Phosphorylation of two regulatory tyrosine residues in the activation of Bruton's tyrosine kinase via alternative receptors.
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.
Wahl
M I
MI
Department of Microbiology and Molecular Genetics, University of California, Los Angeles, CA 90095-1662, USA.
Fluckiger
A C
AC
Kato
R M
RM
Park
H
H
Witte
O N
ON
Rawlings
D J
DJ
D016475:3T3 Cells; D000077329:Agammaglobulinaemia Tyrosine Kinase; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008407:Mast Cells / Q000201:enzymology / Q000276:immunology*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000276:immunology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011817:Rabbits; D011947:Receptors, Antigen, B-Cell / Q000502:physiology*; D017455:Receptors, IgE / Q000502:physiology*; D018123:Receptors, Interleukin / Q000502:physiology*; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine; D014616:Vaccinia virus; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D000906:Antibodies; D010748:Phosphopeptides; D011947:Receptors, Antigen, B-Cell; D017455:Receptors, IgE; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000625951:Btk protein, mouse; D019061:src-Family Kinases