Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells

J Biol Chem. 2001 Aug 17;276(33):31216-28. doi: 10.1074/jbc.M104874200. Epub 2001 Jun 18.


STAT5A is a molecular regulator of proliferation, differentiation, and apoptosis in lymphohematopoietic cells. Here we show that STAT5A can serve as a functional substrate of Bruton's tyrosine kinase (BTK). Purified recombinant BTK was capable of directly binding purified recombinant STAT5A with high affinity (K(d) = 44 nm), as determined by surface plasmon resonance using a BIAcore biosensor system. BTK was also capable of tyrosine-phosphorylating ectopically expressed recombinant STAT5A on Tyr(694) both in vitro and in vivo in a Janus kinase 3-independent fashion. BTK phosphorylated the Y665F, Y668F, and Y682F,Y683F mutants but not the Y694F mutant of STAT5A. STAT5A mutations in the Src homology 2 (SH2) and SH3 domains did not alter the BTK-mediated tyrosine phosphorylation. Recombinant BTK proteins with mutant pleckstrin homology, SH2, or SH3 domains were capable of phosphorylating STAT5A, whereas recombinant BTK proteins with SH1/kinase domain mutations were not. In pull-down experiments, only full-length BTK and its SH1/kinase domain (but not the pleckstrin homology, SH2, or SH3 domains) were capable of binding STAT5A. Ectopically expressed BTK kinase domain was capable of tyrosine-phosphorylating STAT5A both in vitro and in vivo. BTK-mediated tyrosine phosphorylation of ectopically expressed wild type (but not Tyr(694) mutant) STAT5A enhanced its DNA binding activity. In BTK-competent chicken B cells, anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was prevented by pretreatment with the BTK inhibitor LFM-A13 but not by pretreatment with the JAK3 inhibitor HI-P131. B cell antigen receptor ligation resulted in enhanced tyrosine phosphorylation of STAT5 in BTK-deficient chicken B cells reconstituted with wild type human BTK but not in BTK-deficient chicken B cells reconstituted with kinase-inactive mutant BTK. Similarly, anti-IgM stimulation resulted in enhanced tyrosine phosphorylation of STAT5A in BTK-competent B cells from wild type mice but not in BTK-deficient B cells from XID mice. In contrast to B cells from XID mice, B cells from JAK3 knockout mice showed a normal STAT5A phosphorylation response to anti-IgM stimulation. These findings provide unprecedented experimental evidence that BTK plays a nonredundant and pivotal role in B cell antigen receptor-mediated STAT5A activation in B cells.

MeSH terms

  • Agammaglobulinaemia Tyrosine Kinase
  • Animals
  • B-Lymphocytes / metabolism*
  • Cell Line
  • Chickens
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • DNA-Binding Proteins / physiology
  • Humans
  • Janus Kinase 3
  • Mice
  • Milk Proteins*
  • Models, Molecular
  • Phosphorylation
  • Protein-Tyrosine Kinases / physiology*
  • STAT5 Transcription Factor
  • Trans-Activators / chemistry
  • Trans-Activators / metabolism*
  • Transcription Factors / physiology
  • Tumor Suppressor Proteins
  • Tyrosine / metabolism


  • DNA-Binding Proteins
  • Milk Proteins
  • STAT5 Transcription Factor
  • STAT5A protein, human
  • Stat5a protein, mouse
  • Trans-Activators
  • Transcription Factors
  • Tumor Suppressor Proteins
  • Tyrosine
  • Protein-Tyrosine Kinases
  • Agammaglobulinaemia Tyrosine Kinase
  • BTK protein, human
  • Btk protein, mouse
  • JAK3 protein, human
  • Jak3 protein, mouse
  • Janus Kinase 3