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.

Establishment and characterization of a novel imatinib‐sensitive chronic myeloid leukemia cell line MYL, and an imatinib‐resistant subline MYL‐R showing overexpression of Lyn

“…These results were also compatible with the results of RQ‐PCR, and suggest that the mechanism of imatinib‐resistance in MYL‐R may be different from that of K562‐R and LAMA‐R (13), and that the Lyn‐associated resistance mechanism may be diverse among cell types. The expression of STAT5, considered one of the downstream target molecules of BCR‐ABL (29) and of Lyn (30, 31), was slightly elevated in MYL‐R when compared with MYL. Furthermore, phosphorylated STAT5 in MYL‐R was markedly elevated when compared with MYL or K562 cells, indicating that high expression of active Lyn in MYL‐R may have resulted in phosphorylation of its substrate, STAT5.…”

“…Lyn plays a important role in the activation of signaling pathways such as JAK2/STAT5, PI3K, and MAP kinase (30, 40). On the other hand, active Lyn kinase may partially block ERK activation or accelerate ERK inactivation (41).…”

JAK2 activation by growth hormone and other cytokines

“…Thus the biased signalling was not a consequence of altered JAK2 catalytic mechanisms, but rather a result of utilizing an alternate tyrosine kinase. This may well be the case for other class I receptors which have been shown to associate with SFKs such as the prolactin receptor [ 106 , 107 ], EPO receptor [ 108 , 109 ], IL-6 receptor [ 110 ], GM-SCF receptor [ 111 – 113 ], the TPO receptor and others [ 114 ].…”

Origins of the Vertebrate Erythro/Megakaryocytic System

“…In contrast to EPOR, TPOR is a much more potent activator of the MAPK pathway and STAT3 signaling [ 53 ]. Conversely, it has been shown that EPOR interacts with LYN kinase, which can bind to JAK2 and affects STAT5 [ 54 ]. EPOR and TPOR signaling is limited by a negative feedback loop employing SHP1 and SHIP phosphatases and suppressors of cytokine signaling (SOCS1, SOCS3) [ 49 , 55 , 56 ].…”