Mitogen-activated protein kinase plays an essential role in the erythropoietin-dependent proliferation of CTLL-2 cells.
Erythropoietin (EPO) and its receptor (EPOR) are required for development of erythrocytes. It has been shown that the ectopic expression of EPOR confers EPO-dependent proliferation on an interleukin 3 (IL3)-dependent cell line, Ba/F3, whereas the IL2-dependent T cell line, CTLL-2 expressing the EPOR (T-ER), fails to proliferate in response to EPO. However, the molecular basis of the EPO unresponsiveness in CTLL-2 has not been clarified. We found that the expression level of JAK2 in T-ER cells was much lower than that in Ba/F3 cells. Therefore, we examined the effects of forced expression of JAK2 in T-ER cells. In T-ER transformants expressing JAK2 (T-JER), EPO induced tyrosine phosphorylation of the EPOR, JAK2, and STAT5, and consequently STAT5-responsive genes including bcl-X and cis1 were normally induced. Furthermore, T-JER cells were resistant to apoptosis until at least 72 h after switching from IL2 to EPO. Although T-JER cells could not continuously proliferate in the presence of EPO, additional expression of JAK2 in T-JER (T-JJER) to a level similar to that in Ba/F3 cells supported long term proliferation in response to EPO. JAK2 was equally co-immunoprecipitated with the EPOR among T-JER, T-JJER, and Ba/F3 cells expressing the EPOR (BF-ER). However, EPO-dependent mitogen-activated protein (MAP) kinase activation was observed in T-JJER and BF-ER cells but not in T-JER cells. EPO-dependent long term proliferation of T-JER cells was conferred by expression of the constitutively activated form of MEK1. Our results suggest that MAP kinase activation is, at least in part, an important component for mitotic signal from the EPOR, and CTLL-2 cells probably lack signaling molecule(s) in JAK2 and the Ras-MAP kinase pathway.
Sakamoto
H
H
Institute of Life Science, Kurume University, Aikawa-machi 2432-3, Kurume 839-0861, Japan.
Kitamura
T
T
Yoshimura
A
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D053614:Janus Kinase 2; D048369:MAP Kinase Kinase 1; D051379:Mice; D008894:Milk Proteins; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017467:Receptors, Erythropoietin; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004921:Erythropoietin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases