An alternative pathway for STAT activation that is mediated by the direct interaction between JAK and STAT Yoshio Fujitani 1, *, Masahiko Hibi 1 , Toshiyuki Fukada 1 , Mariko Takahashi-Tezuka 1 , Hideyuki Yoshida 1 , Takuya Yamaguchi 1 , Kenji Sugiyama 2 , Yojiro Yamanaka 1 , Koichi Nakajima 1 and Toshio Hirano 1 1 Department of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565; 2 Nippon Boehringer Ingelheim Co., Ltd., 3-10-1 Yato, Kawanishi, Hyogo 666-01, Japan JAK is believed to be an essential tyrosine kinase that mediates signals from the cytokine receptor to its downstream events. JAK associates with the cytoplasmic domain of the type I cytokine receptor superfamily and upon the ligand stimulation it can be activated, resulting in the receptor phosphorylation. In signaling from gp130, a common signal transducer for the IL-6 family cytokines, STAT3, a transcription factor that contains an SH2 domain, is recruited by phosphotyrosines on gp130 and is subsequently phosphorylated by gp130- associated JAKs. In this study, we attempted to find a new target for JAK that is directly activated by JAK, independent of gp130 tyrosine phosphorylation, by using a yeast two-hybrid system. In the process we found that the JH2 domain of JAK1, JAK2 or JAK3 could specifically associate with the carboxy-terminal portion of STAT5, but not with STAT3 or STAT1. The interaction was confirmed using both a transient expression system in a cell line and a GST-fusion protein binding assay. Furthermore, we showed that the activation of STAT5 via gp130 did not need any phosphotyrosines on gp130 while that of STAT3 strictly depended on phosphotyrosines on gp130. Mutations of STAT5 that eliminated the interaction with JAK1 reduced the activation of STAT5 upon the gp130 stimulation, although such mutants could be still activated through erythropoietin receptor. These results indicate that STATs are activated through cytokine receptors by two distinct mechanisms, one dependent on receptor tyrosine phosphorylation and the other mediated by the JAK – STAT direct interaction. Keywords: JAK; STAT; yeast two-hybrid system; JH2 domain Introduction Cytokines exhibit functional pleiotrophy and redun- dancy and play a crucial role in regulating cell growth, dierentiation, and death in various organs (Thomson, 1994). Molecular cloning of the receptors for cytokines has revealed that they constitute the type I cytokine receptor superfamily (Bazan, 1990; Miyajima et al., 1992). In contrast to the receptors for growth factors and the TGF-b superfamily, these cytokine receptors do not contain any intrinsic catalytic domain in their cytoplasmic regions (Heldin, 1995). It has been found that the cytokine receptors constitutively associate with tyrosine kniase JAKs (Janus kinases) (Ziemiecki et al., 1994; Ihle, 1995) and Src-related tyrosine kinases in their cytoplasmic domain (Taniguchi, 1995). JAK family kinases are thought to be implicated in the activation and tyrosine phosphorylation of various known signaling proteins including signal transducers and activators of transcription (STATs) (Darnell et al., 1994; Ihle, 1996). Interleukin-6 (IL-6) and its related cytokine sub- families including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), IL-11, and cardiotrophin-1 (CT-1) are typical cytokines that exhibit such functional pleiotrophy and redun- dancy (Hirano et al., 1986; Sehgel et al., 1989; Hirano and Kishimoto, 1990; Van Snick, 1990; Hirano, 1994; Mackiewicz et al., 1995; Hibi et al., 1996). The receptor complex for IL-6 is composed of a ligand-binding receptor a-subunit and a signal transducing receptor b- subunit, gp130 (Yamasaki et al., 1988; Taga et al., 1989; Hibi et al., 1990). The binding of IL-6 to the receptor a-subunit induces the homodimerization of gp130 (Davis et al., 1993; Murakami et al., 1993), resulting in the clustering and trans-activation of JAKs (JAK1, JAK2, and TYK2), which are constitutively associated with the box 1 region of gp130 (Matsuda et al., 1994; Stahl et al., 1994). It is thought to be likely that the activated JAKs initiate signal transduction by phosphorylating the downstream signaling molecules including gp130, STATs (STAT3 and STAT1) (Zhong et al., 1994; Akira et al., 1994; Fujitani et al., 1994; Nakajima et al., 1995), and other molecules (Boulton et al., 1994; Stahl et al., 1995). STATs were originally identified as IFN-induced transcription factors by Darnell and his colleagues (Darnell et al., 1994). To date, seven members of STAT family, six in mammals (STAT1-6) and one in Drosophila (D-STAT), have been identified and characterized. STAT factors play essential roles in a variety of biological functions; STAT1 is critical for interferons (IFNs) to exert their functions, and is also involved in innate immunity (Durbin et al., 1996; Meraz et al., 1996). STAT6 is essential for IgE class switching and the Th2 response in the immune system (Shimoda et al., 1996; Takeda et al., 1996). D-STAT regulates the expression of pair rule genes and is critically involved in Drosophila early development (Hou et al., 1996; Yan et al., 1996b). We have recently shown that STAT3 plays a crucial role in IL-6- Correspondence: T Hirano *Present address: First Department of Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan Received 25 September 1996; revised 21 October 1996; accepted 21 October 1996 Oncogene (1997) 14, 751 – 761  1997 Stockton Press All rights reserved 0950 – 9232/97 $12.00