STAT6 is a central mediator of IL‐4‐induced gene responses. STAT6‐mediated transcription is depend ent on the C‐terminal transcription activation domain (TAD), but the mechanisms by which STAT6 activates transcription are poorly understood. Here, we have identified the staphylococcal nuclease (SN)‐like domain and tudor domain containing protein p100 as a STAT6 TAD interacting protein. p100 was originally characterized as a transcriptional coactivator for Epstein–Barr virus nuclear antigen 2. STAT6 interacted with p100 in vitro and in vivo. The interaction was mediated by the TAD domain of STAT6 and the SN‐like domain of p100. p100 did not affect the immediate activation events of STAT6, but enhanced STAT6‐mediated transcriptional activation and the IL‐4‐induced Igϵ gene transcription in human B‐cell line. Finally, p100 associated with the large subunit of RNA polymerase II and was mediating interaction between STAT6 and RNA polymerase II. These findings identify p100 as a novel coactivator for STAT6 and suggest that p100 functions as a bridging factor between STAT6 and the basal transcription machinery.

IntroductionRegulation of signal transducer and activator of transcription 1 (STAT1) involves posttranslational modifications such as phosphorylation of Tyr701 and Ser727 as well as arginine methylation. 1,2 STAT1 signaling is negatively regulated by protein tyrosine phosphatases (PTPs), suppressors of cytokine signaling (SOCS), and protein inhibitor of activated STAT1 (PIAS) proteins. 1-3 The family of PIAS proteins consists of 5 members that have been implicated in the regulation of several nuclear proteins. PIAS1 and PIAS3 were identified as interaction partners for STAT1 and STAT3, respectively, and they were found to inhibit the DNA-binding activity of activated STATs. [3][4][5] The other members, PIASx␣/ARIP3 (androgen receptor-interacting protein 3), PIASx␤/ Miz1, and PIASy, function as transcriptional coregulators for steroid hormone receptors. [6][7][8] Recently, several PIAS proteins have been shown to function as E3-type small ubiquitin-like modifier (SUMO) ligases. 9-12 SUMO-1, -2, and -3 are small modifier proteins that are covalently conjugated to specific lysine residues of target proteins. 13 A number of nuclear proteins, such as transcription factors AR, p53, and c-Jun, become modified by SUMO-1 conjugation, and this reversible posttranslational modification has been implicated in regulation of proteinprotein interactions, protein stability, localization, or activity. 9,13,14 However, it is currently unknown whether STAT factors are modified through sumoylation. Study design ReagentsAntibodies used include anti-SUMO-1 (mouse anti-GMP-1) (Zymed, San Francisco, CA); anti-HA (clone 16B12) (Berkeley-Antibody, Richmond, CA); anti-Flag (anti-Flag M2) (Sigma Aldrich, St Louis, MO); anti-STAT1 (N terminus) (Transduction Laboratories, BD Biosciences); anti-STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA) biotinylated antimouse (Dako, Glustrup, Denmark) and streptavidin-biotin horseradish peroxidase conjugate (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Human interferon-␥ (huIFN-␥) was purchased from Immugenex, Los Angeles, CA. Plasmid constructsSUMO-1-Flag and SUMO-1-Flag-Flag were a kind gift from Dr H. Yasuda. 15 The SUMO-1, Flag-PIAS1, Flag-PIAS1mut (PIAS1⌬310-407), Flag-PIAS3, Flag-ARIP3 Flag-ARIP3mut (ARIP3⌬347-418) plasmids 4,9 as well as STAT1-WT-HA and GAS-luc reporter construct have been previously described. 16 The STAT1 Lys703Arg mutation was created from STAT1-WT-HA using direct polymerase chain reaction (PCR) mutagenesis with the following primers: 5ЈGGAACTGGATATATCAGGACTGAGTTGATTTCTGTGTCTG-3Ј and 5Ј-CAGACACAGAAATCAACTCAGTCCTGATATATCCAGTTCC-3Ј. Transfections, luciferase assay, and immunodetectionCOS-7 cells were electroporated using a Bio-Rad (Hercules, CA) gene pulser at 260 V and 960 microfarads (F) and lysed in Triton X lysis buffer supplemented with 5 mM N-ethylmaleimide (NEM) (Sigma Aldrich). HeLa cells were transfected using a calcium phosphate method. For luciferase assay, U3A cells were transfected using a calcium phosphate method as described. 16 Immunodetection wa...

Antibodies that block cytokine function provide a powerful therapeutic tool especially for the treatment of autoimmune diseases. Cytokines are a group of small hydrophilic glycoproteins that bind their receptors on the cell surface and subsequently activate intracellular signalling cascades, such as the JAK/STAT pathway. A bulk of evidence has demonstrated that genetic mutations in signalling molecules can cause immunodeficiencies and malignant cell growth. As a result, several drug companies have begun to develop therapeutics that inhibit the function of JAK tyrosine kinases. Currently, two JAK inhibitors, tofacitinib and ruxolitinib, are used in the clinic for treating rheumatoid arthritis and myeloproliferative diseases, respectively. Inhibiting JAK function has been shown to efficiently prevent the uncontrolled growth of cancerous cells and to harness overly active immune cells. In the future, other small molecule compounds are likely to come into clinical use, and intense work is ongoing to develop inhibitors that specifically target the constitutively active mutant JAKs. This MiniReview will summarize the basic features of the JAK/STAT pathway, its role in human disease and the therapeutic potential of JAK/STAT inhibitors. The JAK/STAT PathwayCytokines are a large family of secreted proteins that play important roles in the regulation of cell growth and differentiation as well as in all aspects of the immune response. A major subgroup of cytokines exert their effects via binding to and activating a family of conserved receptors, type I and II cytokine receptors. This subfamily of cytokines consists of over 50 members in mammals and includes interferons, colonystimulating factors and many interleukins (reviewed in: [1,2]). Some hormones such as prolactin, growth hormone and erythropoietin also signal via type I cytokine receptors. The signal is transduced through a common pathway, the JAK/STAT pathway, discovered roughly 20 years ago ( fig. 1) (reviewed in [3]). JAKs or Janus kinases are a family of four tyrosine kinases (JAK1, JAK2, JAK3 and TYK2) that selectively associate with cytokine receptor chains and mediate signalling by phosphorylating tyrosine residues on various proteins in the pathway, including themselves and the receptor chains, and STAT (signal transducer and activator of transcription) transcription factors [4]. STATs bind to the phosphorylated tyrosine residues on the receptor, and after phosphorylation by JAKs, STATs dimerize and translocate to the nucleus, where they bind to DNA and can either activate or repress transcription [5]. There are seven members of the STAT family in mammals: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. Although they can be activated by partially overlapping sets of cytokines, different STAT molecules have non-redundant biological roles [6,7]. For example, STAT1 and STAT2 mediate interferon signalling, whereas STAT4 functions in IL-12 signalling and is thus instrumental for the differentiation of T helper (Th) 1 cells. STAT6 is responsible f...