Signal transducers and activators of transcription (STAT) proteins are transcription factors activated by phosphorylation on tyrosine residues after cytokine 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 STAT5, it is unclear whether JAK2 alone can tyrosinephosphorylate STAT5 after EPO stimulation. Several studies indicate that STAT activation is caused by members of other families of protein tyrosine kinases such as the Src family. We previously reported that reduction of Src by induction of antisense src RNA expression suppressed EPO-promoted erythroid dierentiation in K562 cells. In the present study, we explored the function of Src downstream of the EPOR-initiated signaling. Reduction of Src diminished tyrosine phosphorylation of STAT5 in K562 cells regardless of EPO treatment. The tyrosine phosphorylation level of STAT5 induced by EPO in F-36P cells was reduced in the presence of PP1 or PP2 selective Src inhibitor. In addition, the expression of dominant negative Src in F-36P cells reduced the tyrosine phosphorylation of STAT5. When Src and STAT5 were co-expressed in COS7 cells, tyrosine phosphorylation of STAT5 was observed, and tyrosine residue 694 (Tyr 694) of STAT5A was identi®ed as the major phosphorylation site by Src. In vitro kinase assay revealed that GST-STAT5 fusion protein with the conserved C-terminal, but not the Cterminal-truncated mutant which lacks Tyr 694, was tyrosine-phosphorylated by Src. Src can thus directly tyrosine-phosphorylate the activation site of STAT5 (Tyr 694 in STAT5A), and Src may contribute to EPOinduced signal transduction via STAT5. Oncogene (2001) 20, 6643 ± 6650.

Previous studies have shown that B-Myb, a conserved member of the Myb transcription factor family, is a potent activator of the promoter of the human HSP70 gene but does not activate promoters containing Myb binding sites. We have now investigated the transactivation properties of B-Myb in more detail. We here report that B-Myb activates the HSP70 promoter by a novel mechanism which involves the heat shock element (HSE). Deletion analysis of B-Myb shows that a speci®c domain in the center of B-Myb, but not the DNA-binding domain is required for HSE-dependent transactivation. We also show that deletion of the C-terminal domain of B-Myb does not a ect HSE-dependent transactivation but allows the protein to activate a promoter containing Myb binding sites. This suggests that the ability to activate Myb binding site containing promoters is repressed in the context of full length B-Myb and that HSE dependent and Myb binding site dependent transactivation are distinct functions of B-Myb. Finally, we report that cyclin D1 like B-Myb strongly activates the HSP70 promoter via the HSE. HSE-dependent transactivation is a novel activity of cyclin D1 and appears to be independent of the phosphorylation of the Rb protein. Our results reveal an interesting and unexpected connection between HSE-dependent gene activation and proteins expressed during the G1/Stransition of the cell cycle.

Abstract

In this study, we examined the molecular mechanism of erythropoietin‐initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3‐kinase (PI3‐kinase). Antisense oligonucleotides against src but not lyn inhibited the formation of erythropoietin‐dependent colonies derived from human bone marrow cells and erythropoietin‐induced differentiation of K562 human erythroleukaemia cells. Antisense p85α oligonucleotide or LY294002, a selective inhibitor of PI3‐kinase, independently inhibited the formation of erythropoietin‐dependent colonies. In K562 cells, Src associated with PI3‐kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibited the erythropoietin‐induced activation of PI3‐kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin‐induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3‐kinase in F‐36P human erythroleukaemia cells. The coexpression experiments and in vitro kinase assay further demonstrated that Src directly tyrosine‐phosphorylated erythropoietin receptor, and associated with PI3‐kinase. In vitro binding experiments proved that glutathione S‐transferase–p85α N‐ or C‐terminal SH2 domains independently bound to erythropoietin receptor, which was tyrosine‐phosphorylated by Src. Taken together, Src transduces the erythropoietin‐induced erythroid differentiation signals by regulating PI3‐kinase activity.

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