PHOSPHORYLATION OF 2 REGULATORY TYROSINE RESIDUES IN THE ACTIVATION OF BRUTON TYROSINE KINASE VIA ALTERNATIVE RECEPTORS

Mutation of Bruton's tyrosine kinase (Btk) impairs B cell maturation a nd function and results in a clinical phenotype of X-linked agammaglob ulinemia, Activation of Btk correlates with an increase in the phospho rylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Sr c family tyrosine kinases. Y223 (site 2) is an autophosphorylation sit e within the Btk SH3 domain, Polyclonal, phosphopeptide-specific antib odies were developed to evaluate the phosphorylation of Btk sites 1 an d 2, Crosslinking of the B cell antigen receptor (BCR) or the mast cel l Fc epsilon receptor, or interleukin 5 receptor stimulation each indu ced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled ma nner. Btk molecules were singly and doubly tyrosine-phosphorylated, Ph osphorylated Btk comprised only a small fraction (less than or equal t o 5%) of the total pool of Btk molecules in the BCR-activated B cells, Increased dosage of Lyn in B cells augmented BCR-induced phosphorylat ion at both sites, Kinetic analysis supports a sequential activation m echanism in which individual Btk molecules undergo serial transphospho rylation (site 1) then autophosphorylation (site 2), followed by succe ssive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kin ases is likely to regulate their activation.