A Stat3-interacting protein (StIP1) regulates cytokine signal transduction Robert G. Collum*, Siska Brutsaert†, Gladys Lee*, and Christian Schindler*†‡ Departments of †Microbiology and *Medicine, Columbia University, New York, NY 10032 Edited by James E. Darnell, Jr., The Rockfeller University, New York, NY, and approved June 28, 2000 (received for review May 12, 1999) Genetic and biochemical studies have led to the identification of Materials and Methods the Stat3-Interacting Protein StIP1. The preferential association of Cell Culture and Transfections. 293T, M1, HepG2, NIH 3T3, and StIP1 with inactive (i.e., unphosphorylated) Stat3 suggests that it SF9 cells were acquired from the American Type Culture may contribute to the regulation of Stat3 activation. Consistent Collection and grown as reported (22–24). Before stimulation with this possibility, StIP1 also exhibits an affinity for members of with IL-6 (15 ng͞ml; PeproTech, Rocky Hill, NJ) or IFN-␥ (66 the Janus kinase family. Overexpression of the Stat3-binding units͞ml for 15 min; Pestka Research Labs, New Brunswick, NJ), domain of StIP1 blocks Stat3 activation, nuclear translocation, and cells were starved in 0.1% serum. SF9 insect cells were infected Stat3-dependent induction of a reporter gene. These studies indi- with a baculovirus encoding Stat3 and Jak1 as described (22). cate that StIP1 regulates the ligand-dependent activation of Stat3, HepG2 and 293T cells were transfected by the calcium phos- potentially by serving as a scaffold protein that promotes the phate method after a 5-min pretreatment with 25 mM Chloro- interaction between Janus kinases and their Stat3 substrate. The quine (Sigma) as described (24). The cells were stimulated with ability of StIP1 to associate with several additional members of the IL-6 24 h later. signal transducer and activator of transcription family suggests that StIP1 may serve a broader role in cytokine-signaling events. Yeast Two-Hybrid Assay. Two-hybrid screens were performed as described in Y153 cells with a ‘‘bait’’ plasmid (pAS-Stat3) he ability of IFN-␣ to rapidly induce new genes led to the encoding amino acids 103–330 of murine Stat3 (25, 26). A mouse Tidentification of the signal transducer and activator of tran- myelomonocytic cDNA library (cloned into pGADNOT) served scription (STAT) family of transcription factors. Subsequent as the ‘‘prey’’ (a generous gift from S. Goff, Columbia Univer- studies determined that IL-6, as well as all other cytokines, sity, New York; ref. 27). Hisϩ͞␤-galactosidaseϩ prey plasmids transduce vital signals through members of the STAT family (1, underwent secondary screens, including retransformation into 2). On binding ligand, receptor-associated kinases from the Y153 cells with pAS1 (empty vector) or a Gal4-Cyclophillin Janus kinase (JAK) family become activated. STATs are in turn fusion (a generous gift from J. Luban, Columbia University, New recruited to the receptor, where they become activated in a York) to eliminate false positives. To confirm interactions, prey JAK-dependent tyrosine phosphorylation event. Activated plasmids were retransformed into CTY10–5d cells along with a STATs are released from the receptor and dimerize. These bait plasmid, where the DNA-binding domain had been changed dimers then translocate to the nucleus and bind to members of to that of LexA (pSH2–1; ref. 28). the interferon-gamma activation site (GAS) family of enhancers. One member of the STAT family, Stat3, has been implicated DNA and RNA. Full-length StIP1 cDNAs were isolated from a in a wide range of biological processes including nephrogenesis, murine library (25). StIP-1 glutathione S-transferase (GST) gliogenesis, hepatogenesis, T cell proliferation, inflammation, fusion proteins were generated by cloning the appropriate and oncogenesis (3–9). Many of these Stat3-dependent re- restriction fragments into pGEX-2T and confirmed by sequenc- sponses are triggered by members of the IL-6 family of cytokines, ing. GST-StIP1A contains the original sequence identified in the which transduce their signals through a common gp130 receptor yeast two-hybrid screen (StIP1 amino acids 511–831; Fig. 1 and chain. Consistent with these pleiotropic effects, Stat3 knockout see Fig. 3D). Additionally, GST-StIP1 fusion proteins included: mice exhibit an embryonic lethal phenotype (10). More recent GST-StIP1B, amino acids 511–724; GST-StIP1C, amino acids studies have begun to identify additional molecules that regulate 511–683; GST-StIP1D, amino acids 554–683; GST-StIP1E, Stat3 signaling. Some of these molecules potentiate the tran- amino acids 554–634; GST-StIP1G, amino acids 254–593; and scriptional activity of Stat3 (11, 12) whereas others antagonize GST-StIP1H, amino acids 254–395. GST-StIP1G and GST- Stat3-dependent signals (13, 14). StIP1H were derived from the cDNA missing WD40 repeat Stat3 consists of several conserved domains that are important number 7. A dominant-negative StIP1 mutant, pCGN0.8 (amino for STAT function. This includes well-characterized DNA bind- acids 554–789) was amino terminally hemagglutinin-tagged ing, linker, Src homology 2, and transcriptional activation do- (pCGN, a generous gift from S. Goff, Columbia University, New mains (15, 16). In addition, there are two well-conserved amino- York; ref. 27). RcCMV-Stat3 and pMT2T-Jak1K896R (‘‘kinase terminal domains whose functions are less well characterized. dead’’) expression constructs were generous gifts from J. Darnell The more amino proximal domain has been shown to promote cooperativity in DNA binding (17). The larger and more amino distal domain has been shown to form a coiled coil that projects This paper was submitted directly (Track II) to the PNAS office. laterally from the core STAT structure (15, 16). This generates Abbreviations: StIP1, Stat3-interacting protein; JAK, Janus kinase; STAT, signal transducer a large exposed surface area. Consistent with this, several recent and activator of transcription; GAS, interferon-gamma activation site; GST, glutathione S-transferase; IRF, IFN regulatory factor. studies have identified proteins that interact with the coiled coil ͞ Data deposition: The sequence reported in this paper has been deposited in the GenBank of Stat3 and or other STATs (12, 18–20). database (accession no. AF291064). In an effort to characterize the conserved coiled-coil domain ‡To whom reprint requests should be addressed. E-mail: [email protected]. in Stat3, a yeast two-hybrid screen was performed. This led to The publication costs of this article were defrayed in part by page charge payment. This identification of StIP1. This protein encodes 12 WD40 repeats, article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. which are known to mediate protein–protein interactions (21). §1734 solely to indicate this fact. Biochemical studies suggest that StIP1 may regulate the activa- Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.170192197. tion of Stat3 as well as other STATs. Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.170192197 10120–10125 ͉ PNAS ͉ August 29, 2000 ͉ vol. 97 ͉ no. 18 Downloaded by guest on October 1, 2021 Fig. 2. Expression pattern of StIP1 transcripts. Expression of StIP1 was evaluated by a reverse transcription–PCR assay (Roche Molecular Biochemi- Fig. 1. The StIP1 sequence. Comparison of conceptually translated murine cals). RNA was prepared from each of the indicated tissues and amplified with (mu) and human (hu) StIP1 cDNA sequences. WD40 motifs are shaded and primers from repeat 6 and 8 (Fig. 3A). PCR products corresponding to the labeled 1–12. Dots represent absence and dashes represent identity of corre- full-length (FL) or ⌬7 transcripts are indicated. Controls samples (Cntl) were sponding amino acids. Arrows indicate borders of the spliced exon in ⌬7 StIP1. amplified from cDNA templates. Murine StIP1 has a predicted molecular mass of 93 kDa. Stat3 (1:250 dilution of C-20; Santa Cruz Biotechnology) and (The Rockefeller University, New York) (25) and J. Krolewski hemagglutinin epitope tag (1:250 dilution of 12CA5; Roche (University of California, Irvine) (29). RNA was prepared by Molecular Biochemicals) primary antibodies and then Cy3- guanidium thiocyanate lysis (30). For reverse transcription– conjugated (1:500; Jackson ImmunoResearch) and Alexa- PCR, RNA was reverse transcribed (AMV-RT; Roche Molec- conjugated (1:500; Molecular Probes) secondary antibodies. ular Biochemicals) with a StIP1-specific oligonucleotide primer NIH 3T3 cells were stained with the StIP1 antiserum (1:100) and corresponding to amino acids 384–391 and then amplified with visualized with the Cy3-conjugated secondary antibody. Because primers flanking WD40 repeat number 7 (i.e., amino acids the StIP1 antibody does not recognize human StIP1, it was first 296–305 and 378–383; see Fig. 3D). preabsorbed on HeLa cells. Cells were observed at ϫ400 under a Nikon Eclipse TE300 microscope after excitation at 495 nm Cell Extracts and Protein Assays. GST pulldown assays were per- (Alexa) or 550 nm (Cy3). formed as described (22). Immobilized GST fusion proteins (i.e., bound to glutathione agarose; Sigma) then were incubated for Results 2–16 h with whole-cell extracts from HepG2 and M1 cells (23, 31) A yeast two-hybrid screen, where the coiled-coil domain of Stat3 ͞ or recombinant Stat3 from insect cells infected with Stat3 and or (amino acids 103–330) served as ‘‘bait’’, identified a novel 1.3-kb Jak1 baculoviruses (22). Bound proteins were eluted either with murine cDNA, referred to as StIP1. Full-length (i.e., Ϸ3 kb) ͞ 25 mM glutathione or by boiling in SDS Laemmli buffer, cDNAs were isolated and found to encode an 831-aa protein ͞ fractionated on 7–8% SDS PAGE, and then immunoblotted whose ORF corresponds to the reading frame identified in the with Stat3 (C-20; Santa Cruz Biotechnology), phospho-specific yeast two-hybrid ‘‘hit’’ (Fig. 1). One notable feature of the StIP1 Stat3 (New England Biolabs), or Jak1 (Upstate Biotechnology, sequence is the presence of 12 WD40 repeat motifs. These motifs IMMUNOLOGY Lake Placid, NY) antibodies.