Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.
A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (IL-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and IL-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while IL-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked 20 amino acids in a region of sequence homology with the IL-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.
Miura
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O
Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
D'Andrea
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A
Kabat
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Ihle
J N
JN
D000595:Amino Acid Sequence; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D004790:Enzyme Induction / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D015151:Immunoblotting; D007377:Interleukin-3 / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017467:Receptors, Erythropoietin; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D007377:Interleukin-3; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D015375:Receptors, Interleukin-2; D004921:Erythropoietin; D014443:Tyrosine