Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells.
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.
Mahajan
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S
Molecular Signal Transduction Laboratory, Parker Hughes Cancer Center and the Department of Biochemistry, Parker Hughes Institute, St. Paul, Minnesota 55113, USA.
Vassilev
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A
Sun
N
N
Ozer
Z
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Mao
C
C
Uckun
F M
FM
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D053616:Janus Kinase 3; D051379:Mice; D008894:Milk Proteins; D008958:Models, Molecular; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000502:physiology; D025521:Tumor Suppressor Proteins; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; D008894:Milk Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494093:Stat5a protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000625951:Btk protein, mouse; C507933:JAK3 protein, human; C507935:Jak3 protein, mouse; D053616:Janus Kinase 3