Oncogene (2000) 19, 2638 ± 2644 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Modulation of STAT signaling by STAT-interacting proteins K Shuai*,1 1Departments of Medicine and Biological Chemistry, University of California, Los Angeles, California, CA 90095, USA STATs (signal transducer and activator of transcription) play important roles in numerous cellular processes Interaction with non-STAT transcription factors including immune responses, cell growth and dierentia- tion, cell survival and apoptosis, and oncogenesis. In Studies on the promoters of a number of IFN-a- contrast to many other cellular signaling cascades, the induced genes identi®ed a conserved DNA sequence STAT pathway is direct: STATs bind to receptors at the named ISRE (interferon-a stimulated response element) cell surface and translocate into the nucleus where they that mediates IFN-a response (Darnell, 1997; Darnell function as transcription factors to trigger gene activa- et al., 1994). Stat1 and Stat2, the ®rst known members tion. However, STATs do not act alone. A number of of the STAT family, were identi®ed in the transcription proteins are found to be associated with STATs. These complex ISGF-3 (interferon-stimulated gene factor 3) STAT-interacting proteins function to modulate STAT that binds to ISRE (Fu et al., 1990, 1992; Schindler et signaling at various steps and mediate the crosstalk of al., 1992). ISGF-3 consists of a Stat1:Stat2 heterodimer STATs with other cellular signaling pathways. This and a non-STAT protein named p48, a member of the article reviews the roles of STAT-interacting proteins in IRF (interferon regulated factor) family (Levy, 1997). the regulation of STAT signaling. Oncogene (2000) 19, p48 alone can bind to ISRE weakly. Although Stat1 2638 ± 2644. and Stat2 can not bind to ISRE without p48, the ISGF3 complex has a high binding anity toward Keywords: STAT-interacting proteins; signal transduc- ISRE. The ISGF3 complex established a paradigm in tion; transcription which STAT proteins may aect transcription by modulating other non-STAT transcription factors. In fact, since STAT proteins were ®rst identi®ed through Introduction the characterization of ISGF3, it had been questioned whether STATs alone can bind to DNA. Characteriza- In unstimulated cells, a STAT protein exists in the tion of IFN-g-induced gene activation demonstrated cytoplasm as a monomer. Upon activation by that Stat1 is a sequence speci®c DNA binding protein tyrosine phosphorylation in response to ligand that drives transcription through GAS (g-activation stimulation, STAT forms a dimer through SH2- site) (Decker et al., 1991; Shuai et al., 1992). It now phosphotyrosyl interactions (Becker et al., 1998; Chen appears that a STAT protein may participate in et al., 1998; Shuai et al., 1994). The STAT dimer transcriptional activation through four distinct me- then translocates into the nucleus to activate chanisms: (1) A STAT protein may bind to its own transcription. It is believed that the STAT dimer is DNA target site to directly drive transcription; (2) A dephosphorylated by an unidenti®ed protein tyrosine STAT protein may form a transcriptional complex phosphatase (PTPase) to form non-phosphorylated with a non-STAT transcription factor to trigger STAT monomers in the nucleus (Darnell, 1997; transcription through a STAT; or (3) a non-STAT Shuai, 1999). The inactivated STAT monomer is DNA binding element; (4) A STAT and a non-STAT relocated back to the cytoplasm where it can be transcription factor may cooperate to activate tran- reactivated, completing an activation-inactivation scription through binding to clustered independent cycle (Figure 1). The hypothesis that a STAT can DNA binding sites (Figure 2). shuttle between cytoplasm and nucleus was initially derived from results of biochemical fractionation and p48 pulse-chase experiments (Haspel and Darnell, 1999; Haspel et al., 1996), and is clearly supported by The molecular basis of ISGF3 complex formation has recent studies using GFP (green ¯uorescence protein) been extensively studied. Although Stat1, Stat2 and p48 fusion proteins to visualize the tracking of Stat1 in can form a stable complex in the presence of DNA, initial living cells (Keoster and Hauser, 1999). A number of attempts to detect association of Stat1 or Stat2 with p48 proteins have been identi®ed that interact with in solution had failed. Subsequently, in vivo coimmuno- STATs and modulate the activity of STATs at precipitation studies using speci®c antibodies against p48 various steps of the activation-inactivation cycle or Stat2 demonstrated that Stat2 can interact with p48 in (Figure 1). The focus of this article is to review the the absence of DNA (Martinez-Moczygemba et al., roles of these STAT-interacting proteins in STAT 1997). The Stat2-p48 interaction was detectable in cells signaling. with or without IFN-a treatment. But similar coimmu- noprecipitation analysis failed to detect an interaction between Stat1 and p48. These results suggest that either Stat1 does not interact with p48 in solution in vivo,or *Correspondence: K Shuai, Division of Hematology-Oncology, 11- alternatively, the interaction of p48 and Stat1 is weak 934 Factor Bldg, 10833 LeConte Avenue, Los Angeles, California and transient in the absence of the DNA target. 90095-1678. Consistent with the latter hypothesis, the Stat1-p48 STAT-interacting proteins K Shuai 2639 Figure 1 The activation and inactivation cycle of STAT. STAT shuttles between cytoplasm and nucleus. STAT-interacting proteins aect various steps of STAT signaling. See text for detailed description Figure 2 Mechanisms of transcriptional activation by STATs. STATs alone or in cooperation with non-STAT transcription factors activate transcription through a STAT site or a non-STAT site in gene promoters. See text for details interaction was observed when analysed by the yeast al., 1997) (Figure 3). The coiled-coil domain of Stat2 is two-hybrid assay, a highly sensitive method to detect essential for Stat2-p48 interaction. A single mutation protein-protein interactions. In addition, in vitro GST (K161A) in the p48 contact region of Stat1 inhibited pull-down experiments suggested that both Stat1 and the Stat1-p48 interaction as well as the IFN-a-triggered Stat2 can interact with p48, although the Stat2-p48 ISG15 gene induction, whereas this mutation had no interaction is several fold stronger than the Stat1-p48 eect on either the Stat1-Stat2 interaction or the IFN-g association (Horvath et al., 1996). response which is independent of p48 (Horvath et al., In vitro reconstitution experiments identi®ed the 1996). These results strongly support the importance of COOH-terminal region of p48 (aa 217 ± 377) is required the Stat1-p48 interaction in IFN-a signaling. for ISGF3 formation (Veals et al., 1992). Detailed domain mapping studies suggest that the NH2-terminal c-Jun and Sp1 region of Stat1 (aa 152 ± 239) or Stat2 (aa 1 ± 324 in Stat2) contacts with the COOH-terminal portion of It has been well established that the transcriptional p48 (Horvath et al., 1996; Martinez-Moczygemba et activation of mammalian genes usually requires the Oncogene STAT-interacting proteins K Shuai 2640 Figure 3 Regions of STATs involved in binding to non-STAT proteins. The domain structure of STAT is shown. The contact regions of STAT-interacting proteins are underlined. ND: the N domain; CCD: Coiled coil domain; DBD: DNA binding domain; LD: Linker domain; SH2: Src homology 2 domain; TAD: Transcription activation domain cooperative eect of many transcription factors. (Figure 3). Point mutations in the interaction regions Studies on a number of genes have shown that of Stat3 abolished Stat3-c-Jun interaction as well as independent but closely spaced DNA binding sites for their cooperation in IL-6-induced transcriptional STAT and other transcription factors are required for activation of the a2-macroglobulin gene. Further- maximal transcriptional activation. Two well studied more, these mutations did not aect the ability of examples involving cooperative interactions between Stat3 to drive noncooperative IL-6-induced transcrip- STAT and Sp1 or STAT and c-Jun are discussed in tion. It is unclear why dierent regions of c-Jun detail here. were observed to interact with Stat3 by dierent The intercellular adhesion molecule-1 (ICAM-1) investigators. Nevertheless, these results demonstrate gene is induced by IFN-g. In the promoter of ICAM- the importance of the Stat3-c-Jun interaction in gene 1 gene, contiguous but independent DNA binding sites activation. for Stat1 and Sp1 are present and are shown to be required for the full transcriptional activation of Glucocortical receptor ICAM-1 gene in response to IFN-g. (Look et al., 1995). Similarly, both a Stat3 binding sequence and an The ®nding that glucocorticoid receptor (GR) is adjacent Sp1 binding site present in the promoter of associated with Stat5 came from studies on the b- the C/EBPd (CCAAT/enhancer binding protein d) gene casein gene. Upon interaction with its steroid ligand, are shown to be required for the induction of C/EBPd GR binds to speci®c DNA sequence, the glucocorti- by IL-6 (Cantwell et al., 1998). In addition, Stat1 and coid response element (GRE) to activate transcrip- Sp1 association in solution has been observed. These tion (Beato et al., 1995). The optimal expression of studies demonstrate that Stat1 or Stat3 can cooperate b-casein gene is achieved when costimulated with with Sp1 to activate genes. glucocorticoids and prolactin (PRL), a growth The involvement of Stat3 and c-Jun cooperation hormone that activates Stat5 (Schmitt-Ney et al., in transcriptional activation has been well documen- 1991). A functional Stat5 binding site and half- ted for a number of genes, including the induction palindromic GREs are present in the promoter of b- of a2-macroglobulin gene by IL-6 (Ito et al., 1989), casein gene. Mutation of the Stat5 binding site in the induction of c-fos by multiple ligands (Robertson the b-casein gene promoter abolishes both glucocor- et al., 1995), the induction of matrix metalloprotei- ticoid- and PRL-induced transcription.