RESUMO
The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation.
Assuntos
Fosfolipase C gama/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos , Ressonância Magnética Nuclear Biomolecular , Fosfatidilinositóis/metabolismo , Fosfolipase C gama/química , Fosfolipase C gama/genética , Fosforilação , Ligação Proteica , Conformação Proteica , Transporte Proteico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Relação Estrutura-Atividade , Transfecção , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Domínios de Homologia de srcRESUMO
A unique heteropolyoxotantalate (hetero-POTa) cluster [P2O7Ta5O14]7- (P2Ta5) was first developed using pyrophosphate as a key to open the ultrastable skeleton of the classical Lindqvist-type [Ta6O19]8- precursor. The P2Ta5 cluster can serve as a general and flexible secondary building unit to create a family of brand-new multidimensional POTa architectures. This work not only promotes the limited structural diversity of hetero-POTa but also provides a practical strategy for new extended POTa architectures.
RESUMO
A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.
Assuntos
Domínio AAA/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Domínio AAA/genética , Domínio Catalítico , Dimerização , Ativação Enzimática , Humanos , Ligantes , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Tirosina/químicaRESUMO
Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here, we report that lysine acetylation of PDHA1 and PDP1 is common in epidermal growth factor (EGF)-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1, and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important in promoting glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct posttranslational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect.
Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Piruvato Desidrogenase (Lipoamida)-Fosfatase/metabolismo , Piruvato Desidrogenase (Lipoamida)/metabolismo , Sirtuína 3/metabolismo , Tirosina/química , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Lisina/química , Masculino , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Transplante de Neoplasias , Neoplasias/metabolismo , FosforilaçãoRESUMO
Recombinant fibroblast growth factor-2 (FGF-2) has been extensively studied and used in several clinical applications including wound healing, bone regeneration, and neuroprotection. Poly(ethylene glycol) (PEG) modification of recombinant human FGF-2 (rhFGF-2) in solution phase has been studied to increase the in vivo biostabilities and therapeutic potency. However, the solution-phase strategy is not site-controlled and the products are often not homogeneous due to the generation of multi-PEGylated proteins. In order to increase mono-PEGylated rhFGF-2 level, a novel solid-phase strategy for rhFGF-2 PEGylation is developed. RhFGF-2 proteins were loaded onto a heparin-sepharose column and the PEGylaton reaction was carried out at the N-terminus by PEG20 kDa butyraldehyde through reductive alkylation. The PEGylated rhFGF-2 was purified to near homogeneity by SP sepharose anion-exchange chromatography and the purity was more than 95% with a yield of mono-PEGylated rhFGF-2 of 58.3%, as confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. In vitro biophysical and biochemical measurements demonstrated that PEGylated rhFGF-2 has an unchanged secondary structure, receptor binding activity, cell proliferation, and MAP kinase stimulating activity, and an improved bio- and thermal stability. Animal assay showed that PEGylated rhFGF-2 has an increased half-life and reduced immunogenicity. Compared to conventional solution-phase PEGylation, the solid-phase PEGylation is advantageous in reaction time, production of mono-PEGylated protein, and improvement of biochemical and biological activity.
Assuntos
Cromatografia por Troca Iônica/métodos , Fator 2 de Crescimento de Fibroblastos/química , Heparina , Mitógenos/química , Polietilenoglicóis/química , Proteínas Recombinantes/química , Sefarose , Animais , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Masculino , Camundongos , Mitógenos/isolamento & purificação , Mitógenos/metabolismo , Mitógenos/farmacologia , Células NIH 3T3 , Estabilidade Proteica , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Soluções , Especificidade por Substrato , TemperaturaRESUMO
OBJECTIVE: To observe the effect of long-term external use of Goupi Gao on renal function and lead accumulation in rats. METHOD: Rats were externally administered with Goupi Gao at different doses (7, 3.5 and 1.75 g x kg(-1)) for 90 d. At 45 days and 90 days after administration, the renal indicator, levels of blood urea nitrogen (BU) and creatinine (Cr) in serum, beta2-microglobulin (beta2-MG) and N-acetyl-beta-D-glucosaminidase (NAG) in urine were determined. Lead content in kidneys was detected by atomic absorption spectrometry. RESULT: A 90-day administration with Goupi Gao significantly enhanced the renal indicator, levels of NAG in urine and lead content in renal, when compared with the normal rats. However, the levels of BUN and beta2-MG as well as renal pathology in Goupi Gao treated rats were not obviously changed. CONCLUSION: Consecutive administration of Goupi Gao for 90 days can increase the renal indicator and levels of NAG in urine, enhance the accumulation of lead in renal, but with no effect on excretory function of kidneys and organic changes.
Assuntos
Medicamentos de Ervas Chinesas/toxicidade , Rim/metabolismo , Chumbo/metabolismo , Acetilglucosaminidase/urina , Animais , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Medicamentos de Ervas Chinesas/administração & dosagem , Feminino , Rim/efeitos dos fármacos , Rim/patologia , Chumbo/análise , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Espectrofotometria Atômica , Microglobulina beta-2/urinaRESUMO
Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 A away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.
Assuntos
Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Tirosina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Síndrome de Kallmann/genética , Mutação , Fosforilação , Estrutura Terciária de Proteína , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Tirosina/química , Tirosina/genéticaRESUMO
We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.
Assuntos
Melanoma/genética , Mutação , Polimorfismo de Nucleotídeo Único , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Neoplasias Cutâneas/genética , Divisão Celular , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Melanoma/patologia , Modelos Moleculares , Conformação Proteica , Neoplasias Cutâneas/patologiaRESUMO
Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the 'molecular brake', 'DFG latch', 'A-loop plug', and 'αC tether'. The first three sites repress the kinase from adopting an active conformation, whereas the αC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs.
Assuntos
Regulação Alostérica , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
The K650E gain-of-function mutation in the tyrosine kinase domain of FGF receptor 3 (FGFR3) causes Thanatophoric Dysplasia type II, a neonatal lethal congenital dwarfism syndrome, and when acquired somatically, it contributes to carcinogenesis. In this report, we determine the crystal structure of the FGFR3 kinase domain harboring this pathogenic mutation and show that the mutation introduces a network of intramolecular hydrogen bonds to stabilize the active-state conformation. In the crystal, the mutant FGFR3 kinases are caught in the act of trans-phosphorylation on a kinase insert autophosphorylation site, emphasizing the fact that the K650E mutation circumvents the requirement for A-loop tyrosine phosphorylation in kinase activation. Analysis of this trans-phosphorylation complex sheds light onto the determinants of tyrosine trans-phosphorylation specificity. We propose that the targeted inhibition of this pathogenic FGFR3 kinase may be achievable by small molecule kinase inhibitors that selectively bind the active-state conformation of FGFR3 kinase.
Assuntos
Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Estabilidade Enzimática , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Mimetismo Molecular , Mutação de Sentido Incorreto , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Crânio/anormalidades , Displasia Tanatofórica/genética , Tirosina/químicaRESUMO
The basal (ligand-independent) kinase activity of receptor tyrosine kinases (RTKs) promotes trans-phosphorylation on activation loop tyrosines upon ligand-induced receptor dimerization, thus upregulating intrinsic kinase activity and triggering intracellular signaling. To understand the molecular determinants of intrinsic kinase activity, we used X-ray crystallography and NMR spectroscopy to analyze pathogenic FGF receptor mutants with gradations in gain-of-function activity. These structural analyses revealed a "two-state" dynamic equilibrium model whereby the kinase toggles between an "inhibited," structurally rigid ground state and a more dynamic and heterogeneous active state. The pathogenic mutations have different abilities to shift this equilibrium toward the active state. The increase in the fractional population of FGF receptors in the active state correlates with the degree of gain-of-function activity and clinical severity. Our data demonstrate that the fractional population of RTKs in the active state determines intrinsic kinase activity and underscore how a slight increase in the active population of kinases can have grave consequences for human health.
Assuntos
Mutação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Transdução de SinaisRESUMO
We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.
Assuntos
Benzimidazóis/farmacologia , Imidazóis/farmacologia , Mutação , Piridazinas/farmacologia , Pirimidinas/farmacologia , Quinolonas/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Eletroforese em Gel de Poliacrilamida , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
CONTEXT: Kallmann syndrome (KS), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) all result from development defects of the anterior midline in the human forebrain. OBJECTIVE: The objective of the study was to investigate whether KS, CPHD, and SOD have shared genetic origins. DESIGN AND PARTICIPANTS: A total of 103 patients with either CPHD (n = 35) or SOD (n = 68) were investigated for mutations in genes implicated in the etiology of KS (FGFR1, FGF8, PROKR2, PROK2, and KAL1). Consequences of identified FGFR1, FGF8, and PROKR2 mutations were investigated in vitro. RESULTS: Three patients with SOD had heterozygous mutations in FGFR1; these were either shown to alter receptor signaling (p.S450F, p.P483S) or predicted to affect splicing (c.336C>T, p.T112T). One patient had a synonymous change in FGF8 (c.216G>A, p.T72T) that was shown to affect splicing and ligand signaling activity. Four patients with CPHD/SOD were found to harbor heterozygous rare loss-of-function variants in PROKR2 (p.R85G, p.R85H, p.R268C). CONCLUSIONS: Mutations in FGFR1/FGF8/PROKR2 contributed to 7.8% of our patients with CPHD/SOD. These data suggest a significant genetic overlap between conditions affecting the development of anterior midline in the human forebrain.
Assuntos
Fator 8 de Crescimento de Fibroblasto/genética , Hipopituitarismo/genética , Síndrome de Kallmann/genética , Mutação , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Displasia Septo-Óptica/genética , Animais , Feminino , Fator 8 de Crescimento de Fibroblasto/metabolismo , Estudos de Associação Genética , Heterozigoto , Humanos , Hipopituitarismo/metabolismo , Síndrome de Kallmann/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neuro-Hipófise/metabolismo , Neuro-Hipófise/patologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Displasia Septo-Óptica/metabolismo , Transdução de Sinais , Reino Unido , Estados UnidosRESUMO
CONTEXT: FGFR1 mutations have been identified in about 10% of patients with Kallmann syndrome. Recently cases of idiopathic hypogonadotropic hypogonadism (IHH) with a normal sense of smell (nIHH) have been reported. AIMS: The objective of the study was to define the frequency of FGFR1 mutations in a large cohort of nIHH, delineate the spectrum of reproductive phenotypes, assess functionality of the FGFR1 mutant alleles in vitro, and investigate genotype-phenotype relationships. DESIGN: FGFR1 sequencing of 134 well-characterized nIHH patients (112 men and 22 women) and 270 healthy controls was performed. The impact of the identified mutations on FGFR1 function was assessed using structural prediction and in vitro studies. RESULTS: Nine nIHH subjects (five males and four females; 7%) harbor a heterozygous mutation in FGFR1 and exhibit a wide spectrum of pubertal development, ranging from absent puberty to reversal of IHH in both sexes. All mutations impair receptor function. The Y99C, Y228D, and I239T mutants impair the tertiary folding, resulting in incomplete glycosylation and reduced cell surface expression. The R250Q mutant reduces receptor affinity for FGF. The K618N, A671P, and Q680X mutants impair tyrosine kinase activity. However, the degree of functional impairment of the mutant receptors did not always correlate with the reproductive phenotype, and variable expressivity of the disease was noted within family members carrying the same FGFR1 mutation. These discrepancies were partially explained by additional mutations in known IHH loci. CONCLUSIONS: Loss-of-function mutations in FGFR1 underlie 7% of nIHH with different degrees of impairment in vitro. These mutations act in concert with other gene defects in several cases, consistent with oligogenicity.
Assuntos
Hipogonadismo/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Animais , Células COS , Chlorocebus aethiops , Estudos de Coortes , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Estradiol/sangue , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Genótipo , Humanos , Masculino , Mutação , Fenótipo , Fosfotirosina/metabolismo , Puberdade Tardia/genética , Valores de Referência , Testosterona/sangueRESUMO
Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory "molecular brake" mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.