Src kinases and not JAKs activate STATs during IL-3 induced myeloid cell proliferation Priya Chaturvedi, MV Ramana Reddy and E Premkumar Reddy Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicne, 3307 N. Broad Street, Philadelphia, Pennsylvania 19140, USA Interaction of IL-3 with its receptor is known to activate STAT-3 via phosphorylation of Tyrosine 701, which facilitates its dimerization and translocation to the nucleus, leading to the transcription of its target genes. In this communication, we have investigated the nature of tyrosine kinases that mediate STAT-3 phosphoryla- tion during IL-3-mediated activation of myeloid cell proliferation. Our results show that interaction of IL-3 with its receptor leads to the activation of c-Src kinase activity, which in turn facilitates the binding of c-Src to STAT-3. This association leads to the phosphorylation of STAT-3, allowing this transcription factor to translocate to the nucleus. Expression of a dominant negative mutant of src (AMSrc) in these cells results in a block to IL-3 mediated phosphorylation of STAT-3, and its ability to bind to DNA. On the other hand, expression of a dominant negative mutant of JAK2 (JAK2KE) had no eect on IL-3-mediated activation of STAT-3. Our results also show that AMSrc does not aect the phosphorylation of JAK2, suggesting that JAK and STAT phosphorylation events are mediated by two independent pathways. Inhibition of c-Src activation by AMSrc, which leads to a block to STAT-3 activation, results in a dramatic inhibition of cell proliferation mediated by IL-3. However, expression of AMSrc does not activate apoptotic pathways. In contrast, expression of JAK2KE results in accelerated apoptosis of 32Dcl3 cells grown in the absence of IL-3 with concomitant down-regulation of Erk-2 kinase activity. These results suggest that Src family kinases mediate the phosphor- ylation of STATs and play a critical role in signal transduction pathways associated with myeloid cell proliferation while JAK kinases mediate the activation of Erk-2 pathway which appears to provide anti- apoptotic signals. Thus the activation of JAKs and STATs appear to be two independent but related events, which dictate two separate biological outcomes, the combination of which results in proliferation and survival of myeloid precursor cells. Keywords: STATs; JAKs; Src kinases; IL-3; myeloid cell proliferation Introduction Hematopoietic cell growth is mediated by a group of soluble growth factors, which bind to their cognate receptors and trigger the activation of latent cytoplas- mic transcription factors termed STATs (Ihle and Kerr, 1995). These transcription factors were originally described by Darnell and his co-workers (Darnell et al., 1994; Darnell, 1996, 1997) as ligand-induced transcrip- tion factors in interferon-treated cells. Subsequent studies by a number of groups showed that STATs play a critical role in signal transduction pathways associated with several cytokines and neurokines including the interleukins, the interferons, erythropoie- tin, prolactin, growth hormone, oncostatin M (OSM), and ciliary neurotrophic factor (CNTF) (Eilers et al., 1994; Lehmann et al., 1994; Silva et al., 1996). To date, seven mammalian genes that code for dierent STATs have been identi®ed, all of which encode for proteins of 750 ± 850 amino acids long and are characterized by the presence of a DNA-binding domain followed by putative SH3 and SH2 domains (Darnell, 1996). These proteins, which are normally localized in the cytoplasm are activated when phosphorylated on a tyrosine located around residue 700, which facilitates their dimerization and translocation to the nucleus (Dar- nell et al., 1994; Schindler and Darnell, 1995). The phosphorylation of STATs is known to occur immediately after the binding of growth factors or interferons to their receptors. Since this receptor-ligand interaction was also found to result in the activation of JAK kinases, which often exist in association with cytokine receptors, it was originally thought that STATs might be substrates for JAK kinases. How- ever, accumulating evidence suggests that STAT activation may not be mediated by JAK kinases. Thus, the activated JAK kinases do not seem to exhibit speci®city for a particular STAT as dierent receptors activate a common STAT, even though they activate distinctively dierent JAK kinases (Kohlhuber et al., 1997; Darnell, 1997). In addition, chimeric receptor molecules with dierent JAK binding sites but containing the same STAT-binding site were found to activate the same STAT (Kotenko et al., 1996; Kohlhuber et al., 1997). Thus, the speci®city for STAT phosphorylation appears to be determined by the docking sites for STATs that are present in the receptor molecules and not JAK kinases (Stahl et al., 1995). This leaves the question open as to which tyrosine kinases mediate the phosphorylation of STATs. A clue to this question is provided by the observation that several oncogenes derived from the src family transform hematopoietic cells and render them cytokine- independent for growth (Pierce et al., 1985; Cook et al., 1985; Mathey-Prevot et al., 1986; Rovera et al., 1987). Recent studies have provided conclusive evidence that these oncogenes bring about these transforming eects Correspondence: E Premkumar Reddy Received 3 March 1998; revised 13 March 1998; accepted 13 March 1998 Oncogene (1998) 16, 1749 ± 1758  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc