1. IFNs,Cytokines & the JAK- STAT Signaling Cascade March 10, 2005 Chris Schindler

2. Innate Viral Interfering Activity is Discovered in Cultured Cells (1950s) X 1960s Initial and intermittent character- ization of IFN activity. 1970s Purification of “Immune”, “Leukocyte” & “Fibroblast” IFNs yields >10 type I IFNs (  &  ) and one type II IFN (IFN-  ). 1980s Cloned IFNs, the first of many disappointments for biotech industry, is a boon to molecular biologists.

3. An Evolving IFN-  Signaling Paradigm IRF-9 antiviral* antiproliferative* chemokine* PKR OAS IP10 iNOS Stat2 Stat1 P P P P P P IFN-  PTK-x

4. Interferon Stimulated Response Elements (ISREs) ISG54TAGTTTCACTTTCCC ISG15CAGTTTCGGTTTCCC GBPTACTTTCAGTTTCAT IP10AGGTTTCACTTTCCA OASTGGTTTCGTTTCCTC HLA-ACAGTTTCTTTTCTCC MXGAGTTTCGTTTCTGA

5. Identification of “High IFN Producing Cells” (HIPCs) in vivo A small subset of WBCs produce most of the circulating IFN-Is

6. IRF-3 The IFN-I Autocrine Loop and Toll Like Receptors (TLRs) TLR3 TLR4 IKK  /  /  dsRNA LPS NF  B ? IRF-7 IFN-  ’s danger TLR7 TLR9 CpG ssDNA IKK  /  /  IRF-8 ? IRF-7 IFN-  ’s danger I FNAR IFN-  St1/St2 P IRF-7

7. Stat1 inflammation* macrophage act.* chemokines* IRF1 MIG GBP iNOS CIITA IFN Type II (a.k.a. IFN-  ) Signaling Paradigm Stat1 IFN-  P P P P IFN  R Jak1 Jak2 Y

8. GeneSpeciesElement GBP Fc  RI ICSBP ICAM-1 c-fos IFP53 IRF-1 Ly6E mig human mouse ATT AC T CT AAA TTT CC C AG AAA TTT CT C GG AAA TTT CC C GG AAA GTT CC C GT CAA ATT CT C AG AAA TTT CC C CG AAA ATT CC T GT AAG CTT AC T AT AAA GAS Binding Elements

9. Tyk2: Member of the Unusual Janus Kinase Family 900 700600 500 400300 100 200 010001100 800 1200 JH1/TK JH2/  K JH3JH4JH5JH6JH7 Tyk2 Jak1 Jak2 Jak3 Expressed in all tissues Expressed in hemato- poietic tissues

10. STATs & Four Helix Bundle Cytokines Stat1 IFN-I ( , , , ) & IFN-  Stat2 IFN-I ( , , , ) Stat3 IL-10 and IL-6 families Stat4 IL-12, IL-23, IL-27 Stat5a/b Single Chain, IL-2 & IL-3 families Stat6 IL-4, IL-13

11. 8007006005004003001002000 TAD Coil-CoilDNASH2 NH 2 YLK Conserved STAT Domains

12. STAT Evolution STAT homologues have been identified in most model metazoans, including Dictyostelium, C. elegans, Drosophila, Anopheles, Xenopus and zebrafish. STAT homologues in C. elegans are very distantly related. The three STAT Dictyostelium homologues are structurally and functionally quite divergent. The Dictyostelium STATs, most closely related to Stat3 & Stat5 may encode the primordial SH2 domain. Homologues of a STAT (37 % identity to Stat5), JAK (27 % identity to Jak2), cytokine and cytokine receptor have each been identified in Drosophila. This pathway play several important developmental roles. Zebra fish encodes a Stat1 and Stat3 homologue. The Stat3 homologue is developmentally expressed and syntenic with both murine and human Stat3. The Stat1 homologue is more distantly related and expressed later in life. Sequence analysis suggests that mammalian STATs evolved from a single syntenic Stat3-Stat5 (murine chromosome 17) tandem cluster that was duplicated to form the Stat1-Stat4 (murine chromosome 2) and Stat2-Stat6 (murine chromosome 12) tandem clusters. This was followed by the duplication of Stat5 into Stat5a and Stat5b.

13. Stat1 Transduces critical signals for all three classes of IFNs. Stat1 is activated by several other ligands, but its role appears to be more secondary. There are two isoforms - Stat1 , which is carboxy terminally truncated before the TAD, is assumed to function as an antagonist of Stat1 . Generic biological activities include: –Promoting the expression of inflammatory or “danger” signals. –Antagonizing proliferation and/or promoting apoptosis. –Stat3 appears to antagonize both of these activities. Stat1 is tyrosine phosphorylated on Y 701 and serine phosphorylated on S 727 (in the TAD). This latter phosphorylation is required for maximal transcription of some genes. Stat1 has also been reported to be acetylated at K 785 and SUMOylated at K 703. Stat1 activity is antagonized by phosphatases directed at the receptor and JAKs, and directly by one or more nuclear phosphatases. Suppressor of Cytokine Signaling 1 (SOCS-1), a Stat1 target gene, directs an important negative feed back loop to suppress IFN  -Stat1 dependent signals.

14. Type I IFN Signaling P P Stat2 IRF-9 Stat1 P P P P P P P P P P Jak1 Tyk2 Y Y Y

15. Murine Human 76 % HomologyDivergent TAD TAD Coil-CoilDNASH2 Coil-CoilDNASH2 923 aa 851 aa Y 689 Y 690 Stat2 Transduces critical signals for type I IFNs and -IFNs. Murine and Human Stat2 are functionally analogous but uncharacteristically divergent in sequence, especially in the TAD. Human Stat2 is tyrosine phosphorylated on Y 690 and murine Stat2 on Y 689. Other modifications have not been reported, although it is likely to be serine phosphorylated. Stat2 is the only STAT that does not appear to homodimerize and bind DNA directly. Stat2 activity is antagonized by phosphatases directed at the receptor and JAKs, and directly by one or more nuclear phosphatases. In contrast to Stat1 and Stat3, the distribution of Stat2 is unstimulated cells is almost exclusively cytoplasmic.

16. Stat3 Transduces signals for type I IFNs,  -IFNs, the critical signals for the extended IL-10 family and the extended IL-6 families. There are two isoforms, Stat3  & Stat3 . Like Stat1 , Stat3  is carboxy terminally truncated before the TAD, but has been shown to direct the expression of a unique set of genes through association with other transcription factors. Generic biological activities include: –Promoting cell growth associated with cell transformation. –Antagonizing the expression of inflammatory or “danger” signals. –Stat1 antagonizes both of these activities (a ying-yang relationship). Stat3 is the only STAT that is essential for development (i.e., the knockout is embryonic lethal). Tissues specific knockouts reveal an important roles in protecting tissues form inflammation and suppressing transformation. Stat3 is tyrosine phosphorylated at Y 705, serine phosphorylated at S 727 and acetylated at K 785. Stat3 activity is antagonized by phosphatases directed at the receptor and JAKs, and directly by one or more nuclear phosphatases. In response to stimulation by the IL-6 family of ligands, Stat3 activity is directly suppressed by SOCS-3, a Stat3 target gene.

17. Stat3 Gene Expression IL-6 Signaling IL-6R  IL-6 Jak1 Jak2 P P P P P gp130 Nucleus Cytoplasm Endosome Cytoplasm Endocytosis Y Y Y P SHP-2 MAPK ERK Jak2 Jak1 Jak2 Jak1

18. Stat5a and Stat5b Transduce critical signals for all members of the IL-2, IL-3 and single chain cytokine receptor families, i.e. is Stat5 is functionally pleiotropic like its ancestral partner Stat3. Consistent with their significant level of homology, Stat5a & Stat5b can functionally overlap, especially in hematopoietic tissues (e.g., in response to IL-2 and IL-3 family ligands). However, gene targeting studies have underscored a specificity for Stat5a in prolactin response and a specificity for Stat5b in GH response. Stat5 has been implicated in the development of some hematopoietic tumors and the regulation of some stem cell populations. SOCS-2 specifically antagonizes Stat5b activation in response to GH (i.e., SOCS-2 knockout mice exhibit gigantism).

19. Stat4 & Stat6 Stat4 and Stat6 exhibit a the most ligand specificity. –Stat4 transduces signals for members of the IL-12 family. (IFN-Is can also promote a partial activation of Stat4). –Stat6 transduces signals for closely related IL-4 and IL-13. Stat4, Stat6 and their activating ligands play an essential role in the development of effector CD4 + T-helper cells. –Stat4 directs the development of Th1 cells. –Stat6 directs the development of Th2 cells. Full Stat4 activation requires both tyrosine phosphorylation at Y 689 and serine phosphorylation at S 721. The ability of IL-18 to direct Stat4 serine phosphorylation accounts for its ability to synergize with some Stat4 activating ligands (e.g., IFN-Is) to direct the activation to Th1 cells.

20. Cytokines and the Th1-Th2 Paradigm IL-2 IFN-  TNF Th 0 Stat6 Th 2 IL-5 IL-4 IL-6 IL-10 TGF-  iDC DC Ag IFN-  Flt-3L GM-CSF IL-12 Stat4 Th 1 IFN- 

21. The Four (  -Helix Bundle Family of Cytokines -There are ~50 members; a.k.a - hematopoietins -They play important roles in development/activation of lymphocytes & myelocytes. - They all transduce pivotal signals through JAKs and STATs -They can be divided into two families based on the receptors they bind: Cytokines that bind type I cytokine receptors: The  C Family - IL-2 family includes IL-2, IL-7, TSLP, IL-9, IL-15, IL-21 IL-4 family includes IL-4 and IL-13 The IL-3 family includes IL-3, IL-5 and GM-CSF The Extended gp130 Family- IL6-gp130 family includes IL-6, IL-11, CNTF, LIF, OSM, CT-1, NP, NNT1/BSF3 gp130-like family includes OSM, IL-31, G-CSF, Leptin IL-12 family includes IL-12, IL-23, IL-27, IL-30 The single chain family includes Epo, GH, PRL, Tpo Cytokines that bind type II cytokine receptors: The human Type I IFN Family includes 12  -IFNs, IFN- , IFN- , limitin Type II IFN Family includes only IFN-  -IFN family includes IL-28a, IL-28b, IL-29 IL-10 family includes IL-10, IL-19, IL-20, IL-22, IL-24, IL-26 (Stat5) (Stat6) (Stat5) (Stat3) (Stat4*) (Stat3) (Stat5) (Stat1-3) (Stat1) (Stat1-3) (Stat3)