RESUMO
The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn(-/-) mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.
Assuntos
Biocatálise , Sequência Conservada , Proteínas Quinases/química , Eletricidade Estática , Quinases da Família src/química , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Benzamidas , Biocatálise/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Mesilato de Imatinib , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Mutação/genética , Filogenia , Piperazinas/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The mechanisms governing positive selection of T cells in the thymus are still incompletely understood. Here, we describe a N-ethyl-N-nitrosourea induced recessive mouse mutant, Ms. T-less, which lacks T cells in the peripheral blood because of a complete block of thymocyte development at the CD4(+)CD8(+) stage. Single nucleotide polymorphism mapping and candidate gene sequencing revealed a nonsense mutation in the inositol (1,4,5) trisphosphate 3 kinase B (Itpkb) gene in Ms. T-less mice. Accordingly, Ms. T-less thymocytes do not show detectable expression of Itpkb protein and have drastically reduced basal inositol (1,4,5) trisphosphate kinase activity. Itpkb converts inositol (1,4,5) trisphosphate to inositol (1,3,4,5) tetrakisphosphate, soluble second messengers that have been implicated in Ca(2+) signaling. Surprisingly, Ca(2+) responses show no significant differences between wild type (WT) and mutant thymocytes. However, extracellular signal-regulated kinase (Erk) activation in response to suboptimal antigen receptor stimulation is attenuated in Ms. T-less thymocytes, suggesting a role for Itpkb in linking T cell receptor signaling to efficient and sustained Erk activation.
