Lyn tyrosine kinase is essential for erythropoietin-induced differentiation of J 2 E erythroid cells and two protein interaction domains

Department of Biochemistry (Laboratory for Cancer Medicine), 1991). Lyn is expressed mainly in haematopoetic cells of University of Western Australia, Royal Perth Hospital, Western myeloid (macrophage, monocyte and platelets) and B Australia 6001 and 1Ludwig Institute for Cancer Research, lymphoid origin (Bolen et al., 1992). It is involved in the Melbourne Tumour Biology Branch, c/o Post Office, transmission of signals from a number of receptors such Royal Melbourne Hospital, Victoria 3050, Australia as the IL-2 receptor, GM-CSF receptor, high-affinity IgE 2Corresponding author receptor (FcεRI), B-cell antigen receptor and thrombin receptor (Burkhardt et al., 1991; Yamanshi et al., 1991a; Erythropoietin stimulates the immature erythroid J2E Campbell and Sefton, 1992; Cichowski et al., 1992; cell line to terminally differentiate and maintains the Torigoe et al., 1992; Corey et al., 1993, 1994; Yamamoto viability of the cells in the absence of serum. In contrast, et al., 1993). Importantly, lyn has been implicated in the a mutant J2E clone (J2E-NR) fails to mature in phosphorylation of a number of signalling molecules, response to erythropoietin; however, it remains viable including phosphoinositol-3 kinase (PI-3K), ras GTPase in the presence of the hormone. We have shown activating protein (GAP), phospholipase C (PLC) γ2 and previously that intracellular signalling is disrupted in mitogen-activated protein (MAP) kinase (Cichowski et al., the J2E-NR cell line and that tyrosine phosphorylation 1992; Yamanashi et al., 1992; Corey et al., 1993; Pleiman is dramatically reduced after erythropoietin stimulaet al., 1993). To date, there have been no links between tion. In this study we investigated the defect in J2Elyn and the erythropoietin receptor (epo-R), and no NR cells that is responsible for their inability to suggestion of an involvement of lyn in signalling within differentiate. Screening of numerous signalling moleerythroid cells. cules revealed that the lyn tyrosine kinase appeared to Erythropoietin (epo) acts primarily on immature erybe absent from J2E-NR cells. On closer examination, throid precursors to maintain viability and promote both lyn mRNA and protein content were reduced proliferation and terminal differentiation (Krantz, 1991; 500-fold. Consistent with a defect in lyn, amphotropic Jelkman, 1992; Koury and Bondurant, 1992). The epo-R retroviral infection of J2E-NR cells with lyn restored and its associated protein Janus kinase 2 (JAK2) are the ability of the cells to synthesize haemoglobin and rapidly phosphorylated (Witthuhn et al., 1993; Miura enabled the cells to mature morphologically. Conet al., 1994) after ligand binding. Subsequently, the Signal versely, the ability of J2E cells to differentiate in Transducers and Activators of Transcription (STAT) and response to epo was severely curtailed when antisense ras/MAP kinase pathways are activated. Proteins known lyn oligonucleotides or a dominant negative lyn were to be phosphorylated after epo stimulation include STAT5, introduced into the cells. However, erythropoietinShc, p92c-fes, PI-3K, SHP2 (Syp), ras GAP and MAP supported viability was unaffected by reducing lyn kinases. Down-regulation of the epo-R signal is achieved activity. The ability of two other erythropoietinvia association of the phosphatase SHP1 (HCP) and deresponsive cell lines (R11 and R24) to differentiate in phosphorylation of the receptor (Klingmuller et al., 1995; response to the hormone was also reduced by dominant Yi et al., 1995). negative lyn. Finally, co-immunoprecipitation and yeast The J2E cell line was generated by transforming immatwo-hybrid analyses indicated that lyn directly associture erythroid cells with the raf/myc-containing J2 retroated with the erythropoietin receptor complex. These virus (Klinken et al., 1988). These cells express ~1000 data indicate for the first time an essential role for lyn epo-R per cell and proliferate and differentiate in response in erythropoietin-initiated differentiation of J2E cells to epo; haemoglobin is synthesized and the cells undergo but not in the maintenance of cell viability. morphological alterations which culminate in a proportion