Clustering of Activating Mutations in c-KIT’s Juxtamembrane Coding Region in Canine Mast Cell Neoplasms
1999; Elsevier BV; Volume: 112; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.1999.00488.x
ISSN1523-1747
AutoresYongsheng Ma, B. Jack Longley, Xiaomei Wang, John L. Blount, Keith Langley, George H. Caughey,
Tópico(s)Platelet Disorders and Treatments
ResumoThe proto-oncogene c-KIT encodes a growth factor receptor, KIT, with ligand-dependent tyrosine kinase activity that is expressed by several cell types including mast cells. c-KIT juxtamembrane coding region mutations causing constitutive activation of KIT are capable of transforming cell lines and have been identified in a human mast cell line and in situ in human gastrointestinal stromal tumors, but have not been demonstrated in situ in neoplastic mast cells from any species. To determine whether c-KIT juxtamembrane mutations occur in the development of mast cell neoplasms, we examined canine mastocytomas, which are among the most common tumors of dogs and which often behave in a malignant fashion, unlike human solitary mastocytomas. Sequencing of c-KIT cDNA generated from tumor tissues removed from seven dogs revealed that three of the tumors contained a total of four mutations in an intracellular juxtamembrane coding region that is completely conserved among vertebrates. In addition, two mutations were found in three mast cell lines derived from two additional dogs. One mutation from one line matched that found in situ in one of the tumors. The second was found in two lines derived from one dog at different times, indicating that the mutation was present in situ in the animal. All five mutations cause high spontaneous tyrosine phosphorylation of KIT. Our study provides in situ evidence that activating c-KIT juxtamembrane mutations are present in, and may therefore contribute to, the pathogenesis of mast cell neoplasia. Our data also suggest an inhibitory role for the KIT juxtamembrane region in controlling the receptor kinase activity. The proto-oncogene c-KIT encodes a growth factor receptor, KIT, with ligand-dependent tyrosine kinase activity that is expressed by several cell types including mast cells. c-KIT juxtamembrane coding region mutations causing constitutive activation of KIT are capable of transforming cell lines and have been identified in a human mast cell line and in situ in human gastrointestinal stromal tumors, but have not been demonstrated in situ in neoplastic mast cells from any species. To determine whether c-KIT juxtamembrane mutations occur in the development of mast cell neoplasms, we examined canine mastocytomas, which are among the most common tumors of dogs and which often behave in a malignant fashion, unlike human solitary mastocytomas. Sequencing of c-KIT cDNA generated from tumor tissues removed from seven dogs revealed that three of the tumors contained a total of four mutations in an intracellular juxtamembrane coding region that is completely conserved among vertebrates. In addition, two mutations were found in three mast cell lines derived from two additional dogs. One mutation from one line matched that found in situ in one of the tumors. The second was found in two lines derived from one dog at different times, indicating that the mutation was present in situ in the animal. All five mutations cause high spontaneous tyrosine phosphorylation of KIT. Our study provides in situ evidence that activating c-KIT juxtamembrane mutations are present in, and may therefore contribute to, the pathogenesis of mast cell neoplasia. Our data also suggest an inhibitory role for the KIT juxtamembrane region in controlling the receptor kinase activity. human and nonhuman kit protein phosphotyrosine stem cell factor wild-type The c-KIT proto-oncogene encodes the KIT cell-surface receptor, which is comprised of an extracellular domain containing five immunoglobulin-like loops, a transmembrane domain, and a cytoplasmic domain consisting of a juxtamembrane region, a kinase domain divided into adenosine triphosphate (ATP)-binding and phosphotransferase subdomains by a kinase insert, and a carboxy-terminal tail (Yarden et al., 1987Yarden Y. Kuang W.-J. Yang-Feng T. et al.Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand.Embo J. 1987; 6: 3341-3351Crossref PubMed Scopus (1323) Google Scholar;Qiu et al., 1988Qiu F. Ray P. Brown K. et al.Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus.Embo J. 1988; 7: 1003-1011Crossref PubMed Scopus (568) Google Scholar). KIT shares sequence and domain organization similarity with the receptors for colony-stimulating factor-1 and platelet-derived growth factor, the three forming the type III receptor tyrosine kinase subfamily (Yarden et al., 1987Yarden Y. Kuang W.-J. Yang-Feng T. et al.Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand.Embo J. 1987; 6: 3341-3351Crossref PubMed Scopus (1323) Google Scholar;Qiu et al., 1988Qiu F. Ray P. Brown K. et al.Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus.Embo J. 1988; 7: 1003-1011Crossref PubMed Scopus (568) Google Scholar;Ullrich and Schlessinger, 1990Ullrich A. Schlessinger J. Signal transduction by receptors with tyrosine kinase activity.Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4605) Google Scholar). The cognate ligand for KIT variously named stem cell factor (SCF), mast cell growth factor, steel factor, and KIT ligand, is synthesized as a transmembrane protein that can be cleaved to yield a soluble, bioactive form (Martin et al., 1990Martin F.H. Suggs S.V. Langley K.E. et al.Primary structure and functional expression of rat and human stem cell factor DNAs.Cell. 1990; 63: 203-211Abstract Full Text PDF PubMed Scopus (597) Google Scholar;Longley et al., 1997Longley B.J. Tyrrell L. Ma Y. et al.Chymase cleavage of stem cell factor yields a bioactive, soluble product.Proc Natl Acad Sci USA. 1997; 94: 9017-9021Crossref PubMed Scopus (168) Google Scholar). An isoform of SCF resulting from alternative splicing of exon 6 mRNA lacks a major protease sensitive site and tends to remain membrane bound. As with activation of other receptor tyrosine kinases (Ullrich and Schlessinger, 1990Ullrich A. Schlessinger J. Signal transduction by receptors with tyrosine kinase activity.Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4605) Google Scholar), SCF binding induces KIT dimerization and activates its intrinsic kinase activity, resulting in receptor autophosphorylation (Blume-Jensen et al., 1991Blume-Jensen P. Claesson-Welsh L. Siegbahn A. et al.Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis.Embo J. 1991; 10: 4121-4128Crossref PubMed Scopus (268) Google Scholar;Lev et al., 1992Lev S. Yarden Y. Givol D. Dimerization and activation of the kit receptor by monovalent and bivalent binding of the stem cell factor.J Biol Chem. 1992; 267: 15970-15977Abstract Full Text PDF PubMed Google Scholar). The activated KIT then binds and phosphorylates a class of intracellular substrate proteins, initiating a signaling cascade that culminates in a wide array of biologic activities, including proliferation, migration, maturation, and survival of hematopoietic stem cell, mast cell, melanocyte, and germ cell lineages (Brizzi et al., 1994Brizzi M.F. Zini M.G. Aronica M.G. et al.Convergence of signaling by interleukin-3, granulocyte-macrophage colony-stimulating factor, and mast cell growth factor on JAK2 tyrosine kinase.J Biol Chem. 1994; 269: 31680-31684Abstract Full Text PDF PubMed Google Scholar;Galli et al., 1994Galli S.J. Zsebo K.M. Geissler E.N. The kit ligand, stem cell factor.Adv Immunol. 1994; 55: 1-96Crossref PubMed Scopus (559) Google Scholar;Serve et al., 1994Serve H. Hsu Y.C. Besmer P. Tyrosine residue 719 of the c-kit receptor is essential for binding of the P85 subunit of phosphatidylinositol (PI) 3-kinase and for c-kit-associated PI 3-kinase activity in COS-1 cells.J Biol Chem. 1994; 269: 6026-6030Abstract Full Text PDF PubMed Google Scholar,Serve et al., 1995Serve H. Yee N.S. Stella G. et al.Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells.Embo J. 1995; 14: 473-483Crossref PubMed Scopus (199) Google Scholar;Linnekin et al., 1997Linnekin D. DeBerry C.S. Mou S. Lyn associates with the juxtamemebrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells.J Biol Chem. 1997; 272: 27450-27455Crossref PubMed Scopus (142) Google Scholar;Price et al., 1997Price D.J. Rivnay B. Fu Y. et al.Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocites.J Biol Chem. 1997; 272: 5915-5920Crossref PubMed Scopus (81) Google Scholar). KIT can be constitutively activated by autocrine SCF stimulation (Kondoh et al., 1995Kondoh G. Hayasaka N. Li Q. Nishimune Y. Hakaura A. An in vivo model for receptor tyrosine kinase autocrine/paracrine activation: auto-stimulated KIT receptor acts as a tumor promoting factor in papillomavirus-induced tumorigenesis.Oncogene. 1995; 10: 341-347PubMed Google Scholar) or in a SCF-independent manner involving mutations in the receptor (Furitsu et al., 1993Furitsu T. Tsujimura T. Tono T. et al.Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.J Clin Invest. 1993; 92: 1736-1744Crossref PubMed Scopus (736) Google Scholar;Tsujimura et al., 1994Tsujimura T. Furitsu T. Morimoto M. et al.Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation.Blood. 1994; 83: 2619-2626Crossref PubMed Google Scholar,Tsujimura et al., 1996Tsujimura T. Morimoto M. Hashimoto K. et al.Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain.Blood. 1996; 87: 273-283Crossref PubMed Google Scholar;Hirota et al., 1998Hirota S. Isozaki K. Moriyama Y. et al.Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.Science. 1998; 279: 577-580Crossref PubMed Scopus (3818) Google Scholar). Expression of the activating KIT mutants in murine interleukin-3-dependent cell lines leads to factor-independent growth and aggressive in vivo behavior of the cells (Kitayama et al., 1995Kitayama H. Kanakura Y. Furitsu T. et al.Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines.Blood. 1995; 85: 790-798Crossref PubMed Google Scholar;Tsujimura et al., 1996Tsujimura T. Morimoto M. Hashimoto K. et al.Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain.Blood. 1996; 87: 273-283Crossref PubMed Google Scholar;Hirota et al., 1998Hirota S. Isozaki K. Moriyama Y. et al.Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.Science. 1998; 279: 577-580Crossref PubMed Scopus (3818) Google Scholar), suggesting that constitutively activated KIT is oncogenic. Human mastocytosis, or mast cell disease, is a heterogeneous set of conditions characterized by increased numbers of mast cells in various organs (Longley et al., 1995Longley J. Duffy T.P. Kohn S. The mast cell and mast cell disease.J Am Acad Dermatol. 1995; 32: 545-561Abstract Full Text PDF PubMed Scopus (214) Google Scholar). Mast cell neoplasms occur in both humans and animals. In dogs, mast cell neoplasms are called mastocytomas, and the disease is common, representing 7%–21% of canine tumors (Priester, 1973Priester W.A. Skin tumors in domestic animals: data from 21 US and Canadian colleges of veterinary medicine.J Natl Cancer Inst. 1973; 50: 457-466PubMed Google Scholar;Cohen et al., 1974Cohen D. Reif J.S. Brodey R.S. Keiser H. Epidemiological analysis of the most prevalent sites and types of canine neoplasia observed in a veterinary hospital.Cancer Res. 1974; 34: 2859-2868PubMed Google Scholar). A distinction must be drawn between human mastocytosis, which is usually transient or indolent (Longley et al., 1995Longley J. Duffy T.P. Kohn S. The mast cell and mast cell disease.J Am Acad Dermatol. 1995; 32: 545-561Abstract Full Text PDF PubMed Scopus (214) Google Scholar), and canine mast cell neoplasia, which behaves unpredictably and is often aggressive and metastatic (Macy and MacEwen, 1989Macy D.W. MacEwen E.G. Mast cell tumors.in: Withrow S.J. MacEwen E.G. Clinical Veterinary Oncology. J.B. Lippincott, Philadelphia1989: 156-166Google Scholar). For instance, human solitary mastocytomas essentially never metastasize; in contrast, ≈50% of canine mastocytomas behave in a malignant fashion, as estimated byHottendorf and Nielsen, 1969Hottendorf G.H. Nielsen S.W. Canine mastocytoma—a review of clinical aspects.J Am Vet Med Assoc. 1969; 154: 917-924PubMed Google Scholar after review of 46 published reports of tumors in 938 dogs. KIT's involvement in the pathogenesis of mastocytosis is suggested by the observation that several mutations resulting in constitutive activation of KIT have been detected in a number of mast cell lines. For instance, a point mutation in human c-KIT (causing substitution of Val for Asp816 in the phosphotransferase domain and receptor autoactivation) occurs in HMC-1, a long-term human mast cell leukemia line (Furitsu et al., 1993Furitsu T. Tsujimura T. Tono T. et al.Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.J Clin Invest. 1993; 92: 1736-1744Crossref PubMed Scopus (736) Google Scholar) and in the corresponding codon in two rodent mast cell lines (Tsujimura et al., 1994Tsujimura T. Furitsu T. Morimoto M. et al.Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation.Blood. 1994; 83: 2619-2626Crossref PubMed Google Scholar,Tsujimura et al., 1995Tsujimura T. Furitsu T. Morimoto M. et al.Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3.Int Arch Allergy Immunol. 1995; 106: 377-385Crossref PubMed Scopus (115) Google Scholar). Moreover, this activating mutation has been identified in situ in some cases of human mastocytosis (Nagata et al., 1995Nagata H. Worobec A.S. Oh C.K. et al.Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.Proc Natl Acad Sci USA. 1995; 92: 10560-10564Crossref PubMed Scopus (797) Google Scholar;Longley et al., 1996Longley B.J. Tyrrell L. Lu S. et al.Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm.Nature Genet. 1996; 12: 312-314Crossref PubMed Scopus (553) Google Scholar). Two other activating mutations have been found in another region of KIT, the intracellular juxtamembrane region. One results in Val560Gly substitution in the human HMC-1 mast cell line (Furitsu et al., 1993Furitsu T. Tsujimura T. Tono T. et al.Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.J Clin Invest. 1993; 92: 1736-1744Crossref PubMed Scopus (736) Google Scholar), and the other eliminates seven amino acids (Thr573–His579) in a rodent mast cell line called FMA3 (Tsujimura et al., 1996Tsujimura T. Morimoto M. Hashimoto K. et al.Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain.Blood. 1996; 87: 273-283Crossref PubMed Google Scholar). The significance of these two juxtamembrane region mutations is unclear, as they have not been identified in situ. To determine whether c-KIT juxtamembrane mutations are present in mast cell neoplasms in situ and may therefore contribute to their pathogenesis, we examined c-KIT cDNA from dog mastocytoma tissues and cell lines. We report herein the complete sequence of canine c-KIT, and the development of a model of KIT structure. We also identify five novel juxtamembrane mutations causing constitutive activation of KIT, which suggest a key region involved in negative control of the receptor kinase activity, and show that activating c-KIT juxtamembrane mutations are common in canine mast cell neoplasms. This provides the first in situ evidence that such mutations may play a part in the pathogenesis of mast cell neoplasia. Recombinant human (rh) and canine (rc) SCF, murine monoclonal and rabbit polyclonal anti-human KIT antibody, and wild-type (WT) human full-length c-KIT cDNA/pSPORT1 plasmid were provided by Amgen (Thousand Oaks, CA). Mouse anti-phosphotyrosine (pTyr) monoclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Human SCF cDNA/pBluescript plasmid was provided by Dr. Douglas Williams (Immunex, Seattle, WA). Six mastocytomas freshly excised from six dogs were snap-frozen in OCT medium and cryostat-sectioned. An additional mastocytoma removed from a seventh dog was grown as a solid tumor in the dermis of a BALB/C athymic mouse prior to being frozen. Three dog mastocytoma cell lines (BR, C1, and C2) were established in long-term culture after initial serial passaging in the skin of BALB/C athymic mice from skin mastocytomas of two additional dogs (Lazarus et al., 1986Lazarus S.C. DeVinney R. McCabe L.J. Finkbeiner W.E. Elias D.J. Gold W.M. Isolated canine mastocytoma cells: propagation and characterization of two cell lines.Am J Physiol. 1986; 251: C935-C944PubMed Google Scholar;DeVinney and Gold, 1990DeVinney R. Gold W.V. Establishment of two dog mastocytoma cell lines in continuous culture.Am J Respir Cell Mol Biol. 1990; 3: 413-420Crossref PubMed Scopus (50) Google Scholar). C1 and C2 originated from tumors harvested at separate times from one dog. These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 2% bovine calf serum and 0.25 mg per ml histidine. As control KIT-positive cells, normal human melanocytes provided by Yale Skin Disease Research Center (New Haven, CT) were grown in F10 medium with 10% bovine calf serum. COS-7 cells were cultured in Dulbecco's modified Eagle's medium with 10% bovine calf serum. Follow-up is only available on the dog from which the C1 and C2 cell lines were derived which died of its tumor. The sources of the BR cell line and the individual tumors were all lost to follow-up. After RNA extraction, cDNA was synthesized by reverse transcriptase using random hexamers as previously described (Longley et al., 1991Longley J. Ding T. Cuono C. et al.Isolation, detection, and amplification of intact mRNA from dermatome strips, epidermal sheets, and sorted epidermal cells.J Invest Dermatol. 1991; 97: 974-979Abstract Full Text PDF PubMed Google Scholar,Longley et al., 1996Longley B.J. Tyrrell L. Lu S. et al.Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm.Nature Genet. 1996; 12: 312-314Crossref PubMed Scopus (553) Google Scholar). c-KIT cDNA were amplified in the presence of 1 μM of primers (see below) with Taq DNA polymerase for 30 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. Dideoxysequencing was performed directly from gel-purified polymerase chain reaction amplimers, or individual cDNA subcloned into plasmids using the TA Cloning Kit (Invitrogen, Carlsbad, CA) by the W.M. Keck Foundation Biotechnology Resource Laboratories at Yale University, the Biomolecular Resource Center at the University of California at San Francisco, and the Skin Disease Research Center of the Department of Dermatology of Columbia University. Sequencing of mastocytoma c-KIT focused on the juxtamembrane and phosphotransferase coding regions using primer pairs: 5′-CAAATCCATCCCCACACCCTGTTCAC-3′ (1564–1589) and 5′-CCATAAGCAGTTGCCTCAAC-3′ (1850–1831); 5′-TGTATTCACAGAGACTTGGC-3′ (2383–2402) and 5′-GGCTGAGCATCCGGAAGCCT-3′ (2695–2676). The numbers in parentheses refer to human c-KIT cDNA (Yarden et al., 1987Yarden Y. Kuang W.-J. Yang-Feng T. et al.Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand.Embo J. 1987; 6: 3341-3351Crossref PubMed Scopus (1323) Google Scholar). The entire KIT coding region was sequenced from cDNA prepared from normal dog lung mRNA. Mutations identified in dog mastocytomas (Trp556Arg, Leu575Pro, ΔTrp556–Lys557, and ΔVal558) were reproduced in human c-KIT cDNA in the pcDNA3 mammalian expression vector (Invitrogen) using the Quikchange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Human c-KIT was used as the juxtamembrane amino acids are completely conserved in mammals (see Results section). COS-7 cells (90% confluent in 10-cm plate) were transfected with 5 μg plasmid using 15 μl LipofectAMINE (Life Technologies, Gaithersburg, MD) in serum-free medium for 5 h. An equal volume of medium with 20% bovine calf serum was then added and cells incubated overnight, followed by 8–24 h culture in regular medium prior to protein expression and tyrosine phosphorylation assay. For SCF stimulation experiments, cells were serum-starved for 18 h before incubation with SCF. Cells were harvested in lysis buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg leupeptin per ml, 10 μg aprotinin per ml, and 1 mM Na orthovanadate. Centrifugation-clarified cell lysates were immunoblotted as total cell lysates (see below) or immunoprecipitated for 1.5 h at 4°C with mouse anti-human KIT antibody and protein A agarose. Immunoprecipitates were washed with lysis buffer and heat-eluted in sodium dodecyl sulfate sample buffer. Samples were fractionated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred on to polyvinylidene difluoride membrane, blocked by 5% bovine serum albumin in Tris-buffered saline (50 mM Tris–HCl, pH 7.5, and 150 mM NaCl) plus 0.1% Tween-20 (TBST), and probed with rabbit anti-human KIT or mouse anti-pTyr antibody for 1 h, followed by washing with TBST. Membranes were incubated in TBST with horseradish peroxidase-linked secondary antibody for 45 min, washed, and antigen–antibody complexes detected using the ECL System (Amersham, Arlington Heights, IL). Anti-pTyr blots were stripped in 100 mM β-mercaptoethanol, 2% sodium dodecyl sulfate, and 62.5 mM Tris–HCl (pH 6.7) at 50°C for 30 min, then reprobed with rabbit anti-human KIT antibody. SCF mRNA was detected by reverse transcriptase–polymerase chain reaction, followed by Southern blotting with a SCF-specific oligonucleotide probe and by direct amplimer sequencing. Canine SCF primers correspond to nucleotides 191–209 and 607–588 (Shull et al., 1992Shull R.M. Suggs S.V. Langley K.E. et al.Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture.Exp Hematol. 1992; 20: 1118-1124PubMed Google Scholar), and the probe to human bases 301–367 (Martin et al., 1990Martin F.H. Suggs S.V. Langley K.E. et al.Primary structure and functional expression of rat and human stem cell factor DNAs.Cell. 1990; 63: 203-211Abstract Full Text PDF PubMed Scopus (597) Google Scholar). A low-resolution homology model of the intracellular canine KIT tyrosine kinase domains was constructed with assistance from an automated protein modeling tool (ProMod) and server (Swiss-Model) (Peitsch, 1996Peitsch M.C. ProMod and Swiss-Model: internal-based tools for automated comparative protein modeling.Biochem Soc Trans. 1996; 24: 274-279Crossref PubMed Scopus (899) Google Scholar). The X-ray diffraction-derived coordinates of the fibroblast growth factor (FGF) receptor tyrosine kinase domain (Mohammadi et al., 1996Mohammadi M. Schlessinger J. Hubbard S.R. Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism.Cell. 1996; 86: 577-587Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar) (1FGK in the Brookhaven Protein Data Bank) served as a template for the model. Among available experimentally defined tyrosine kinase structures, 1FGK is most homologous to KIT, as determined by alignments with BLAST and FastA algorithms (Altschul et al., 1990Altschul S.F. Gish W. Miller W. Myers E.M. Lipman D.J. Basic local alignment search tool.J Mol Biol. 1990; 215: 403-410Crossref PubMed Scopus (70338) Google Scholar). First-approach alignments were checked in a process that included a comparison of sequences conserved in other tyrosine kinases. The model was then optimized by Swiss-Model, which idealizes bond geometry and removes unfavorable nonbonded contacts by energy minimization with CHARMM. The resulting coordinate files were displayed using the RasMol molecular visualization program. Overall, the deduced amino acid sequence of normal dog KIT shows identity of 93%, 88%, 81%, and 64% compared with cat, human, mouse, and chicken KIT, respectively. Conservation is less in the extracellular domain than in the intracellular domains. For example, dog and human KIT are 78% identical in the 519/520 extracellular domain residues but are 97% identical in the 433 intracellular domain residues. In mammalian KIT (human, dog, cat, cow, goat, rat, and mouse), the 38 residues of the intracellular juxtamembrane region (amino acids 543–580 in Figure 1 ) are identical as are all but one (Ile/Val661) of the adjacent 104 residues in the ATP-binding domain. We used Trp581–Glu582 as the beginning of the kinase domain, as defined by the crystal structure of FGF receptor kinase (Mohammadi et al., 1996Mohammadi M. Schlessinger J. Hubbard S.R. Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism.Cell. 1996; 86: 577-587Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Residues 768–914 comprise another high-homology region (96% identical in alignments of mammalian KIT), corresponding to the phosphotransferase domain. These lengthy regions, which are intolerant of sequence variation among different species, appear fundamental for concerted receptor function. The 12 extracellular domain cysteines (plus another in the signal peptide) are conserved in dog and human KIT, as are nine of 11 predicted canine N-linked glycosylation sites. We sequenced c-KIT cDNA generated from mastocytoma tissues from seven dogs (DS, AG, SW, RT, KR, LL, MN) and three mast cell lines derived from two additional dogs (BR, C1, C2). Three of the tissue samples showed four mutations clustered in the intracellular juxtamembrane coding region (Figure 1 and Table 1 ). Sample DS had a 6 bp deletion eliminating Trp556–Lys557. Sample AG contained a 3 bp deletion eliminating Val558. We found no WT sequence in either sample, suggesting functional loss of one (presumably WT) allele. Sample SW was heterozygous for point mutations in separate alleles: one substituting Arg for Trp556 and the other substituting Pro for Leu575. The Leu575Pro mutation also was present in cells of the BR line, which was derived from a different dog. The C1 and C2 cell lines, derived, respectively, from early- and late-stage tumors of one dog, both contained a 48 bp insertion between nucleotides 1784 and 1785, which are, respectively, the first and second base of codon 586 in the proximal ATP-binding domain (Figure 1). Except for the first 3 bp, the inserted nucleotide sequence is a direct repeat of the segment immediately preceding the site of insertion. Deduced amino acids 3–16 of the inserted sequence (Table 1) are a repeat of residues 572–585 of normal dog KIT (Figure 1). All the three cell lines (BR, C1, and C2) were heterozygous for their respective mutations, with the other allele being WT in the region of the identified mutations. The mutations in the mastocytoma tissues clearly developed in situ and could contribute to the pathogenesis of mast cell neoplasms in the animals in which they developed. Likewise, the detection of the same 48 bp insertion in both C1 and C2 cell lines, which were derived separately from the same animal at different times, indicates that the mutation was present in the original tumors. Therefore, we link all five mutations to dog mastocytomas in situ.Table 1c-KIT juxtamembrane mutations in canine mastocytomasWTBase changeAmino acid changeTissue DS-6 bp deletion (1694–1699)ΔTrp556–Lys557 AG-3 bp deletion (1700–1702)ΔVal558 SW-T→C transition (1694)Trp556ArgT→C transition (1752)Leu575ProCell line BR+T→C transition (1752)Leu575Pro C1, C2+48 bp insertion (1784–1785)+TYPTQLPYDHKWEFPR Open table in a new tab Limited sequencing of the region encompassing codons 514–609 in the other four dog mastocytomas showed only WT sequences. As an activating point mutation has previously been identified in codon 816 in the phosphotransferase domain in some human patients with mastocytosis (Nagata et al., 1995Nagata H. Worobec A.S. Oh C.K. et al.Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.Proc Natl Acad Sci USA. 1995; 92: 10560-10564Crossref PubMed Scopus (797) Google Scholar;Longley et al., 1996Longley B.J. Tyrrell L. Lu S. et al.Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm.Nature Genet. 1996; 12: 312-314Crossref PubMed Scopus (553) Google Scholar), we also sequenced cDNA corresponding to codon
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