Abstract
Loss of function of Bruton's tyrosine kinase (Btk) causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice (xid). By using MS analysis and phosphopeptide-specific antibodies, we identified a tyrosine phosphorylation site (Y617) near the carboxyl terminus of the Btk domain from Btk expressed in 293T as well as DT-40 cells. Y617 is conserved in all Tec family kinases except murine Tec. Replacement of Y617 with a negatively charged glutamic acid (E) suppressed Btk-mediated phospholipase Cgamma2 activation and calcium response in DT-40 cells, whereas Akt activation was not affected. The Btk Y617E mutant could partially restore conventional B cell development and proliferation in Btk(-)/Tec(-) mice but failed to rescue CD5(+) B-1 cell development and the TI-II immune response to 2,4,6,-trinitrophenyl-Ficoll. These data suggest that Y617 phosphorylation or a negative charge at this site may down-regulate the function of Btk by selectively suppressing the B cell calcium signaling pathway.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Agammaglobulinaemia Tyrosine Kinase
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Amino Acid Sequence
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Amino Acid Substitution
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Animals
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B-Lymphocytes / cytology
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B-Lymphocytes / immunology
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B-Lymphocytes / metabolism*
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Binding Sites
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CD5 Antigens / immunology
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Calcium Signaling / physiology*
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Cell Line
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Cell Line, Tumor
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Cell Proliferation / drug effects
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Enzyme Activation / physiology
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Glutamic Acid / genetics
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Glutamic Acid / metabolism
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Humans
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Immunoprecipitation / methods
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Mice
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Phospholipase C gamma
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Phosphorylation
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Protein Structure, Tertiary
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Protein-Tyrosine Kinases / chemistry
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Protein-Tyrosine Kinases / deficiency
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Protein-Tyrosine Kinases / genetics
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Protein-Tyrosine Kinases / physiology*
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Trinitrobenzenes / chemistry
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Trinitrobenzenes / immunology
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Trinitrobenzenes / pharmacology
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Type C Phospholipases / antagonists & inhibitors
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Type C Phospholipases / metabolism*
Substances
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CD5 Antigens
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Recombinant Proteins
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Trinitrobenzenes
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Glutamic Acid
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Tec protein-tyrosine kinase
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Protein-Tyrosine Kinases
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Agammaglobulinaemia Tyrosine Kinase
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BTK protein, human
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Btk protein, mouse
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Type C Phospholipases
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Phospholipase C gamma