RESUMO
To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Mutação , Proteínas Proto-Oncogênicas c-ret/química , Proteínas Proto-Oncogênicas c-ret/genética , Homologia de Sequência de Aminoácidos , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Insetos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Especificidade por Substrato , Fatores de Tempo , Tirosina/químicaRESUMO
The aPKC [atypical PKC (protein kinase C)] isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCι kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, CRT0066854 displaces a crucial Asn-Phe-Asp motif that is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors.
Assuntos
Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Tiofenos/farmacologia , Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Proteínas do Citoesqueleto/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Mimetismo Molecular , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Pirimidinas/farmacologia , Tiofenos/químicaRESUMO
Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with ß-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.
Assuntos
Citoesqueleto/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas do Citoesqueleto , Citoesqueleto/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Domínio LIM , Masculino , Proteínas dos Microfilamentos/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Ratos , Proteínas Supressoras de Tumor/genéticaRESUMO
Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely unexplored. In this study, oligopeptide arrays were used to screen substrates directly phosphorylated by purified recombinant wild-type and oncogenic RET kinase domain in the presence or absence of small molecule inhibitors. The results of the peptide array were validated by enzyme kinetics, in vitro kinase, and cell-based experiments. The identification of focal adhesion kinase (FAK) as a direct substrate for RET kinase revealed (i) a RET-FAK transactivation mechanism consisting of direct phosphorylation of FAK Tyr-576/577 by RET and a reciprocal phosphorylation of RET by FAK, which crucially is able to rescue the kinase-impaired RET K758M mutant and (ii) that FAK binds RET via its FERM domain. Interestingly, this interaction is abolished upon RET phosphorylation, indicating that RET binding to the FERM domain of FAK is a priming step for RET-FAK transactivation. Finally, our data indicate that FAK inhibitors could be used as potential therapeutic agents for patients with multiple endocrine neoplasia type 2 tumors because both, treatment with the FAK kinase inhibitor NVP-TAE226 and FAK down-regulation by siRNA reduced RET phosphorylation and signaling as well as the proliferation and survival of tumor and transfected cell lines expressing oncogenic RET.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ativação Transcricional , Antineoplásicos/farmacologia , Proliferação de Células , Proteína-Tirosina Quinases de Adesão Focal/genética , Glutationa Transferase/metabolismo , Humanos , Cinética , Oligopeptídeos/química , Fenótipo , Fosforilação , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ret/genética , Transdução de SinaisRESUMO
Mutations of the rearranged during transfection (RET) kinase are frequently reported in cancer, which make it as an attractive therapeutic target. Herein, we discovered a series of N-trisubstituted pyrimidine derivatives as potent inhibitors for both wild-type (wt) RET and RETV804M, which is a resistant mutant for several FDA-approved inhibitors. The X-ray structure of a representative inhibitor with RET revealed that the compound binds in a unique pose that bifurcates beneath the P-loop and confirmed the compound as a type I inhibitor. Through the structure-activity relationship (SAR) study, compound 20 was identified as a lead compound, showing potent inhibition of both RET and RETV804M. Additionally, compound 20 displayed potent antiproliferative activity of CCDC6-RET-driven LC-2/ad cells. Analysis of RET phosphorylation indicated that biological activity was mediated by RET inhibition. Collectively, N-trisubstituted pyrimidine derivatives could serve as scaffolds for the discovery and development of potent inhibitors of type I RET and its gatekeeper mutant for the treatment of RET-driven cancers.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Pirimidinas/química , Adenocarcinoma de Pulmão/patologia , Apoptose , Proliferação de Células , Humanos , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-ret/genética , Relação Estrutura-Atividade , Células Tumorais Cultivadas , CicatrizaçãoRESUMO
RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.
Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Caderinas/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-ret/química , Proteínas de Peixe-Zebra/químicaRESUMO
The synthesis, structure-activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.
Assuntos
Indóis/síntese química , Indóis/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Cristalografia por Raios X , Ligação de Hidrogênio , Indóis/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Recombinantes/antagonistas & inibidores , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
The cysteine-based protein phosphatase H1L was the first reported dual-specificity protein phosphatase. H1L is encapsidated within the vaccinia virus and is required for successful host infection and for the production of viable vaccinia progeny. H1L has therefore been proposed as a target candidate for antiviral compounds. Recombinant H1L has been expressed in a catalytically inactive form using an Escherichia coli host, leading to purification and crystallization by the microbatch method. The crystals diffract to 2.1 A resolution using synchrotron radiation. These crystals belong to space group P422, with unit-cell parameters a = b = 98.31, c = 169.15 A, and are likely to contain four molecules in the asymmetric unit. A sulfur SAD data set was collected to 2.8 A resolution on beamline BM14 at the ESRF to facilitate structure determination. Attempts to derivatize these crystals with xenon gas changed the space group to I422, with unit-cell parameters a = b = 63.28, c = 169.68 A and a single molecule in the asymmetric unit. The relationship between these two crystal forms is discussed.
Assuntos
Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Vaccinia virus/enzimologia , Cristalização , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/genética , Vaccinia virus/genética , Difração de Raios XRESUMO
Resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic lung adenocarcinoma harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in a serine-to-phenylalanine substitution at codon 904 in the activation loop of the RET kinase domain. The S904F mutation confers resistance to vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase. A reduced interaction with the drug is also observed in vitro for the S904F mutant by thermal shift assay. A crystal structure of the S904F mutant reveals a small hydrophobic core around F904 likely to enhance basal kinase activity by stabilizing an active conformer. Our findings indicate that missense mutations in the activation loop of the kinase domain are able to increase kinase activity and confer drug resistance through allosteric effects.
Assuntos
Adenocarcinoma/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto , Piperidinas/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/genética , Quinazolinas/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Pessoa de Meia-Idade , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Quinazolinas/farmacologiaRESUMO
Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. We provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors, such as AD80 or ponatinib, that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells, we identify the CCDC6-RETI788N mutation and drug-induced mitogen-activated protein kinase pathway reactivation as possible mechanisms by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors.
Assuntos
Adenocarcinoma/metabolismo , Rearranjo Gênico/genética , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/genética , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Imidazóis/farmacologia , Camundongos , Mutação , Células NIH 3T3 , Piridazinas/farmacologiaRESUMO
The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RET(ECD)), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RET(ECD) envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RET(ECD) cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation.
Assuntos
Membrana Celular/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Epitopos/imunologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ret/química , Proteínas Proto-Oncogênicas c-ret/genética , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
Atypical protein kinase Cι (PKCι) has roles in cell growth, cellular polarity, and migration, and its abundance is frequently increased in cancer. We identified a protein interaction surface containing a dibasic motif (RIPR) that bound a distinct subset of PKCι substrates including lethal giant larvae 2 (LLGL2) and myosin X, but not other substrates such as Par3. Further characterization demonstrated that Arg471 in this motif was important for binding to LLGL2, whereas Arg474 was critical for interaction with myosin X, indicating that multiple complexes could be formed through this motif. A somatic mutation of the dibasic motif (R471C) was the most frequent mutation of PKCι in human cancer, and the intact dibasic motif was required for normal polarized epithelial morphogenesis in three-dimensional cysts. Thus, the R471C substitution is a change-of-function mutation acting at this substrate-specific recruitment site to selectively disrupt the polarizing activity of PKCι.
Assuntos
Polaridade Celular/genética , Proteínas do Citoesqueleto/metabolismo , Isoenzimas/química , Isoenzimas/genética , Modelos Moleculares , Proteínas de Neoplasias/genética , Conformação Proteica , Proteína Quinase C/química , Proteína Quinase C/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Diamino Aminoácidos/genética , Análise de Variância , Animais , Proteínas do Citoesqueleto/genética , Cães , Células HEK293 , Humanos , Immunoblotting , Células Madin Darby de Rim Canino , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCF(Fbxo7) E3 ligase, and the proteasome inhibitor PI31. SCF(Fbxo7) is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7.Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel alpha/beta-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7.Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCF(Fbxo7). We present a model for FP domain-mediated dimerization of SCF(Fbxo7) and PI31.
Assuntos
Proteínas F-Box/química , Proteínas F-Box/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Dimerização , Humanos , Células Jurkat , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma , Mapeamento de Interação de Proteínas , Alinhamento de SequênciaRESUMO
The RET proto-oncogene encodes a receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family of ligands. Loss-of-function mutations in RET are implicated in Hirschsprung disease, whereas activating mutations in RET are found in human cancers, including familial medullar thyroid carcinoma and multiple endocrine neoplasias 2A and 2B. We report here the biochemical characterization of the human RET tyrosine kinase domain and the structure determination of the non-phosphorylated and phosphorylated forms. Both structures adopt the same active kinase conformation competent to bind ATP and substrate and have a pre-organized activation loop conformation that is independent of phosphorylation status. In agreement with the structural data, enzyme kinetic data show that autophosphorylation produces only a modest increase in activity. Longer forms of RET containing the juxtamembrane domain and C-terminal tail exhibited similar kinetic behavior, implying that there is no cis-inhibitory mechanism within the RET intracellular domain. Our results suggest the existence of alternative inhibitory mechanisms, possibly in trans, for the autoregulation of RET kinase activity. We also present the structures of the RET tyrosine kinase domain bound to two inhibitors, the pyrazolopyrimidine PP1 and the clinically relevant 4-anilinoquinazoline ZD6474. These structures explain why certain multiple endocrine neoplasia 2-associated RET mutants found in patients are resistant to inhibition and form the basis for design of more effective inhibitors.
Assuntos
Proteínas Proto-Oncogênicas c-ret/química , Proteínas Proto-Oncogênicas c-ret/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Dimerização , Cinética , Ligantes , Modelos Moleculares , Mutação/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
D cyclins (D1, D2 and D3) and their catalytic subunits (cyclin-dependent kinases cdk4 and cdk6) have a facilitating, but nonessential, role in cell cycle entry. Tissue-specific functions for D-type cyclins and cdks have been reported; however, the biochemical properties of these kinases are indistinguishable. We report that an F box protein, Fbxo7, interacted with cellular and viral D cyclins and distinguished among the cdks that bind D-type cyclins, specifically binding cdk6, in vitro and in vivo. Fbxo7 specifically regulated D cyclin/cdk6 complexes: Fbxo7 knockdown decreased cdk6 association with cyclin and its overexpression increased D cyclin/cdk6 activity and E2F activity. Fbxo7 interacted with p27, but its enhancement of cyclin D/cdk6 activity was p21/p27 independent. Fbxo7 overexpression transformed murine fibroblasts, rendering them tumorigenic in athymic nude mice. Transformed phenotypes were dependent on cdk6, as knockdown of cdk6 reversed them. Fbxo7 was highly expressed in epithelial tumors, but not in normal tissues, suggesting that it may have a proto-oncogenic role in human cancers.