. Reynolds M.G. Carlson E. Lieuw K.H. Cheng J.W. Lee C.M. Stokoe D. Bonifas J.M. Curtiss N.P. Gotlib J. Meschinchi S. LeBeau M.M. Emanuel P.D. Shannon K.M. Blood. 2004; 103: 2325-2331Crossref PubMed Scopus (353) Google Scholar, 7Bentires-Alj M. Paez J.G. David F.S. Keilhack H. Halmos B. Naoki K. Maris J.M. Richardson A. Bardelli A. Sugarbaker D.J. Richards W.G. Du J. Girard L. Minna J.D. Loh M.L. Fischer D.E. Velculescu V. Vogelstein B. Myerson M. Sellers W.R. Neel B.G. Cancer. Res. 2004; 64: 8816-8820Crossref PubMed Scopus (421) Google Scholar, 8Tartaglia M. Martinelli S. Cazzaniga G. Cordeddu V. Iavarone I. Spinelli M. Palmi C. Carta C. Pession A. Arico M. Masera G. Basso G. Sorcini M. Gelb B.D. Biondi A. Blood. 2004; 104: 307-313Crossref PubMed Scopus (234) Google Scholar). LS (MIM 151100) and NS (MIM 163950) are developmental disorders with many shared features. NS is characterized by facial dysmorphia, typically ocular hypertelorism, cardiac defects, most commonly pulmonary valve stenosis, and proportionate short stature. Cryptorchidism and deafness are also reported in NS patients (9Allanson J.E. Hall J.G. Hughes H.E. Preus M. Witt R.D. Am. J. Med. Genet. 1985; 21: 507-514Crossref PubMed Scopus (194) Google Scholar). However, lentigines, a hallmark of LS (10Gorlin R.J. Anderson R.C. Blaw M. Am. J. Dis. Child. 1969; 117: 652-662Crossref PubMed Scopus (249) Google Scholar), are uncommon in NS, and less penetrant NS abnormalities, such as webbed neck, skeletal defects, and bleeding/coagulation abnormalities, are typically absent in LS (10Gorlin R.J. Anderson R.C. Blaw M. Am. J. Dis. Child. 1969; 117: 652-662Crossref PubMed Scopus (249) Google Scholar, 11Sarkozy A. Conti E. Digilio M.C. Marino B. Morini E. Pacileo G. Wilson M. Calabro R. Pizzuti A. Dallapiccola B. J. Med. Genet. 2004; 41: e68-e74Crossref PubMed Scopus (119) Google Scholar, 12Keren B. Hadchouel A. Saba S. Sznajer Y. Bonneau D. Leheup B. Boute O. Gaillard D. Lacombe D. Layet V. Marlin S. Mortier G. Toutain A. Beylot C. Baumann C. Verloes A. Cave H. J. Med. Genet. 2004; 41: 117-119Crossref PubMed Scopus (72) Google Scholar). Both LS and NS are associated with increased risk of malignancy. However, acute myelogenous leukemia and neuroblastoma are associated with LS (12Keren B. Hadchouel A. Saba S. Sznajer Y. Bonneau D. Leheup B. Boute O. Gaillard D. Lacombe D. Layet V. Marlin S. Mortier G. Toutain A. Beylot C. Baumann C. Verloes A. Cave H. J. Med. Genet. 2004; 41: 117-119Crossref PubMed Scopus (72) Google Scholar, 13Merks J.H. Caron H.N. Hennekam R.C. Am. J. Med. Genet. 2005; 134: 132-143Crossref Scopus (129) Google Scholar), whereas various childhood hematological disorders, most notably juvenile myelomonocytic leukemia and possibly acute lymphoblastic leukemia, are found at increased incidence in NS (1Tartaglia M. Gelb B.D. Annu. Rev. Genomics Hum. Genet. 2005; 6: 45-68Crossref PubMed Scopus (279) Google Scholar). Somatic PTPN11 mutations are found in sporadic juvenile myelomonocytic leukemia (∼35%), B cell acute lymphoblastic leukemia (∼7%), other childhood myeloproliferative or myelodysplastic disorders (1-5%), adult acute myelogenous leukemia (∼5%), and occasionally in neuroblastoma and other solid tumors (5Tartaglia M. Niemeyer C.M. Fragale A. Song X. Buechner J. Jung A. Hahlen K. Hasle H. Licht J.D. Gelb B.D. Nat. Genet. 2003; 34: 148-150Crossref PubMed Scopus (830) Google Scholar, 6Loh M.L. Vattikuti S. Schubbert S. Reynolds M.G. Carlson E. Lieuw K.H. Cheng J.W. Lee C.M. Stokoe D. Bonifas J.M. Curtiss N.P. Gotlib J. Meschinchi S. LeBeau M.M. Emanuel P.D. Shannon K.M. Blood. 2004; 103: 2325-2331Crossref PubMed Scopus (353) Google Scholar, 7Bentires-Alj M. Paez J.G. David F.S. Keilhack H. Halmos B. Naoki K. Maris J.M. Richardson A. Bardelli A. Sugarbaker D.J. Richards W.G. Du J. Girard L. Minna J.D. Loh M.L. Fischer D.E. Velculescu V. Vogelstein B. Myerson M. Sellers W.R. Neel B.G. Cancer. Res. 2004; 64: 8816-8820Crossref PubMed Scopus (421) Google Scholar, 8Tartaglia M. Martinelli S. Cazzaniga G. Cordeddu V. Iavarone I. Spinelli M. Palmi C. Carta C. Pession A. Arico M. Masera G. Basso G. Sorcini M. Gelb B.D. Biondi A. Blood. 2004; 104: 307-313Crossref PubMed Scopus (234) Google Scholar). Shp2 is a ubiquitously expressed, non-receptor protein-tyrosine phosphatase (PTP) comprising two N-terminal SH2 domains, a catalytic (PTP) domain, and a C terminus with tyrosyl phosphorylation sites and a proline-rich stretch (14Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). Via its SH2 domains, Shp2 binds directly to some growth factor receptors, such as the platelet-derived growth factor (PDGF) receptor, as well as to several scaffolding adapters, including IRS, FGF receptor substrate (FRS), and Gab proteins. Formation of such complexes is required for full activation of the Ras/Erk cascade in most, if not all receptor-tyrosine kinase, cytokine receptor, and integrin signaling pathways. Consequently, Shp2 plays an important role in mediating multiple downstream biological responses, such as proliferation and/or survival, adhesion, and migration. All known biological functions of Shp2 require its catalytic activity (14Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). Shp2 activity is tightly regulated by an elegant "molecular switch" mechanism that couples activation with recruitment of Shp2 to its binding proteins (14Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar, 15Hof P. Pluskey S. Dhe-Paganon S. Eck M.J. Shoelson S.E. Cell. 1998; 98: 441-450Abstract Full Text Full Text PDF Scopus (756) Google Scholar, 16Barford D. Neel B.G. Structure. 1998; 6: 249-254Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In the basal state (i.e. in unstimulated cells when Shp2 is found primarily in the cytoplasm), the "backside loop" of the N-SH2 domain (the side opposite to its phosphotyrosyl peptide binding pocket) interacts with the PTP domain, preventing substrate access to the active site (15Hof P. Pluskey S. Dhe-Paganon S. Eck M.J. Shoelson S.E. Cell. 1998; 98: 441-450Abstract Full Text Full Text PDF Scopus (756) Google Scholar). Binding to a phosphotyrosyl (Tyr(P)) protein ligand (e.g. in a receptor-tyrosine kinase or scaffolding adapter) alters the conformation of the N-SH2 domain, rendering it unable to bind the PTP domain and activating the enzyme (Fig. 1A). Earlier studies showed that mutating key contacts between the N-SH2 and the PTP domains led to biochemically and biologically "activated mutants" of Shp2 (17O'Reilly A.M. Pluskey S. Shoelson S.E. Neel B.G. Mol. Cell. Biol. 1999; 20: 299-311Crossref Scopus (101) Google Scholar). A large number of NS mutants have been identified (1Tartaglia M. Gelb B.D. Annu. Rev. Genomics Hum. Genet. 2005; 6: 45-68Crossref PubMed Scopus (279) Google Scholar, 4Tartaglia M. Mehler E.L. Goldberg R. Zampino G. Brunner H.G. Kremer H. van der Burgt I. Crosby A.H. Ion A. Jeffery S. Kalidas K. Patton M.A. Kucherlapati R.S. Gelb B.D. Nat. Genet. 2001; 29: 465-468Crossref PubMed Scopus (1341) Google Scholar). Most of these disrupt key contacts between the N-SH2 and PTP domains, resulting in increased basal and stimulated PTP activity (18Fragale A. Tartaglia M. Wu J. Gelb B.D. Hum. Mutat. 2004; 23: 267-277Crossref PubMed Scopus (167) Google Scholar, 19Niihori T. Aoki Y. Ohashi H. Kurosawa K. Kondoh T. Ishikiriyama S. Kawame H. Kamasaki H. Yamanaka T. Takada F. Nishio K. Sakurai M. Tamai H. Nagashima T. Suzuki Y. Kure S. Fujii K. Imaizumi M. Matsubara Y. J. Hum. Genet. 2005; 50: 192-202Crossref PubMed Scopus (96) Google Scholar, 20Keilhack H. David F.S. McGregor M. Cantley L.C. Neel B.G. J. Biol. Chem. 2005; 280: 30984-30993Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). NS mutants (when co-transfected with the scaffolding adapter Gab1), enhance Erk mitogen-activated protein kinase activation in transient transfection assays (18Fragale A. Tartaglia M. Wu J. Gelb B.D. Hum. Mutat. 2004; 23: 267-277Crossref PubMed Scopus (167) Google Scholar). Erk is also hyperactivated in selected embryonic tissues from a mouse model of NS (21Araki T. Mohi M.G. F.A. I. Bronson R.T. Williams I.R. Kutok J.L. Pao L.I. Gilliland D.G. Epstein J.A. Neel B.G. Nat. Med. 2004; 10: 849-857Crossref PubMed Scopus (350) Google Scholar). Neoplasia-associated PTPN11 mutations affect many of the same residues but typically are less conservative, resulting in greater catalytic activation (5Tartaglia M. Niemeyer C.M. Fragale A. Song X. Buechner J. Jung A. Hahlen K. Hasle H. Licht J.D. Gelb B.D. Nat. Genet. 2003; 34: 148-150Crossref PubMed Scopus (830) Google Scholar, 7Bentires-Alj M. Paez J.G. David F.S. Keilhack H. Halmos B. Naoki K. Maris J.M. Richardson A. Bardelli A. Sugarbaker D.J. Richards W.G. Du J. Girard L. Minna J.D. Loh M.L. Fischer D.E. Velculescu V. Vogelstein B. Myerson M. Sellers W.R. Neel B.G. Cancer. Res. 2004; 64: 8816-8820Crossref PubMed Scopus (421) Google Scholar, 19Niihori T. Aoki Y. Ohashi H. Kurosawa K. Kondoh T. Ishikiriyama S. Kawame H. Kamasaki H. Yamanaka T. Takada F. Nishio K. Sakurai M. Tamai H. Nagashima T. Suzuki Y. Kure S. Fujii K. Imaizumi M. Matsubara Y. J. Hum. Genet. 2005; 50: 192-202Crossref PubMed Scopus (96) Google Scholar, 20Keilhack H. David F.S. McGregor M. Cantley L.C. Neel B.G. J. Biol. Chem. 2005; 280: 30984-30993Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). When expressed in hematopoietic cells, such mutants enhance basal and cytokine-evoked Erk, Akt, and Stat 5 activation (22Mohi M.G. Williams I.R. Dearolf C.R. Chan G. Kutok J.L. Cohen S. Morgan K. Boulton C. Shigematsu H. Keilhack H. Akashi K. Gilliland D.G. Neel B.G. Cancer Cell. 2005; 7: 1-14Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 23Chan R.J. Leedy M.B. Manugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (133) Google Scholar). Because NS and LS share multiple phenotypic features and are caused by PTPN11 mutations, they have been viewed as overlap syndromes (1Tartaglia M. Gelb B.D. Annu. Rev. Genomics Hum. Genet. 2005; 6: 45-68Crossref PubMed Scopus (279) Google Scholar). We examined the enzymatic properties of LS mutants and their effects on receptor-tyrosine kinase signaling. Surprisingly, despite its phenotypic and genetic similarities to NS, we found that LS is caused by catalytically defective, loss-of-function mutations in PTPN11. Structural Interpretation and Molecular Modeling—The Shp2 structure 2SHP (Protein Data Bank), comprising the two SH2 domains plus the PTP domain and corresponding to the basal, inactive form of the enzyme in the absence of Tyr(P) peptide (15Hof P. Pluskey S. Dhe-Paganon S. Eck M.J. Shoelson S.E. Cell. 1998; 98: 441-450Abstract Full Text Full Text PDF Scopus (756) Google Scholar), and the predicted effects of various mutants were visualized using the PyMol Molecular Graphic System, Version 0.97 (DeLano Scientific). Expression Constructs and Protein Purification—Point mutations were introduced using the QuikChange kit (Stratagene). Wild type Shp2 (WT) and mutant Shp2 in pGEX-4T-2F, a modified version of pGEX-4T (Amersham Biosciences) that generates proteins with N-terminal GST- and C-terminal FLAG tags, were produced and purified on glutathione-Sepharose (Amersham Biosciences), as described previously (20Keilhack H. David F.S. McGregor M. Cantley L.C. Neel B.G. J. Biol. Chem. 2005; 280: 30984-30993Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Protein concentrations were determined by densitometric analysis of Coomassie-stained SDS-PAGE gels using bovine serum albumin as the standard. Purified enzymes were stored in the presence of 33% glycerol at -80 °C. PTP Assays—PTP assays using 32P-labeled carboxamido-methylated and -maleylated lysozyme (RCML) (typical specific activity ∼4000 cpm/pmol) as substrate were conducted in assay buffer (50 mm Hepes (pH 7.4), 150 mm NaCl, 0.1 mg/ml bovine serum albumin, 10 mm dithiothreitol, 5 mm EDTA, 2 μm RCML, and 5 nm GST-enzyme) with or without IRS-1 Tyr(P)-1172 (SLNpYIDLDLVK; pY is Tyr(P)) or Tyr(P)-1222 (LSTpYASINFQK) peptides as described (20Keilhack H. David F.S. McGregor M. Cantley L.C. Neel B.G. J. Biol. Chem. 2005; 280: 30984-30993Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Assays using para-nitrophenyl phosphate (pNPP, obtained from Sigma) as substrate were carried out in 30 mm Hepes (pH 7.4), 120 mm NaCl, 5 mm dithiothreitol, 10 mm pNPP, and 5 nm enzyme with or without various concentrations of Tyr(P)-peptide at 30 °C for 10 min and terminated with 0.2 n NaOH. Phosphate release was determined by measuring A410. Immune complex PTP assays were performed on lysates of transiently transfected 293 cells. Forty-eight hours after transfection, lysates from serum-starved or epidermal growth factor (EGF)-stimulated (50 ng/ml) cells were prepared, and Shp2 proteins were immunoprecipitated by using anti-Shp2 polyclonal antibodies (Santa Cruz) coupled to protein A-Sepharose. Shp2 immune complexes were washed 3 times in 1% Nonidet P-40 lysis buffer (see below) without sodium orthovanadate and once in wash buffer (30 mm HEPES (pH 7.4), 120 mm NaCl without pNPP). PTP assays were performed at 37 °C in 50 μl of pNPP assay buffer as described above. Recovered immune complexes were boiled in 2× SDS-PAGE sample buffer, resolved by SDS-PAGE, and immunoblotted for Shp2 to control for equal Shp2 expression. All assays were carried out in triplicate. Cell Culture—293T cells were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Sigma), 1% sodium pyruvate, and 1% penicillin-streptomycin. Transient Transfections—WT or mutant Shp2 cloned in the mammalian expression vector pBABE-puro (Invitrogen) were transfected into 293T cells using Lipofectamine 2000 (Invitrogen). For some experiments, WT or mutant Shp2 (5 μg), were co-transfected with HA-Erk (0.5 μg) and/or HA-Gab-1 (2.0 μg). Twenty-four hours post-transfection cells were serum-starved for an additional 24 h and then either left unstimulated or stimulated with EGF (50 ng/ml), fibroblast growth factor (FGF)-2 (20 ng/ml), or PDGF (100 ng/ml) for various times. All growth factors were obtained from Calbiochem. Cells were lysed in 1 ml of Nonidet P-40 (1.0% Nonidet P-40, 50 mm Tris ·HCl (pH 7.4), 150 mm NaCl, 5 mm EDTA, 2 mm NaVO3) or radioimmune precipitation assay buffer (1.0% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 25 mm Tris ·HCl (pH 7.4), 150 mm NaCl, 2 mm NaVO3) plus a protease mixture (5 μg/ml leupeptin, 5 μg/ml aprotinin; 1 μg/ml pepstatin A, 1 mmphenylmethylsulfonyl fluoride, 1 mm benzamidine) and clarified in a microcentrifuge. Protein concentrations were measured by using Coomassie protein reagent. Proteins were resolved by SDS-PAGE. Immunoblotting onto Immobilon P and detection by enhanced chemiluminescence (ECL) were performed essentially as described (21Araki T. Mohi M.G. F.A. I. Bronson R.T. Williams I.R. Kutok J.L. Pao L.I. Gilliland D.G. Epstein J.A. Neel B.G. Nat. Med. 2004; 10: 849-857Crossref PubMed Scopus (350) Google Scholar). Rabbit polyclonal anti-Shp2 and anti-total Erk antibodies (Santa Cruz Biotechnology, Inc.), anti-Gab1 antibodies (Upstate Biotechnology, Inc.), anti-phospho-Erk1/2 antibodies (Cell Signaling Technology), and horseradish peroxidase-conjugated anti-rabbit secondary antibodies (Amersham Biosciences) were all used according to their manufacturer's instructions. Stable Cell Lines—Retroviruses expressing WT or mutant Shp2 were generated by transient co-transfection of 293T cells with pBABE-puro constructs and AmphoPac (Invitrogen). Viral supernatants were collected 48 h post-transfection, passed through a 0.45-μm filter, and used to infect fresh 293T cells in the presence of 4 μg/ml Polybrene (Sigma). Forty-eight hours later cells were selected in 1 μg/ml puromycin (Sigma). Cell stimulations and analyses were performed as described above. LS Mutations Are Catalytically Inactive—In contrast to NS mutations, which are scattered throughout the Shp2 molecule, LS mutants are confined to the PTP domain (11Sarkozy A. Conti E. Digilio M.C. Marino B. Morini E. Pacileo G. Wilson M. Calabro R. Pizzuti A. Dallapiccola B. J. Med. Genet. 2004; 41: e68-e74Crossref PubMed Scopus (119) Google Scholar, 12Keren B. Hadchouel A. Saba S. Sznajer Y. Bonneau D. Leheup B. Boute O. Gaillard D. Lacombe D. Layet V. Marlin S. Mortier G. Toutain A. Beylot C. Baumann C. Verloes A. Cave H. J. Med. Genet. 2004; 41: 117-119Crossref PubMed Scopus (72) Google Scholar, 23Chan R.J. Leedy M.B. Manugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (133) Google Scholar) (Figs. 1, B-D). Based on structural and enzymologic studies of other PTPs (e.g. PTP1B (24Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (688) Google Scholar, 25Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar)), the six mutations specifically linked to LS (Y279(C/S), T468M, A461T, G464A, Q506P, Q510P) involve residues predicted to affect catalysis (26Denu J.E. Stuckey J.A. Saper M.A. Dixon J.E. Cell. 1996; 87: 361-364Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Three (Ala-461, Gly-464, and Thr-468) reside within the "signature motif" ((I/V)HCXAGXGR(S/T)GT) that defines the PTP family (26Denu J.E. Stuckey J.A. Saper M.A. Dixon J.E. Cell. 1996; 87: 361-364Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) and comprises the "PTP loop" in PTP structures (24Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (688) Google Scholar, 25Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar). The conserved cysteinyl residue within this motif carries out a nucleophilic attack on the substrate phosphotyrosyl residue, generating a thiophosphate intermediate that subsequently undergoes hydrolysis by a bound water molecule. Other signature motif residues are important for lowering the pKa (i.e. enhancing the nucleophilicity) of the catalytic cysteine, orienting the substrate for nucleophilic attack, maintaining the structural integrity of the catalytic pocket, and/or neutralizing the charge of the phosphotyrosine in the substrate (26Denu J.E. Stuckey J.A. Saper M.A. Dixon J.E. Cell. 1996; 87: 361-364Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Shp2 Tyr-279 is cognate to Tyr-46 in PTP1B, which sets the depth of the catalytic cleft and, thus, confers specificity for phosphotyrosine-containing substrates (24Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (688) Google Scholar). Gln-506 or possibly Gln-510 is the Shp2 analog of Gln-262 in PTP1B, which helps position the water molecule to hydrolyze the thiophosphate intermediate (25Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar, 27Pannifer A.D.B. Flint A.J. Tonks N.K. Barford D. J. Biol. Chem. 1998; 273: 10454-10462Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Based on their locations in the PTP catalytic cleft, we hypothesized that the LS mutations might have fundamentally different biochemical and biological properties than other disease-associated PTPN11 mutants. To begin to test this hypothesis, we expressed and purified GST-tagged wild type Shp2 (WT), the NS mutation D61G, the leukemia-associated mutant E76K, and several LS mutants as recombinant proteins in bacteria, as described previously (20Keilhack H. David F.S. McGregor M. Cantley L.C. Neel B.G. J. Biol. Chem. 2005; 280: 30984-30993Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Purified proteins were assayed against the artificial substrates pNPP and reduced carboxamido-methylated and -maleylated lysozyme (RCML). As expected (17O'Reilly A.M. Pluskey S. Shoelson S.E. Neel B.G. Mol. Cell. Biol. 1999; 20: 299-311Crossref Scopus (101) Google Scholar, 18Fragale A. Tartaglia M. Wu J. Gelb B.D. Hum. Mutat. 2004; 23: 267-277Crossref PubMed Scopus (167) Google Scholar, 19Niihori T. Aoki Y. Ohashi H. Kurosawa K. Kondoh T. Ishikiriyama S. Kawame H. Kamasaki H. Yamanaka T. Takada F. Nishio K. Sakurai M. Tamai H. Nagashima T. Suzuki Y. Kure S. Fujii K. Imaizumi M. Matsubara Y. J. Hum. Genet. 2005; 50: 192-202Crossref PubMed Scopus (96) Google Scholar, 20Keilhack H. David F.S. McGregor M. Cantley L.C. Neel B.G. J. Biol. Chem. 2005; 280: 30984-30993Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), WT Shp2 exhibited low basal activity that was stimulated after the addition of a Tyr(P) peptide from IRS-1 (Tyr(P)-1172) that binds the N-SH2 domain (Fig. 2A). D61G had increased basal activity (compared with WT) that was further enhanced upon the addition of Tyr(P)-1172, whereas E76K was maximally activated even in the absence of Tyr(P) peptide. In contrast, none of the LS mutants exhibited detectable PTP activity against either substrate, even in the presence of saturating amounts of Tyr(P)-1172 (Fig. 2A) or over a wide range of doses of another activating phosphopeptide, Tyr(P)-1222, of IRS-1 (Fig. 2B). To exclude the possibility that the recombinant LS proteins produced in bacteria folded inappropriately and/or were unstable, mammalian expression constructs for (untagged) WT Shp2 and the indicated mutants were transiently transfected into 293T cells, which were then starved and either left unstimulated or stimulated with EGF. Endogenous (i.e. empty vector-transfected) Shp2 activity, as measured by immune complex PTP assay, was enhanced slightly upon growth factor stimulation (Fig. 2C). Cells transfected with WT Shp2 showed slightly higher basal and stimulated Shp2 activity, whereas those expressing the leukemia-associated E76K mutant showed markedly enhanced and constitutive Shp2 activity. However, transient expression of LS mutants led to no increase in PTP activity even though these mutants were expressed at levels comparable with WT Shp2 or E76K (Fig. 2C, bottom panel). Instead, LS mutants tended to cause decreased EGF-stimulated Shp2 activity (as measured by immune complex assay), raising the possibility that such mutants have dominant negative effects. LS Mutations Disrupt the Catalytic Pocket of Shp2—Examination of the Shp2 crystal structure (Fig. 1D) provided potential explanations for these observations. Because Tyr-279 sets the depth of the catalytic cleft, substitution by cysteine (or serine as in another LS patient (12Keren B. Hadchouel A. Saba S. Sznajer Y. Bonneau D. Leheup B. Boute O. Gaillard D. Lacombe D. Layet V. Marlin S. Mortier G. Toutain A. Beylot C. Baumann C. Verloes A. Cave H. J. Med. Genet. 2004; 41: 117-119Crossref PubMed Scopus (72) Google Scholar)) should alter the depth of the cleft and might also perturb the orientation of the catalytic cysteine (Figs. 3, A and B). Ala-461 is a highly conserved (see ptp.cshl.edu) PTP loop residue that also contacts Tyr-279; placing a bulkier threonyl substitution here should contort the catalytic site and interfere with substrate phosphotyrosine binding (Figs. 3, C and D). Gly-464 is an invariant signature motif residue in PTP superfamily members. Its replacement by alanine also should disrupt the PTP loop and sterically clash with the phosphate group of an incoming substrate phosphotyrosyl residue (Figs. 3, E and F). Thr-468 is a buried residue on the αG (α-4 in PTP1B) helix. Methionine substitution here probably would destabilize the entire PTP catalytic domain, with associated loss of enzymatic activity (Figs. 3, G and H). LS Mutants Are Dominant Negative—We next examined the effects of LS mutants on receptor-tyrosine kinase signaling. Expression constructs for WT or mutant Shp2, Gab1, and HA epitope-tagged Erk1 were co-transfected into 293T cells, as described previously (18Fragale A. Tartaglia M. Wu J. Gelb B.D. Hum. Mutat. 2004; 23: 267-277Crossref PubMed Scopus (167) Google Scholar). Transiently transfected cells were starved or stimulated with EGF, and total Tyr(P) (data not shown) and Erk activation was monitored by antiphospho-Erk immunoblotting (Fig. 4A). As expected, EGF stimulation activated Erk in WT-expressing cells, and Erk activation was enhanced to a much greater extent in cells expressing E76K or D61G. Expression of a mutant of the essential catalytic cysteinyl residue (C459S) inhibited Erk activation, as reported (28Bennett A.M. Hausdorff S.F. O'Reilly A.M. Freeman R.M. Neel B.G. Mol. Cell. Biol. 1996; 16: 1189-1202Crossref PubMed Scopus (226) Google Scholar). Notably, the two most common LS mutants, Y279C and T468M, also had dominant negative effects, strongly inhibiting EGF-evoked Erk activation (Fig. 4A). Likewise, in stable cell pools expressing each mutant, D61G enhanced, whereas LS mutants strongly impaired, EGF-evoked Erk activation (Fig. 4B). Similar to the effects of Shp2 deficiency in fibroblasts (29Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.-S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (406) Google Scholar, 30Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (191) Google Scholar, 31Zhang S.Q. Tsiaris W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (212) Google Scholar), the major effect of LS mutants was to inhibit sustained EGF-evoked Erk activation (Fig. 4C). LS mutants also impaired Erk activation in response to FGF or PDGF stimulation (Fig. 4, D and E), consistent with a general
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