Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis.
Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.
Tauchi
T
T
First Department of Internal Medicine, Tokyo Medical College, Tokyo, Japan.
Damen
J E
JE
Toyama
K
K
Feng
G S
GS
Broxmeyer
H E
HE
Krystal
G
G
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D004921:Erythropoietin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053614:Janus Kinase 2; D051379:Mice; D008938:Mitosis / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry*
D047908:Intracellular Signaling Peptides and Proteins; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C509248:Ptpn6 protein, mouse