Modulation of STAT3 Folding and Function by TRiC/CCT Chaperonin

Citation: Kasembeli M, Lau WCY, Roh S-H, Eckols TK, Frydman J, et al. ( Modulation of STAT3 Folding and Function by TRiC/CCT Chaperonin Moses Kasembeli 0 Wilson Chun Yu Lau 0 Soung-Hun Roh 0 T. Kris Eckols 0 Judith Frydman 0 Wah Chiu 0 David J. Tweardy 0 Hidde L. Ploegh, Whitehead Institute, United States of America 0 1 Section of Infectious Diseases, Department of Medicine, Baylor College of Medicine , Houston , Texas, United States of America, 2 Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine , Houston , Texas, United States of America, 3 Department of Biology and the BioX Program, Stanford University, Stanford, California, United States of America, 4 Department of Cellular and Molecular Biology, Baylor College of Medicine , Houston, Texas , United States of America Signal transducer and activator of transcription 3 (Stat3) transduces signals of many peptide hormones from the cell surface to the nucleus and functions as an oncoprotein in many types of cancers, yet little is known about how it achieves its native folded state within the cell. Here we show that Stat3 is a novel substrate of the ring-shaped hetero-oligomeric eukaryotic chaperonin, TRiC/CCT, which contributes to its biosynthesis and activity in vitro and in vivo. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped predominantly to the b-strand rich, DNAbinding domain of Stat3. Notably, enhancing Stat3 binding to TRiC by engineering an additional TRiC-binding domain from the von Hippel-Lindau protein (vTBD), at the N-terminus of Stat3, further increased its affinity for TRiC as well as its function, as determined by Stat3's ability to bind to its phosphotyrosyl-peptide ligand, an interaction critical for Stat3 activation. Thus, Stat3 levels and function are regulated by TRiC and can be modulated by manipulating its interaction with TRiC. - Funding: This work is supported by the National Institutes of Health (P41GM103832 and PN2EY016525, http://www.nih.gov). The Cancer Prevention and Research Institute of Texas (RP110291), and the Cancer Prevention and Research Institute of Texas postdoctoral training grants (RP101499 and RP101489, http:// www.cprit.state.tx.us). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing Interests: The authors have declared that no competing interests exist. Abbreviations: BSA, bovine serum albumin; CCT, chaperonin containing TCP-1; DBD, DNA-binding domain; GFP, green fluorescent protein; MEFs, mouse embryonic fibroblasts; pVHL, von Hippel-Lindau protein; RRLs, rabbit reticulocyte lysates; Stat3, signal transducer and activator of transcription 3; TBD, TRiC binding domain; TRiC, tailless complex protein-1 (TCP-1) ring complex Signal transducer and activator of transcription 3 (Stat3) is a member of a family of seven closely related proteins responsible for the transmission of peptide hormone signals from the extracellular surface of cells to the nucleus [1,2]. Mice deficient in Stat3 died during early embryogenesis, indicating its critical role in development [3]. Conditional gene targeting of Stat3 in several types of tissues revealed important roles for Stat3 in key biological functions including cell survival and growth, immunity, and inflammation [3,4]. These findings in mice have been recapitulated, in part, in patients with autosomal-dominant hyper-IgE syndrome (AD-HIES) or Jobs syndrome, a rare immunodeficiency syndrome, which results from loss-of-function mutations in the Stat3 gene [5]. At the opposite end of the spectrum, gain-offunction mutations or increased wild-type Stat3 activity has been implicated in the pathogenesis of up to 50% of hematological and solid tumors [68]. Despite its importance to normal physiology and pathophysiology, however, little is known about how Stat3 achieves its native state within the cell, information that potentially could be exploited to develop novel therapies for AD-HIES and/ or cancer. For the majority of eukaryotic proteins, folding to the native and functional conformation requires the assistance of elaborate cellular machinery composed of proteins known as molecular chaperones [9,10]. The chaperonins comprise a class of oligomeric, double-ring, high molecular weight, ATP-dependent chaperones with the unique ability to fold certain proteins that cannot be folded by simpler chaperone systems [11,12]. The primary cytosolic eukaryotic chaperonin is the tailless complex protein-1 (TCP-1) ring complex (commonly abbreviated TRiC, and also known as complex containing TCP-1 or CCT). TRiC is a large complex (1 MDa) composed of eight homologous but distinct subunits (CCT 18), arranged in two stacked octameric rings to form two interior chambers in which substrate proteins can be encapsulated and folded. TRiC is essential for the de novo folding of approximately 10% of newly synthesized proteins in the eukaryotic cell and for refolding proteins that become denatured following stress [13]. These substrate proteins extend up to 120 kDa in size, and most appear to contain regions with b-strands [14]. Well-characterized clients of TRiC include the cytosolic proteins actin, tubulin, and the tumor-suppressor von Hippel-Lindau protein (pVHL). In contrast, details regarding the contribution of TRiC to the folding and function of transcription factors are limited. Here we demonstrate that TRiC binds the oncogenic transcription factor, Stat3, and contributes to its biosynthesis, refolding, and activity in vitro and within cells. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 Stat3 is a multidomain transcription factor that contributes to many cellular functions by transmitting signals for over 40 peptide hormones from the cell surface to the nucleus. Understanding how multidomain proteins achieve their fully folded and functional state is of substantial biological interest. As Stat3 signaling is up-regulated in many pathological conditions, including cancer and inflammatory diseases, insight into what controls its folding may be useful for the identification of vulnerabilities that can be therapeutically exploited. We demonstrate that the major protein-folding machine or chaperonin within eukaryotic cells, TRiC/CCT, is required for Stat3 to fold during its synthesis and for Stat3 to be fully functional within the cell. We also find that TRiC can refold chemically denatured Stat3 and provide evidence that the CCT3 subunit of TRiC binds to the DNA-binding domain of Stat3. We also show that Stat3 activity is decreased by down-modulating levels of TRiC and can be increased by increasing Stat3s interaction with TRiC. TRiC therefore regulates both Stat3 protein levels and its function, making Stat3 modulation by manipulation of its interaction with TRiC a potential approach for the treatment of cancer and inflammatory disease (...truncated)