Y. Okutani et al., Src directly tyrosine-phosphorylates STAT5 on its activation site and is involved in erythropoietin-induced signaling pathway, ONCOGENE, 20(45), 2001, pp. 6643-6650
Signal transducers and activators of transcription (STAT) proteins are tran
scription factors activated by phosphorylation on tyrosine residues after c
ytokine stimulation. In erythropoietin receptor (EPOR)-mediated signaling,
STAT5 is tyrosine-phosphorylated by EPO stimulation. Although Janus Kinase
2 (JAK2) is reported to play a crucial role in EPO-induced activation of ST
AT5, it is unclear whether JAK2 alone can tyrosine-phosphorylate STAT5 afte
r EPO stimulation. Several studies indicate that STAT activation is caused
by members of other families of protein tyrosine kinases such as the Src fa
mily. We previously reported that reduction of Src by induction of antisens
e src RNA expression suppressed EPO-promoted erythroid differentiation in K
562 cells. In the present study, we explored the function of Src downstream
of the EPOR-initiated signaling. Reduction of Src diminished tyrosine phos
phorylation of STAT5 in K562 cells regardless of EPO treatment. The tyrosin
e phosphorylation level of STAT5 induced by EPO in F-36P cells was reduced
in the presence of PPI or PP2 selective Src inhibitor. In addition, the exp
ression of dominant negative Src in F-36P cells reduced the tyrosine phosph
orylation of STAT5. When Src and STAT5 were co-expressed in COS7 cells, tyr
osine phosphorylation of STAT5 was observed, and tyrosine residue 694 (Tyr
694) of STAT5A was identified as the major phosphorylation site by Src. In
vitro kinase assay revealed that GST-STAT5 fusion protein with the conserve
d C-terminal, but not the C-terminal-truncated mutant which lacks Tyr 694,
was tyrosine-phosphorylated by Src. Src can thus directly tyrosine-phosphor
ylate the activation site of STAT5 (Tyr 694 in STAT5A), and Src may contrib
ute to EPO-induced signal transduction via STAT5.