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

STAT5A is a molecular regulator of proliferation, differentiation, and apop tosis in lymphohematopoietic cells. Here we show that STAT5A can serve as a functional substrate of Bruton's tyrosine kinase (BTK). Purified recombina nt BTK was capable of directly binding purified recombinant STAT5A with hig h affinity (K-d = 44 nm), as determined by surface plasmon resonance using a BIA-core biosensor system. BTK was also capable of tyrosine-phosphorylati ng ectopically expressed recombinant STAT5A on Tyr(694) both in vitro and i n vivo in a Janus kinase 3-independent fashion. BTK phosphorylated the Y665 F, Y668F, and Y682F,Y683F mutants but not the Y694F mutant of STAT5A. STAT5 A mutations in the Src homology 2 (SH2) and SH3 domains did not alter the B TK-mediated tyrosine phosphorylation. Recombinant BTK proteins with mutant pleckstrin homology, SH2, or SH3 domains were capable of phosphorylating ST AT5A, whereas recombinant BTK proteins with SH1/kinase domain mutations wer e not. In pull-down experiments, only full-length BTK and its SH1/kinase do main (but not the pleckstrin homology, SH2, or SH3 domains) were capable of binding STAT5A. Ectopically expressed BTK kinase domain was capable of tyr osine-phosphorylating STAT5A both in vitro and in vivo. BTK-mediated tyrosi ne phosphorylation of ectopically expressed wild type (but not Tyr694 mutan t) STAT5A enhanced its DNA binding activity. In BTK-competent chicken B cel ls, anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was preve nted 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 c ells reconstituted with wild type human BTK but not in BTK-deficient chicke n 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 fro m XID mice. In contrast to B cells from XID mice, B cells from JAK3 knockou t mice showed a normal STAT5A phosphorylation response to anti-IgM stimulat ion. These findings provide unprecedented experimental evidence that BTK pl ays a nonredundant and pivotal role in B cell antigen receptor-mediated STA T5A activation in B cells.