Not just another meeting: the coming of age of JAKs and STATs

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Not just another meeting: the coming of age of JAKs and STATs Elbert H. Chen, Massimo Gadina, Jérôme Galon, Min Chen and John J. O’Shea The discovery of JAKs and STATs provided long-awaited clues to the mechanism of cytokine signal transduction. A recent meeting*

T

he first Janus protein tyrosine kinases or JAKs were cloned about eight years ago. Because their function was not readily apparent, a circulating joke was that the acronym JAK actually stood for Ôjust another kinaseÕ. That pejorative perception vanished with the discovery that the JAK Tyk2 is essential for interferon (IFN) signaling (reviewed in Ref. 1). By contrast, since the STATs (signal transducers and activators of transcription) were isolated as DNAbinding proteins involved in the regulation of IFN-inducible genes, their importance was recognized immediately (reviewed in Refs 1Ð3). Following the identification of these two key players, many laboratories have contributed to our understanding of the role of JAKs and STATs in cytokine signaling (Fig. 1; reviewed in Ref. 4). This first international meeting on these signaling molecules marks the coming of age of JAKs and STATs. Among the highlights were new information on JAKand STAT-knockout mice, the role of JAKs in the pathogenesis of cancer, and the emerging families of JAK and STAT inhibitors. Because of space limitations, only immunological topics will be discussed here.

Structure and function of JAKs and STATs In sharp contrast to the great strides that have been made in deciphering the various cytokine signal transduction pathways, surprisingly little is known about the structure and function of either JAKs or STATs. JAKs are unique among protein tyrosine kinases in having a C-terminal catalytic domain (JH1) juxtaposed with a kinase-like or pseudokinase domain (JH2) of previously unknown function. J. OÕShea (Bethesda, MD) reported that a subset of patients with

reviewed the current state of JAK/STAT biology and discussed new advances that offer novel insights into cytokine signaling.

JAK3 severe combined immunodeficiency have mutations in the JH2 domain. In vitro experiments suggest that the pseudokinase domain has a complex regulatory function; it is required for full catalytic activity but appears also to inhibit kinase activity. Two groups presented data demonstrating the importance of the N-terminal JH7ÐJH6 domains of Tyk2 and JAK3 in receptor binding (S. Pellegrini, Paris; OÕShea). Although the complete structure of a STAT is not yet known, the structure of the N-terminal proteinÐprotein interaction domain has now been revealed5. The socalled ÔN-domainÕ mediates cooperative STAT binding to adjacent DNA recognition sites as well as interaction with other transactivators. The crystal structure of the STAT4 N-domain described by J. Darnell (New York, NY) comprises eight a-helices that form a hook-like structure. His group also identified a conserved tryptophan residue (Trp37) that is crucial to oligomerization and transcriptional activation. Another theme that was discussed is the existence and function of naturally occurring STAT isoforms; variants of STAT4 and STAT6 were presented by T. Hoey (San Francisco, CA) and W. LaRochelle (Bethesda, MD), respectively. In addition, C. Schindler (New York, NY) reported the existence of STAT5 isoforms generated through proteolysis by an unknown, developmentally regulated protease. These STAT isoforms appear to act as negative regulators of signaling, but it is clear that much

Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0167-5699/98/$19.00

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has yet to be learned about the regulation of their expression and their physiological function. T. Decker (Vienna) and D. Cantrell (London) presented provocative data on STAT serine phosphorylation. Although the identity of the serine kinase that phosphorylates STATs remains a mystery, serine phosphorylation is likely to be a point at which multiple signaling pathways converge. On a related note, B. Groner (Freiburg) discussed the interaction of STATs with other transcription factors, including molecules such as CBP/p300, p53 and the glucocorticoid receptor. Interaction with these other factors can regulate transcription either positively or negatively.

Biology of JAKs JAK3 remains the only JAK or STAT for which mutations in humans have been identified (Table 1; reviewed in Ref. 4). JAK3-deficient humans and JAK3-knockout mice demonstrate the essential role of JAK3 in the development and function of the immune system6. L. Berg (Worcester, MA) reported that T cells from JAK32/2 mice fail to produce interleukin 2 (IL-2) in response to T-cell receptor (TCR) crosslinking or phorbol 12-myristate 13-acetate/ionomycin. However, since JAK32/2 T cells are already activated in vivo, it is conceivable that they might not respond to further activation in vitro. To resolve this issue, BergÕs group generated TCR-transgenic/JAK32/2 mice. Although the TCR-transgenic/ JAK32/2 T cells are not activated, nevertheless, they are defective in IL-2 production, confirming that the inability of T cells from JAK32/2 mice to respond to TCRmediated signals is an intrinsic defect. Berg went on to suggest that one explanation might be that JAK3 is essential for PII: S0167-5699(98)01295-X

*The Keystone Symposium on Signal Transduction by JAKs and STATs was held at Tamarron, CO, USA, on 3Ð8 February 1998.

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Cytokine

granulocyteÐmacrophage CSF (GM-CSF), IL-5 and monocyteÐmacrophage CSF signaling is normal in these mice, whereas signaling by cytokines whose receptors use the gp130 subunit is not. Thus, JAK1 appears to be essential for signaling by gp130-containing cytokine receptors, despite the fact that gp130 can associate with other JAKs. Cells from JAK12/2 mice also fail to respond to any cytokines that bind to class II cytokine receptors (IFN-a/b, IFN-g, IL-10). JAK12/2 mice have small thymi with reduced numbers of thymocytes and their B-cell development is arrested at the early pro-B stage, indicating that, similar to JAK3, JAK1 has an essential function in lymphocyte development. JAK2 deficiency is lethal due to defective erythropoiesis (J. Ihle, Memphis, TN). JAK2 is not essential for signaling by GCSF, IFN-a/b, and IL-6, whereas erythropoietin, IL-3 and thrombopoietin signaling was abrogated in JAK22/2 cells. In addition, when JAK32/2 mice were reconstituted with cells from JAK22/2 mice, T- and B-cell development and lymphocyte proliferation were normal, confirming that JAK2 is not essential for lymphoid function. Tyk2-knockout mice have not yet been produced but this effort is under way (M. Muller, Vienna). Several groups described TelÐJAK fusion proteins found in human leukemias (G. Gilliland, Boston, MA; D. Barber, Toronto). Chromosomal translocations found in acute lymphocytic and chronic myelogenous leukemia result in the fusion of the 3' part of JAK2 (which includes the JH1 catalytic domain) to the 5' dimerization region of Tel (an Ets family transcription factor), resulting in a constitutively active JAK (Refs 7, 8). BarberÕs group has also found putative TelÐJAK3 and TelÐTyk2 fusions.

Receptor

P

Y

Y

P

JAK

JAK

STAT

SOCS/JAB/SSI

PIAS

P Y

P Y

STAT

P Y

Y

P

STAT

SOCS/JAB/SSI Other cytokineinducible genes

Fig. 1. Mechanism of JAKÐSTAT signal transduction. JAKs bind to cytokine receptor subunits belonging to the class I and class II cytokine receptor families. Ligand binding induces dimerization of receptor subunits and JAK activation resulting in receptor phosphorylation. Phosphotyrosines on the cytokine receptors are recognized by the SH2 domains of the STATs. The recruited STATs are themselves phosphorylated and dimerize through reciprocal SH2Ðphosphotyrosine interactions. Activated STATs translocate to the nucleus where they bind DNA and modulate gene expression. The SOCS/JAB/SSI proteins are induced by cytokine stimulation; some family members bind to JAKs and block kinase activity. Thus, SOCS/JAB/SSI regulation of cytokine signaling appears to be an example of negative-feedback regulation. PIAS proteins bind STATs and inhibit cytokine signaling by an unknown mechanism. How these inhibitors are regulated is less-well understood. Abbreviations: JAB, JAK-binding protein; JAK, Janus protein tyrosine kinase; PIAS, protein inhibitor of activated STAT; SH2, src homology 2 domain; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; SSI, STAT-induced STAT inhibitor.

maintaining levels of key lymphocyte transcription factors. JAK12/2 mice are largely normal in terms of organogenesis and myeloid, erythroid and megakaryocytic development (R.

Biology of STATs

Schreiber, St Louis, MO). However, they have reduced birth weight, are unable to nurse, and die shortly after birth; the etiology of this remains unclear. Granulocyte colony-stimulating factor (G-CSF),

All the STATs have now been targeted for disruption in mice, and some revealing double-knockouts have been generated (Table 1). STAT2 was the final STAT to be knocked out (Schindler). Not only were no viable knockout mice obtained, there was a total absence of homozygous null embryos.

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Table 1. Phenotypes of mice deficient in various JAKs and STATs Knockout

Phenotype

JAK knockouts JAK1a

Perinatal lethality

JAK2a

Embryonic lethality, defective hematopoiesis

JAK3

Severe combined immunodeficiency

Tyk2

Not yet reported

STAT knockouts STAT1

Defective interferon signaling

STAT2a

No embryos obtained

STAT3

Embryonic lethality

STAT4

Defective Th1 differentiation

STAT5a

Impaired mammary gland development

STAT5b

Defective sexually dimorphic growth

STAT6

Defective Th2 differentiation

STAT4–STAT6a

Default Th1-like development

STAT5a–STAT5ba

Female sterility, smaller size, splenomegaly, early death

Abbreviation: Th1, T helper 1. a Reported at the symposium.

Furthermore, among the surviving mice, STAT21/2 mice outnumber STAT21/1 mice at a far greater ratio than 2:1. These highly unusual findings are surprising given that only IFN-a/b has been shown to activate STAT2. Through reconstitution experiments with Rag1-deficient mice, it has been shown that STAT2 is not necessary for T- and B-cell development. D. Levy (New York, NY) reported some new findings on the immune response of STAT12/2 mice. It had previously been shown that these mice have defective IFN signaling and absent innate responses to viral or bacterial infection. They also have impaired induction of IFN-a production and poor IFN-g production, but produce relatively more IL-5 and IL-4 upon challenge. Accordingly, when infected with virus, the histological features of the infections are atypical and correspond more to a T helper 2 (Th2)-type response (i.e. eosinophilic infiltration). In addition, the mice make more IgE and less IgG2a in response

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to infection. Data were also presented concerning enhanced survival and proliferation of STAT12/2 lymphocytes, supporting the recent report that STAT1 promotes apoptosis by regulating caspase expression9. A fascinating observation, made by both Schreiber and Levy, is that STAT12/2 mice have a higher incidence of tumors. A similar phenomenon was noted in IFN-g2/2 mice, indicating that IFN-g responsiveness is required for the development of host antitumor responses (Schreiber). Furthermore, SchreiberÕs group has discovered human tumors that are insensitive to IFN-g. This suggests that the inability to respond to IFN-g provides a selective advantage to the tumor that permits it to evade tumor immune surveillance and, thus, enhances its tumorigenicity. The importance of STATs in regulating the balance of Th1 versus Th2 differentiation was addressed by M. Grusby (Boston, MA) (Table 1). It appears that many, but not all, IL-4 responses are blocked in STAT62/2

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mice; IL-4 mediated induction of c-myc and protection from apoptosis appear to be STAT6-independent. New studies show that STAT6-deficient mice are protected in a model of asthma whereas STAT42/2 mice are resistant to experimentally induced arthritis. Finally, examination of STAT4Ð STAT6 double-knockout mice revealed that they produce increased levels of IFN-g, comparable with those of STAT62/2 mice. Consequently, while STAT6 is essential for Th2 development, there is default Th1-like development, even in the absence of STAT4. IhleÕs group has now succeeded in producing STAT5aÐSTAT5b double-knockout mice. This causes high, but not uniform, perinatal lethality; the mice that survive have female sterility, growth impairment and splenomegaly, and die prematurely. Importantly, T cells from STAT5aÐSTAT5bknockout mice have an activated phenotype and are capable of producing cytokines in response to TCR engagement; this phenotype is not seen in the individual knockouts. Moreover, these mice have a profound deficiency in peripheral T-cell proliferation; however, the basis of this defect has not been well characterized yet.

Inhibitors of cytokine signaling One of the most exciting developments in the JAK/STAT field has been the recent discovery of two different classes of negative regulators of cytokine signaling. The first family of proteins includes members with various names: suppressor of cytokine signaling (SOCS), JAK-binding protein (JAB), STAT-induced STAT inhibitor (SSI), and cytokine-inducible Src homology 2 domain (SH2)-containing protein (CIS) (reviewed in Ref. 10). There are currently eight members, which share common structural elements: a variable N-terminal region, a central SH2 domain, and a conserved C-terminal motif termed the ÔSOCS boxÕ (D. Hilton, Victoria). While the precise patterns of expression in response to various cytokine stimuli remain to be worked out, it is already clear that the various family members are regulated differently and presumably have nonoverlapping functions (A. Yoshimura, Kurume; M. Narazaki, Osaka). These proteins are able to bind to JAKs and

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inhibit kinase activity, although not all of the structural requirements have been resolved. Moreover, it is not clear that all family members function similarly. Interestingly, as these proteins are induced by cytokines and function to inhibit cytokine signaling, this strongly suggests a model of classical negative-feedback regulation (Fig. 1). Although these molecules were only recently identified, two knockout mice have been generated already and the generation of others is in progress. CIS-12/2 mice have no discernible defects thus far (Ihle), but SOCS-1/JAB/SSI-1-knockout mice are growth retarded, have small thymi, livers and spleens, and die within three weeks of birth (Narazaki). The second family of proteins was identified through the use of the yeast twohybrid assay. Protein inhibitor of activated STAT1 (PIAS1) was isolated by specific interaction with STAT1; PIAS3 was identified by sequence homology and was subsequently found to associate with STAT3 (Ref. 11). K. Shuai (Los Angeles, CA) reported that upon IL-6 stimulation, PIAS3 interacts specifically with STAT3 and not STAT1, whereas PIAS1 binds STAT1 but not STAT3. Furthermore, the PIAS proteins inhibit DNA binding and transcriptional activation by their respective STAT partners. The PIAS proteins do not appear to exert their function by blocking STAT tyrosine phosphorylation or by affecting the protein stability of STATs (Fig. 1). Shuai indicated that other PIAS molecules do exist and hinted that they may have specificity for the other STATs. The precise role of these proteins and

the means by which they inhibit signaling remain to be elucidated.

Conclusions Perhaps the most exciting aspect of working in a relatively young field like JAKÐSTAT signal transduction is that every illuminating answer prompts many, more-provocative questions. Cytokine-inducible genes are still relatively poorly characterized. Little is known about the mechanism of STAT dephosphorylation, and the identity of the STAT serine kinase(s) still eludes us. The role of ubiquitination and the DUB family of deubiquitinating enzymes, although fascinating, remains enigmatic (A. DÕAndrea, Boston, MA). The paucity of structural information on both JAKs and STATs needs to be rectified. It will also be fascinating to see how both JAKs and STATs are coupled to other key intracellular signaling pathways. Finally, understanding the biology and biochemistry of the recently described inhibitors will be of tremendous interest.

Note added in proof: subsequent to submission of this manuscript, the full characterization of JAK1-, JAK2- and STAT5a/STAT5b-knockout mice together with the crystal structure of STAT1 has been published12Ð16. We thank A. DÕAndrea and D. Levy for organizing the conference, and all the conferees who have allowed us to comment on unpublished results. We also apologize to those whose work we could not discuss because of space limitations and the immunological focus of this article. E.H.C. is

Howard Hughes Medical InstituteÐNational Institutes of Health Research Scholar.

Elbert Chen, Massimo Gadina, JŽr™me Galon, Min Chen and John OÕShea (osheajo@ arb.niams.nih.gov) are at the Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA. References 1 Darnell, J.E., Jr, Kerr, I.M. and Stark, G.R. (1994) Science 264, 1415Ð1421 2 Ihle, J.N. (1995) Nature 377, 591Ð594 3 Darnell, J.E., Jr (1997) Science 277, 1630Ð1635 4 OÕShea, J.J. (1997) Immunity 7, 1Ð11 5 Vinkemeier, U., Moarefi, I., Darnell, J.E., Jr and Kuriyan, J. (1998) Science 279, 1048Ð1052 6 Thomis, D.C. and Berg, L.J. (1997) Curr. Opin. Immunol. 9, 541Ð547 7 Peeters, P., Raynaud, S.D., Cools, J. et al. (1997) Blood 90, 2535Ð2540 8 Lacronique, V., Boureux, A., Valle, V.D. et al. (1997) Science 278, 1309Ð1312 9 Kumar, A., Commane, M., Flickinger, T.W. et al. (1997) Science 278, 1630Ð1632 10 Aman, M.J. and Leonard, W.J. (1997) Curr. Biol. 7, R784ÐR788 11 Chung, C.D., Liao, J., Liu, B. et al. (1997) Science 278, 1803Ð1805 12 Rodig, S.J., Meraz, M.A., White, J.M. et al. (1998) Cell 93, 373Ð383 13 Parganas, E., Wang, D., Stravopodid, D. et al. (1998) Cell 93, 385Ð395 14 Neubauer, H., Cumano, A., Muller, M., Wu, H., Huffstadt, U. and Pfeffer, K. (1998) Cell 93, 397Ð409 15 Chen, X., Vinkemeier, U., Zhao, Y., Jeruzalmi, D., Darnell, J.E., Jr and Kuriyan, J. (1998) Cell 93, 827Ð839 16 Teglund, S., McKay, C., Schuetz, E. et al. (1998) Cell 93, 841Ð850

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