Phosphorylation of two regulatory tyrosine residues in the activation of Bruton's tyrosine kinase via alternative receptors

Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11526-33. doi: 10.1073/pnas.94.21.11526.

Abstract

Mutation of Bruton's tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcepsilon receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (</=5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Agammaglobulinaemia Tyrosine Kinase
  • Amino Acid Sequence
  • Animals
  • Antibodies / isolation & purification
  • Antibody Specificity
  • B-Lymphocytes / enzymology
  • B-Lymphocytes / immunology*
  • Cell Line
  • Enzyme Activation
  • Humans
  • Mast Cells / enzymology
  • Mast Cells / immunology*
  • Mice
  • Models, Biological
  • Molecular Sequence Data
  • Phosphopeptides / chemistry
  • Phosphopeptides / immunology
  • Phosphorylation
  • Protein-Tyrosine Kinases / metabolism*
  • Rabbits
  • Receptors, Antigen, B-Cell / physiology*
  • Receptors, IgE / physiology*
  • Receptors, Interleukin / physiology*
  • Receptors, Interleukin-5
  • Recombinant Proteins / metabolism
  • Transfection
  • Tyrosine
  • Vaccinia virus
  • src Homology Domains
  • src-Family Kinases / metabolism*

Substances

  • Antibodies
  • Phosphopeptides
  • Receptors, Antigen, B-Cell
  • Receptors, IgE
  • Receptors, Interleukin
  • Receptors, Interleukin-5
  • Recombinant Proteins
  • Tyrosine
  • Protein-Tyrosine Kinases
  • Agammaglobulinaemia Tyrosine Kinase
  • BTK protein, human
  • Btk protein, mouse
  • src-Family Kinases