Lyn Physically Associates With the Erythropoietin Receptor and May Play a Role in Activation of the Stat5 Pathway

Abstract

Protein tyrosine phosphorylation plays a crucial role in signaling from the receptor for erythropoietin (Epo), although the Epo receptor (EpoR) lacks the tyrosine kinase domain. We have previously shown that the Jak2 tyrosine kinase couples with the EpoR to transduce a growth signal. In the present study, we demonstrate that Lyn, a Src family tyrosine kinase, physically associates with the EpoR in Epo-dependent hematopoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of the EpoR and that both LynA and LynB, alternatively spliced forms of Lyn, bind with the membrane-proximal 91-amino acid region of the EpoR cytoplasmic domain. In vitro binding studies using GST-Lyn fusion proteins further showed that the Src homology (SH)-2 domain of Lyn specifically binds with the tyrosine-phosphorylated EpoR in lysate from Epo-stimulated cells, whereas the tyrosine kinase domain of Lyn binds with the unphosphorylated EpoR. Far-Western blotting and synthetic phosphopeptide competition assays further indicated that the Lyn SH2 domain directly binds to the tyrosine-phosphorylated EpoR, most likely through its interaction with phosphorylated Y-464 or Y-479 in the carboxy-terminal region of the EpoR. In vitro binding studies also demonstrated that the Lyn SH2 domain directly binds to tyrosine-phosphorylated Jak2. In vitro reconstitution experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of Stat5, mainly on Y-694, and activates the DNA-binding and transcription-activating abilities of Stat5. In agreement with this, Lyn enhanced the Stat5-dependent transcriptional activation when overexpressed in 32D/EpoR-Wt cells. In addition, Lyn was demonstrated to phosphorylate the EpoR and Stat5 on tyrosines in vitro. These results suggest that Lyn may play a role in activation of the Jak2/Stat5 and other signaling pathways by the EpoR.

Association of Lyn tyrosine kinase to the GM‐CSF and IL‐3 receptor common βc subunit and role of Src tyrosine kinases in DNA synthesis and anti‐apoptosis

“…We could only detect a maximum increase of 1.23-fold activity in BA/F3 cells expressing bc mutants, possibly due to our experimental conditions or to differences in the cell types used by other investigators. Similar observations have been reported previously by numerous researchers and their inability to detect distinct Lyn activation may also be attributed to the speci®c activation of Lyn bound to the receptor complex, which represents only a small fraction of the total amount of Lyn analysed from whole cell lysates (Chin et al 1998). Alternatively, the rapid turnover of Lyn phosphorylation within the whole cell may contribute to a high background level of Lyn activity.…”

“…The cytoplasmic membrane proximal region of bc and the erythropoietin (Epo) receptor both share a conserved Box 1 motif. Lyn has recently been reported as interacting constitutively with the Epo receptor (Tilbrook et al 1997;Chin et al 1998). Among these, contradicting results were shown.…”

“…Among these, contradicting results were shown. One showed that Lyn could bind to the Epo receptor via its Unique-SH3 domain in the yeast twohybrid system while the other showed that Lyn could associate with a membrane proximal 91 amino acid region of the Epo receptor through the Lyn kinase domain (Chin et al 1998). Acknowledging the possibility that bc and the Epo receptor share a common Src kinase binding mechanism, the previous ®ndings support our observations that this interaction consists of multiple interaction sites on both the receptor and Lyn, and that the constitutive binding observed is regulated through the Lyn Unique-SH3 and SH2 domains rather than the kinase domain.…”

“…It is thus possible that JAK2 plays an essential role in the full activation of the Src kinases, either through direct phosphorylation or through an association that stabilizes its activity. Indeed, it was shown that Lyn binds to tyrosine phosphorylated JAK2 in vitro (Chin et al 1998), and that Fyn associates via its SH2 domain with Tyk2 or JAK2 in cells following interferon a or interferon g stimulation, respectively (Uddin et al 1997). Although Lyn kinase activation by GM-CSF and IL-3 has been reported by several groups (Corey et al 1993;Anderson & Jorgensen 1995;Li et al 1995;Sheng et al 1996;Youse® et al 1996) we could not detect a de®nite increase in Lyn kinase activity in an in vitro kinase assay (Table 1).…”

Direct activation of STAT5 by ETV6‐LYN fusion protein promotes induction of myeloproliferative neoplasm with myelofibrosis

“…Among SRC family kinases, LYN has been characterized to play a major role in B cells (Xu et al, 2005), but recent studies revealed that LYN is also involved in cytokine receptor signalling such as erythropoietin receptor signalling (Tilbrook et al, 1997). LYN physically interacts with the erythropoietin receptor and is proposed to play a role in activation of the STAT5 pathway (Chin et al, 1998). Our findings of ETV6-LYN in this study support the role of the SRC family kinases in the regulation of STAT pathways and implicate active LYN in the alternative pathway for STAT activation in pathological cytokine signalling.…”