Structure of a c-Kit Product Complex Reveals the Basis for Kinase Transactivation
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.c300186200
ISSN1083-351X
AutoresClifford D. Mol, Kheng B. Lim, Vandana Sridhar, Hua Zou, Ellen Y. T. Chien, Bi‐Ching Sang, Jacek Nowakowski, Daniel B. Kassel, Ciarán N. Cronin, Duncan E. McRee,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoThe c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes. The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes. Receptor protein-tyrosine kinases (RPTKs) 1The abbreviations used are: RPTK, receptor protein-tyrosine kinase; SCF, stem cell factor; LC, liquid chromatography; MS, mass spectrometry; MES, 2[N-morpholinoethanesulfonic acid; PTR, phosphotyrosine.1The abbreviations used are: RPTK, receptor protein-tyrosine kinase; SCF, stem cell factor; LC, liquid chromatography; MS, mass spectrometry; MES, 2[N-morpholinoethanesulfonic acid; PTR, phosphotyrosine. regulate key signal transduction cascades that control cellular growth and proliferation. The stem cell factor (SCF) receptor c-Kit is a type III transmembrane RPTK comprised of five extracellular immunoglobulin domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (1Yarden Y. Escobedo J.A. Kuang W.-J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Crossref PubMed Scopus (764) Google Scholar, 2Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4583) Google Scholar). The type III RPTK family includes c-Kit (3Yarden Y. Kuang W.-J. Yang-Feng T. Coussens L. Munemitsu S. Dull T.J. Chen E. Schlessinger J. Francke U. Ullrich A. EMBO J. 1987; 6: 3341-3351Crossref PubMed Scopus (1317) Google Scholar), the colonystimulating factor-1 (formerly FMS) (4Coussens L. Van Beveren C. Smith D. Chen E. Mitchell R.L. Isacke C.M. Verma I.M. Ullrich A. Nature. 1986; 320: 277-280Crossref PubMed Scopus (289) Google Scholar), the platelet-derived growth factor α and β receptors (1Yarden Y. Escobedo J.A. Kuang W.-J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Crossref PubMed Scopus (764) Google Scholar, 5Claesson-Welsh L. Eriksson A. Westermark B. Heldin C.-H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4917-4921Crossref PubMed Scopus (307) Google Scholar), and the FMS-related receptor FLT-3 (6Rosnet O. Schiff C. Pebusque M.-J. Marchetto S. Tonnelle C. Toiron Y. Birg F. Birnbaum D. Blood. 1993; 82: 1110-1119Crossref PubMed Google Scholar). Signaling by RPTKs occurs via ligand binding to the extracellular IG domains, inducing the receptors to form dimers, and thereby activating intrinsic tyrosine kinase activity through the transphosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains (7Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar, 8Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Ligand binding both activates kinase activity and creates tyrosine-phosphorylated receptors that mediate the specific binding of intracellular signaling proteins. Src homology 2 and protein tyrosine binding domains (9Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3099) Google Scholar), including the protein-tyrosine phosphatase SHP-1, act as negative regulators of c-Kit activity (10Kozlowski M. Larose L. Lee F. Le D.M. Rottapel R. Siminovitch K.A. Mol. Cell. Biol. 1998; 18: 2089-2099Crossref PubMed Scopus (176) Google Scholar). These cytoplasmic signaling proteins initiate serine/threonine phosphorylation cascades that activate transcription factors to determine specific cellular responses (Fig. 1).The human c-Kit gene is the cellular homologue of the v-kit oncogene found in the transforming Hardy-Zuckerman 4 feline sarcoma virus (11Snyder Jr., H.W. Broudeur D. Zuckerman E.E. Hardy W.D. Nature. 1986; 320: 415-421Crossref PubMed Scopus (450) Google Scholar) and encodes a 976-amino acid residue RPTK. Loss-of-function c-Kit mutations establish its importance for the normal growth of hematopoietic progenitor cells, mast cells, melanocytes, primordial germ cells, and the interstitial cells of Cajal (12Besmer P. Curr. Opin. Cell Biol. 1991; 3: 939-946Crossref PubMed Scopus (152) Google Scholar, 13Lyman S.D. Jacobsen S.E.W. Blood. 1998; 91: 1101-1134Crossref PubMed Google Scholar, 14Ashman L.K. Int. J. Biochem. Cell Biol. 1999; 31: 1037-1051Crossref PubMed Scopus (475) Google Scholar, 15Kitamura Y. Hirota S. Nishida T. Mutat. Res. 2001; 477: 165-171Crossref PubMed Scopus (60) Google Scholar). Gain-of-function mutations, resulting in SCF-independent, constitutive activation of c-Kit, are found in several highly malignant cancers. Mutations in the c-Kit juxtamembrane region cluster around the two main autophosphorylation sites that mediate protein tyrosine binding, Tyr-568 and Tyr-570, and are associated with human gastrointestinal stromal tumors (16Hirota S. Isozaki K. Moriyama Y. Hashimoto K. Nishida T. Ishiguro S. Kawano K. Hanada M. Kurata A. Takeda M. Tunio G.M. Matsuzawa Y. Kanakura Y. Sinomura Y. Kitamura Y. Science. 1998; 279: 577-580Crossref PubMed Scopus (3789) Google Scholar, 17Hirota S. Taniguchi M. Hashimoto K. Isozaki K. Nakamura H. Kanakura Y. Tanaka T. Takabayashi A. Matsuda H. Kitamura Y. Nishida T. Nat. Genet. 1998; 19: 323-324Crossref PubMed Scopus (497) Google Scholar). Mutations in the kinase domain are found in mast cell and myeloid leukemias (15Kitamura Y. Hirota S. Nishida T. Mutat. Res. 2001; 477: 165-171Crossref PubMed Scopus (60) Google Scholar) and in human germ cell tumors (18Tian Q. Frierson Jr., H.F. Krystal G.W. Moskaluk C.A. Am. J. Pathol. 1999; 154: 1643-1647Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar).These activating, oncogenic mutations transform cells through enhanced c-Kit dimer formation: bringing two c-Kit kinases into close proximity to enable them to act as substrate and enzyme for one another (9Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3099) Google Scholar, 19Hubbard S.R. Mohammadi M. Schlessinger J. J. Biol. Chem. 1998; 273: 11987-11990Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). The juxtamembrane domain c-Kit mutants likely disrupt the binding of regulatory proteins, resulting in autophosphorylation and kinase activation. The transforming kinase domain mutants likely stabilize the active conformation of the c-Kit activation loop (A-loop). Similar mutations in A-loop residues in the MET and fibroblast growth factor 3 receptors are highly phosphorylated when expressed in vivo without added ligand (20Naski M.C. Wang Q. Xu J. Ornitz D.M. Nat. Genet. 1996; 13: 233-237Crossref PubMed Scopus (416) Google Scholar, 21Webster M.K. D'avis P.Y. Robertson S.C. Donoghue D.J. Mol. Cell. Biol. 1996; 16: 4081-4087Crossref PubMed Scopus (160) Google Scholar) and are implicated in kidney and bone cancers, respectively (22Jeffers M. Schmidt L. Nakaigawa N. Webb C.P. Weirich G. Kishida T. Zbar B. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11445-11450Crossref PubMed Scopus (384) Google Scholar, 23Tavormina P.L. Shiang R. Thompson L.M. Zhu Y.Z. Wilkin D.J. Lachman R.S. Wilcox W.R. Rimoin D.L. Cohn D.H. Wasmuth J.J. Nat. Genet. 1995; 9: 321-328Crossref PubMed Scopus (517) Google Scholar).This direct involvement of mutant c-Kit RPTKs in human cancers has encouraged the direct targeting of the c-Kit kinase domain for structure-guided, rational drug design. The success of STI-571 (also known by the trade names Gleevec and Imatinib) in the treatment of chronic myelogenous leukemia is based on the inhibition of the c-Abl kinase, whose activity, like c-Kit's, is tightly controlled in normal cells. STI-571 targets the inactive kinase structure with unphosphorylated A-loop residues, particularly the kinase DFG motif, occupying the binding sites for substrate tyrosine-containing polypeptides (24Schindler T. Bornmann W. Pellicena P. Miller W.T. Clarkson B. Kuriyan J. Science. 2000; 289: 1938-1942Crossref PubMed Scopus (1611) Google Scholar). STI-571 can also be an effective treatment for other human cancers including gastrointestinal stromal tumors and stems from STI-571 inhibition of c-Kit kinase, particularly those with activating mutations in the juxtamembrane domain. Regrettably, STI-571 is less effective in treating cancers with activating mutations in the c-Kit kinase domain (25Zermati Y. De Sepulveda P. Feger F. Letard S. Kersual J. Casteran N. Gorochev G. Dy M. Ribadeau Dumas A. Dorgham K. Parizot C. Bieche Y. Vidaud M. Lortholary O. Arock M. Hermine O. Dubreuil P. Oncogene. 2003; 22: 660-664Crossref PubMed Scopus (163) Google Scholar). We report here on the enzymatic activity, phosphopeptide mapping, and the crystal structure of a c-Kit kinase-product complex. These results provide the molecular basis for understanding the mechanism of c-Kit kinase transactivation and is an essential step for the structure-guided design of specific c-Kit kinase inhibitors.MATERIALS AND METHODSEnzyme Expression and Purification—The catalytic domain of the human c-kit gene (residues 544–935; GenBank™ accession number NM_000222 (4Coussens L. Van Beveren C. Smith D. Chen E. Mitchell R.L. Isacke C.M. Verma I.M. Ullrich A. Nature. 1986; 320: 277-280Crossref PubMed Scopus (289) Google Scholar)) was amplified by PCR from a bone marrow cDNA library (Invitrogen) and cloned into the BamHI and XbaI restriction enzyme sites of pSXB1 (pFastBacHTa baculovirus transfer vector (Invitrogen) modified to include a SmaI site between the NcoI and BamH I sites. The kinase insertion domain residues 694–753 were deleted and replaced with 6 nucleotides encoding Thr-Ser. Recombinant c-Kit protein with a 6x-histidine tag followed by an rTEV protease site was expressed in 5L of Spodoptera frugiperda (Sf9) insect cells using 10L Wave BioReactors (Wave Biotech) and ESF-921 protein-free medium (Expression Systems). Recombinant c-Kit protein was obtained at a yield of 1.8 mg/liter of cell culture and purified by binding to ProBond resin (Invitrogen) and washing with 20 mm imidazole to remove contaminating proteins, and c-Kit protein was eluted with 200 mm imidazole buffer. The His6 tag was removed with rTEV protease (Invitrogen) and uncleaved material removed by a second passage over ProBond. The purified c-Kit protein was not phosphorylated as judged by mass spectrometry (data not shown) and was concentrated to 6 mg/ml in 25 mm Tris, pH 7.6, 250 mm NaCl, 5 mm dithiothreitol, 1 mm EDTA, flash-frozen in liquid nitrogen, and stored at –80 °C.Phosphorylation Sites Mapping by LC/MS/MS—The autophosphorylation reaction was initiated by addition of 5 mm ATP and 10 mm MgCl2 at 20 °C and quenched after 1 h with 20 mm EDTA, and the sample was digested at 20:1 (w/w) ratio using a modified trypsin (Promega) in Tris buffer at 37 °C for 16 h. Tryptic digests were analyzed using a 0.3 × 150-mm 5-μm C18 PepMap capillary column (LC Packings). A 0.3 × 5-mm 5-μm C18 u-Precolumn cartridge (LCPackings) was used to trap and rapidly desalt the tryptic peptides. A capillary flow rate at 10 μl/min was generated using a Paradigm MS4 multidimensional separations module (Michrom BioResources) operated at 150 μL/min and split to 10 μl/min with the built-in variable splitter. MS spectra (m/z 150–2000) and data-dependent MS/MS spectra were acquired on a LCQ DECA ion trap (Finnigan Corp.). Turbo Sequest protein identification software was used to locate the phosphorylation sites on the peptides.Crystallization and Structure Determination—Crystals of c-Kit kinase were obtained by preincubating active enzyme samples (6 mg/ml, 150 mm NaCl, 25 mm Tris, pH 7.9) with 2.5 mm ATP and 5 mm MgCl2, and were grown at room temperature by sitting-drop vapor diffusion using 50 nl of protein solution and 50 nl of reservoir (18% polyethylene glycol 8000, 0.1 m MES, pH 7.1). Crystals were harvested in reservoir solution supplemented with 25% ethylene glycol and flash-frozen by direct immersion in liquid nitrogen. X-ray diffraction data were collected at the Advanced Light Source Beam Line 5.0.3 and were integrated and scaled using HKL2000 (26Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar). The crystals belong to the orthorhombic space group P21212, a = 92.7Å, b = 116.4 Å, and c = 60.1 Å, and have two molecules in the asymmetric unit. Data collection and structure refinement statistics are listed in Table I. The structure was determined by molecular replacement using AMoRe (27Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar) with the VEGFR2 receptor kinase (Protein Data Bank code 1VR2) used as a search model. The correct solutions yielded the highest correlation coefficients in the rotation and translation searches. The model was refined without non-crystallographic symmetry restraints in REFMAC (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13779) Google Scholar) and inspected, built, and rebuilt using Xfit (29McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2019) Google Scholar). The atomic coordinates have been deposited with the Protein Data Bank (accession code 1PKG.pdb).Table IX-ray data collection and refinement statisticsData collectionResolution (Å)30.0-2.9Observations49,361Unique reflections14,652Completeness (%)97.0I/σI11.2 (3.0)R symaR sym = ΣhΣj|〈I(h)〉 - I(h)j | / ΣhΣj 〈I(h)〉, where 〈I(h)〉 is the mean intensity of symmetry-related reflections.0.072 (0.424)RefinementResolution (Å)10.0-2.9Reflections used13,437r.m.s. bonds (Å)0.014r.m.s. angles (°)1.61Average B value (Å2)17.0R-value, R freebR-value = Σ ∥F obs| - |F calc∥/ Σ |F obs|. R free for 5% of reflections excluded from refinement. Values in parentheses are for the 2.9-3.0-Å shell.0.22, 0.31a R sym = ΣhΣj|〈I(h)〉 - I(h)j | / ΣhΣj 〈I(h)〉, where 〈I(h)〉 is the mean intensity of symmetry-related reflections.b R-value = Σ ∥F obs| - |F calc∥/ Σ |F obs|. R free for 5% of reflections excluded from refinement. Values in parentheses are for the 2.9-3.0-Å shell. Open table in a new tab RESULTSPhosphotyrosine Peptide Mapping—To map the phosphorylation sites, LC/MS/MS experiments were performed with targeted MS/MS acquisitions. Since the +2 precursor ions usually yield better MS/MS fragmentation patterns than the +3 precursor ions, we programmed the mass spectrometer to switch automatically to acquire MS/MS spectra if the +2 of the diphosphorylated peptide precursor ion (m/z 1441.4) was detected. The LC/MS/MS spectrum for the diphosphotryptic peptide shows the presence of a series of y- and b-type ions to confirm the sequence assignment (Fig. 2). The y and b ion assignments localize the phosphorylation sites to Tyr-568 and Tyr-570.Fig. 2LC/MS/MS spectrum of the diphosphorylated peptide, VVEEINGNN(pY)V(pY)IDPTQLPYDHK. Phosphotryptic peptides were identified on the basis of a predicted mass difference of 80 Da. Assignments of the y and b ions for the diphosphorylated peptide (precursor ion m/z 1441.4), suggesting two phosphotyrosine residues each at Tyr-568 and Tyr-570 based on the mass difference between the y13/y14 and b9/b10 ions for Tyr-568 and y11/y12 and b11/b12 ions for Tyr-570. The inset shows the LC/MS scan of the precursor ion.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Structure Quality and Global Enzyme Architecture—The structure of active c-Kit kinase is of superb quality and bears most of the defining features of an active kinase configuration (Fig. 3). The electron density is clear and unambiguous for the active site and for most of the enzyme secondary structure and connecting loops. Over 97% of the residues in both enzyme molecules in the asymmetric unit are within the allowed regions of the Ramachandran plot of main chain (ϕ/φ) torsion angles, and a significant portion of both structures is ordered. The amino-terminal ∼20 amino acid residues of both enzymes are disordered, as are the residues comprising the truncated kinase insertion domain. The conformations of both the A-loop and flexible, glycine-rich P-loop, which forms a key part of the active site, are consistent with an active kinase structure (Fig. 3).Fig. 3Structure of active c-Kit kinase. The Cα ribbon illustrates the two-domain kinase fold and key structural elements, including the C-helix, phosphate-binding P-loop, adenine-recognition hinge loop, and kinase activation A-loop. The positions of the ADP, metal ion, and substrate peptide are also shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The global architecture of c-Kit kinase resembles the conserved protein serine/threonine kinase two-domain fold, with a smaller, amino-terminal N-lobe comprised of mostly β-strands, and a larger predominantly α-helical carboxy-terminal C-lobe (Fig. 3). The N-lobe contains a single α-helix, designated the control or C-helix, which directly contacts the A-loop DFG motif and nucleotide binding site and commonly modulates kinase activity in regulatory mechanisms. The c-Kit C-helix is in a conformation consistent with productive nucleotide binding. The conserved C-helix glutamic acid, Glu-640, forms a critical interaction with the side chain of buried Lys-623, which orients and positions the Lys-623 Nϵ atom to bridge the α- and β-phosphates of the bound ADP. The DFG motif is also in an active state consistent with binding a Mg2+ ion that coordinates Asn-797, the ADP α- and β-phosphates, and the phosphate of phosphotyrosine (PTR)-568 from an adjacent molecule (Fig. 4).Fig. 4The c-Kit active site. Interactions at the c-Kit active site with Mg2+, ADP, and phosphotyrosine are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nucleotide Binding and Active Kinase Structure—The c-Kit active site centers around the bound ADP and Mg2+ ion. ADP binds in the interdomain cleft between the N- and C-lobes, inserting the adenine base into a hydrophobic pocket with the adenine N1 atom and exocyclic N6 amino group forming hydrogen bonds to the polypeptide backbone amide and carbonyl groups of the hinge region residues Cys-673 and Glu-671, respectively (Fig. 4). The ribose sugar O2′ hydroxyl contacts Asp-677, and the O3′ group forms a hydrogen bond with the Arg-796 carbonyl. The O4′ side of the ribose sugar packs with Gly-596, the first residue of the c-Kit P-loop sequence. The other P-loop residues contact the ADP phosphates, with Phe-600 capping the active site to shield the transphosphorylation reaction from bulk solvent (Fig. 4).The c-Kit kinase enzyme active site is fully competent for autophosphorylation as evidenced by the presence of both phosphotyrosine residues identified from the MS analyses (PTR-568 and PTR-570) in the bound substrate peptides. These peptides, from adjacent enzyme molecules in the crystal, bind in trans by inserting between the backside of the A-loop and conserved structural elements of the C-lobe (Figs. 3 and 4). Consistent with the known biology of the Type III RPTK family, the target tyrosine residues are immediately adjacent to the conserved kinase domain fold, and the length of the polypeptide chain is insufficient for the enzyme to perform the autophosphorylation reaction in cis. Binding of the substrate is enhanced by a salt link between Arg-830 and PTR-570 and by hydrophobic interactions between Val-569, Ile-571, and Ile-576 with hydrophobic residues emanating from the C-lobe of the kinase. Substrate binding may stabilize the active conformation of the kinase A loop, despite the fact that the target A loop tyrosine, Tyr-823, is not phosphorylated. In support of the conclusion that the c-Kit A loop is in a biologically relevant active conformation, Tyr-823 is within hydrogen-bonding distance of the side chain of the conserved A loop residue Arg-815, with the side chain of Arg-791 also in the vicinity. The positive charge of the side chains on these arginine residues would counterbalance the negative charge of a phosphorylated Tyr-823 and further stabilize the A loop in an active conformation.DISCUSSIONInterest in c-Kit stems from the disease-causing effects of its constitutive activation in cells. In the normal "off" state c-Kit receptor exists as a monomer in the cell membrane. Binding of SCF induces the c-Kit receptor to dimerize and, acting as both enzyme and substrate for itself, c-Kit autophosphorylates specific NH2-terminal tyrosine residues in trans. Unlike most other protein kinases whose targets are separate and distinct cellular proteins, these autophosphorylated phosphotyrosine residues are themselves the signal that activates intracellular serine/threonine protein kinase signaling cascades to determine specific cellular responses in normal cellular growth and development (Fig. 1). We have characterized and determined the crystal structure of active c-Kit kinase to gain an understanding of the molecular basis underlying oncogenic transformation by activating c-Kit mutations. We performed autophosphorylation reactions with a c-Kit construct that contained the critical NH2-terminal target tyrosine residues. For c-Kit autophosphorylated to the +2 diphosphotyrosine state, only Tyr-568 and Tyr-570 are significantly phosphorylated, and thus these two residues are likely among the first to be phosphorylated. Interestingly, when longer incubation times are employed we observe higher levels of autophosphorylation, and our data suggest that the A loop residue Tyr-823 is the last tyrosine residue to be autophosphorylated. As supported by the crystal structure of the active c-Kit kinase, in which the A loop is ordered and in an active conformation, phosphorylation of Tyr-823 is apparently not required to activate the kinase. These results are in contrast with those obtained for most other kinases in which phosphorylation of target A-loop residues is required to stabilize the kinase in an active state (reviewed in Ref. 30Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1346) Google Scholar). Thus, in normal non-phosphorylated, monomeric c-Kit receptor protein-tyrosine kinases, the kinase domain may be poised at or near its active configuration, and that it is only the physical separation of two individual kinase domains that prevents their transactivation. Thus, the active c-Kit kinase domain structure reported here represents the kinase conformation that is resistant to inhibition by STI-571 and will facilitate the design of specific and potent c-Kit kinase inhibitors. Receptor protein-tyrosine kinases (RPTKs) 1The abbreviations used are: RPTK, receptor protein-tyrosine kinase; SCF, stem cell factor; LC, liquid chromatography; MS, mass spectrometry; MES, 2[N-morpholinoethanesulfonic acid; PTR, phosphotyrosine.1The abbreviations used are: RPTK, receptor protein-tyrosine kinase; SCF, stem cell factor; LC, liquid chromatography; MS, mass spectrometry; MES, 2[N-morpholinoethanesulfonic acid; PTR, phosphotyrosine. regulate key signal transduction cascades that control cellular growth and proliferation. The stem cell factor (SCF) receptor c-Kit is a type III transmembrane RPTK comprised of five extracellular immunoglobulin domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (1Yarden Y. Escobedo J.A. Kuang W.-J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Crossref PubMed Scopus (764) Google Scholar, 2Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4583) Google Scholar). The type III RPTK family includes c-Kit (3Yarden Y. Kuang W.-J. Yang-Feng T. Coussens L. Munemitsu S. Dull T.J. Chen E. Schlessinger J. Francke U. Ullrich A. EMBO J. 1987; 6: 3341-3351Crossref PubMed Scopus (1317) Google Scholar), the colonystimulating factor-1 (formerly FMS) (4Coussens L. Van Beveren C. Smith D. Chen E. Mitchell R.L. Isacke C.M. Verma I.M. Ullrich A. Nature. 1986; 320: 277-280Crossref PubMed Scopus (289) Google Scholar), the platelet-derived growth factor α and β receptors (1Yarden Y. Escobedo J.A. Kuang W.-J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Crossref PubMed Scopus (764) Google Scholar, 5Claesson-Welsh L. Eriksson A. Westermark B. Heldin C.-H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4917-4921Crossref PubMed Scopus (307) Google Scholar), and the FMS-related receptor FLT-3 (6Rosnet O. Schiff C. Pebusque M.-J. Marchetto S. Tonnelle C. Toiron Y. Birg F. Birnbaum D. Blood. 1993; 82: 1110-1119Crossref PubMed Google Scholar). Signaling by RPTKs occurs via ligand binding to the extracellular IG domains, inducing the receptors to form dimers, and thereby activating intrinsic tyrosine kinase activity through the transphosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains (7Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar, 8Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Ligand binding both activates kinase activity and creates tyrosine-phosphorylated receptors that mediate the specific binding of intracellular signaling proteins. Src homology 2 and protein tyrosine binding domains (9Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3099) Google Scholar), including the protein-tyrosine phosphatase SHP-1, act as negative regulators of c-Kit activity (10Kozlowski M. Larose L. Lee F. Le D.M. Rottapel R. Siminovitch K.A. Mol. Cell. Biol. 1998; 18: 2089-2099Crossref PubMed Scopus (176) Google Scholar). These cytoplasmic signaling proteins initiate serine/threonine phosphorylation cascades that activate transcription factors to determine specific cellular responses (Fig. 1). The human c-Kit gene is the cellular homologue of the v-kit oncogene found in the transforming Hardy-Zuckerman 4 feline sarcoma virus (11Snyder Jr., H.W. Broudeur D. Zuckerman E.E. Hardy W.D. Nature. 1986; 320: 415-421Crossref PubMed Scopus (450) Google Scholar) and encodes a 976-amino acid residue RPTK. Loss-of-function c-Kit mutations establish its importance for the normal growth of hematopoietic progenitor cells, mast cells, melanocytes, primordial germ cells, and the interstitial cells of Cajal (12Besmer P. Curr. Opin. Cell Biol. 1991; 3: 939-946Crossref PubMed Scopus (152) Google Scholar, 13Lyman S.D. Jacobsen S.E.W. Blood. 1998; 91: 1101-1134Crossref PubMed Google Scholar, 14Ashman L.K. Int. J. Biochem. Cell Biol. 1999; 31: 1037-1051Crossref PubMed Scopus (475) Google Scholar, 15Kitamura Y. Hirota S. Nishida T. Mutat. Res. 2001; 477: 165-171Crossref PubMed Scopus (60) Google Scholar). Gain-of-function mutations, resulting in SCF-independent, constitutive activation of c-Kit, are found in several highly malignant cancers. Mutations in the c-Kit juxtamembrane region cluster around the two main autophosphorylation sites that mediate protein tyrosine binding, Tyr-568 and Tyr-570, and are associated with human gastrointestinal stromal tumors (16Hirota S. Isozaki K. Moriyama Y. Hashimoto K. Nishida T. Ishiguro S. Kawano K. Hanada M. Kurata A. Takeda M. Tunio G.M. Matsuzawa Y. Kanakura Y. Sinomura Y. Kitamura Y. Science. 1998; 279: 577-580Crossref PubMed Scopus (3789) Google Scholar, 17Hirota S. Taniguchi M. Hashimoto K. Isozaki K. Nakamura H. Kanakura Y. Tanaka T. Takabayashi A. Matsuda H. Kitamura Y. Nishida T. Nat. Genet. 1998; 19: 323-324Crossref PubMed Scopus (49
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