JAKs and STATs branch out

JAKs and STATS branch out

The JAK-STAT signal-transduction pathway is utilized by a wide range of cytokines to regulategene expression.Cytokines activate members of the JAK family ofprotein tyrosine kinases, which in turn activate, by tyrosinephosphorylation, one or more STAT transcription factor family members.Activated STATS form dimers, translocate to the nucleus and bind to responseelements to induce transcription. Recent findings are beginning to connect JAKs and STATS with other signallingpathways: JAKs may phosphorylate and activate signallingproteins other than STATS, and STATS can be phosphorylated by non-JAK tyrosine kinases. STAT activity can also be modulated by serinephosphorylation.

lames Briscoe and Franz Kohlhuber are at the Imperial Cancer Research Fund Laboratories, PO Box 123, 44 Lincoln’s Inn Fields, London, UK WCZA 3PX; and Mathias Mijller is at the lnstitut ffir Tierzucht und Cenetik, VMU Wien, Link Bahngasse 11, Al 030, Vienna, Austria.

336

Cytokines and growth factors bind to receptors on the cell surface and activate signal-transduction pathways to elicit a number of responses, including changes in cell function, growth and differentiation. These effects are mediated substantially through altered gene activity in the nucleus. One such pathway is the ‘JAK-STAT’ pathway. STATS(signal transducers and activators of transcription) comprise a family of transcription factors (Box 1) that are activated, in cytokine receptor complexes at the cell membrane, by members of the JAK (Janus kinase) family of tyrosine kinases (Box 2). The JAK-STAT pathway was first identified in the study of interferon (IFN) signal transduction, but it is now clear that JAKs and STATS mediate responses to a wide variety of cytokines and growth factors (reviewed in Refs l-4). Recently, a JAK and STAT have also been identified in Drosophila: they are involved in cell proliferation and patterning (Box 3). The mechanisms of cytokine signal transduction and STAT activation have been reviewed recently3f4, and a general scheme is shown in Figure 1. In unstimulated cells, JAKs are associated with cytokine receptors and are inactive. Binding of a cytokine to its receptor results in the aggregation of receptor subunits juxtaposing the associated JAKs, which are then activated, presumably by transphosphorylation. The activated JAKs phosphorylate multiple tyrosine residues in the cytosolic tails of receptor subunits. In the simplest scenario, these phosphorylated tyrosines act as docking sites for the Src homology 2 (SH2) 0 1996 Elsevier Science Ltd PII: SO962.8924(96)10028-3

domains of STAT family members, thus recruiting STATSinto the receptor complex. The recruited STATS are activated, presumably by JAKs, by phosphorylation of a tyrosine residue just C-terminal of their SH2 domains. Phosphorylated STATSdissociate from the receptor complex and form homo- or heterodimers through reciprocal interactions between their SH2 domains and the phosphorylated tyrosine residue. The precise order of these events is not known. The dimers translocate to the nucleus where they bind, either alone or with additional proteins, to specific DNA elements in the promoters of genes to induce transcription. Each cytokine activates a subset of STATS, and, in many cases, a particular STAT is activated by more than one cytokine. The biological function of the activation of a particular STAT can be examined using knockout mice, and this has been done, for example, for STAT15j6.STAT1 is activated in response to a variety of cytokines, including IFNs. STATl-deficient mice display a complete lack of responsiveness to IFNs and, therefore, are highly sensitive to infection by viruses. This observation indicates a central role for STAT1 in responses to IFN. However, although STAT1 is activated by other cytokines, mice lacking STAT1 do not show any abnormalities in their response to these cytokines, suggesting that STAT1 activation is either redundant or aberrant in these situations. The specificity of STAT activation is achieved principally by the specific STAT-receptor interaction3. This is essentially determined by the SH2 domain of the STAT, which can discriminate between different receptor phosphotyrosines depending on the sequence surrounding the tyrosine. For example, IFN-y, which normally activates STATl, activates STAT2 if the SH2 domain of STAT2 is swapped for that of STAT1 (Ref. 7). Similarly, chimeric receptor constructs containing a ‘,,

;, 3,$jQE 1 - STATS:

SIGNAL

TRANSDUCERS

AND ‘~x.,j~~~‘~,~~~~,,,~~~~,

,,,,lil,,,,i;i,li,~~n!l-~;.~cslnVAToRS OF TRANSC~~ION ~;.,,;~&~P -2

” ”” ” ~‘;

The STATS..a.rea family of transcription factors’. There are seven STATgenes, encoding STATI-STAT4, STATSA, STAT56 and STAT6, ,,,proteins, ‘which vary in size from 734,#5;1 art@@ i.nijil! ~,~,~,‘ I” ,’acids. The complexity of th,ls, family is further i.n& ’dreasec&Mhe differential splicing and processing of some members. AllIthe STATS have a similar structure in terms of. conserved domains’sand motifs, as indicated in,#$Tg,ure~ (, ,\b., .,!;?M& ” li I),(p&fp,;g’ : , DNA-binding domain

Transactivation domain

N

TYr ‘,‘, j;jji: ’“I

-“~ ,’ : “1 ‘ll:il@ll’ll

Ser ‘::ir 3) ,“7

I p]#&, _ Differe,nt cytoktnes,a@@& different subset&of STATs.,Actil I $$ed ST&f home- o@eterodiqri bi,nd to DNA elements wiW.d”ldyg~i,!~~‘;nmetry. ,In the c&e c$#N-cx, an additional protein interacts tiit!#i;&hsSTAT dimer to alte~i~~p,specifici~,, of,&$$jbinding. STAT bind,ingo D&i usually correlates ,ql#i’” wit Induction of gene.,fr@scri@ort; the C-terminal domain is required for, or,inf!uen,ces, transcriptQ&Qvation. ,::’ /( ,,I: ,,, trends in CELL BIOLOGY (Vol. 6) September 1996

BOX 2 -JAR FAMILY TYROSINE KtNASES

tyrosine residue within a consensus sequence sufficient to recruit STAT1 or STAT3 are able to direct the activation of STAT1 or STAT3rr8. However, in some cases,although the SHZphosphotyrosine interaction is necessary, it is not sufficient to direct activation’. A second layer of specificity is determined by the ability of different STATSto bind to distinct DNA response elementsg. Some response elements are able to bind more than one type of STAT dimer and respond to a number of cytokines. Other response elements bind only one type of STAT dimer and are induced only by those cytokines that activate that particular STAT. The region of STATSthat is required for DNA binding has been localized to the centre of the STAT proteins4. Swapping this domain between STATSalters their ability to bind to specific DNA sequences and, presumably, the genes that the STATS induce, although this has yet to be demonstrated. The JAI&STAT pathway does not function in isolation, and recent findings have begun to connect JAKs and STATSinto the network of signalling pathways present in cells (see Fig. 2). In particular, it has emerged that JAKs activate additional signalling pathways, STATScan be activated by other tyrosine kinases, and serine phosphorylation can modulate STAT activity. JAKs activate

additional

pathways

Cytokine receptors normally activate a number of signalling pathways by recruiting and activating signalling molecules2. The mitogen-activated protein (MAP) kinase pathway, for example, is thought to be activated by many cytokines through the recruitment and phosphorylation of SHC. A number of studies indicate that JAKs are required in the activation of the MAP kinase pathwayiOzn. What is the role of JAKs in the activation of this and other pathways? Cytokine receptor mutants that can no longer bind JAKs are not tyrosine phosphorylated in response to their ligands and are defective in the activation of signal-transduction pathways (e.g. see Ref. 12). Furthermore, in cells lacking individual JAK family members, phosphorylation of particular cytokine receptors is abrogated. Cells lacking JAKl, for example, are defective in the phosphorylation of the interleukin 6 (IL-6) and IFN-y receptor13. These data suggest that the JAKs are involved in the phosphorylation of receptor tyrosines, providing docking sites for signalling molecules. In fact, this may be the case for the recruitment of SHC to some receptors of haematopoietic growth factorslO. Presumably, the JAKs are also required for the phosphorylation of these signalling molecules. JAKs also appear to recruit and activate signalling components directly. For example, an erythropoietin receptor lacking all the tyrosines of its cytosolic domain, but retaining its ability to activate JAK2, supports erythropoietin-induced SHC phosphorylation14. Indeed, dimerization of a chimeric protein containing the extracellular domain of CD16 fused to JAK2 results in the tyrosine phosphorylation of a number of proteins, including SHC, and MAP kinase and STAT activation15. These data imply a direct association between JAKs and signalling proteins, and a trends in CELL BIOLOGY (Vol. 6) September 1996

Pseudokinase

Kinase

N

C JH7

JH6

JH5 JH4

JH3

JH2

JH1

There are four mammalian members of this family, JAW (135 kDa), JAKZ (130 kDa), JAK3 (120 kDa) and Tyk2 (140 kDa), and one member identified from Drosophila. The JAKs lack transmembrane domains and have no apparent Src homology 2 (SH2), SH3, PTB (phosphotyrosine binding) or PH (pleckstrin homology) domains. However, seven regions of conserved homology among the JAK family proteins have been identified (see figure) and are named JHl-JH7 (JH, JAK homology). The most C-terminal (JHl] is a tyrosine kinase domain. N-terminal of this is another conserved domain (JH2), which has an overall similarity to a kinase domain but lacks specific conserved motifs and residues characteristic of kinases, so almost certainly lacks catalytic activity. The functions of this domain and the other conserved domains (JH3-JH7) are not known, but some at IeastwSJIpresumably be involved in the interaction with cytokine receptors and association with other signalling proteins. The JAKs are activated by many cytokines, and which particular JAKs are activated depends on the individual cytokine’. JAKs are associated with type I and type II cytokine receptors through a membrane-proximal region of the receptor; different receptor subunits interact with diierent IAKs1t2.

BOX 3 - jAKS AND STATS IN DRCiSOiWlA A JAK homologue encoded by the hopscotch4y gene and a STAT homologue named MARELLE or DWSTAT50,51have been found in Drosophila. Embryos from mothers lacking either germline hopscotch or mare//e have similar developmental defects that are due to deficiencies in the expression of a number of genes. MARELLE is activated, like other STATS, by tyrosine phosphorylation and binds to a DNA sequence similar to that bound by mammalian STATS. Potential binding sites are found in the promoter of the eve&skipped gene, and these sites are required for evenskippedexpression in certain regions of the embryo. These data suggest that hopscotch and murelle are required for the correct expression of some genes necessary for embryonic development. The loss of hopscotch or mareEle also affects the proliferation of cells in imaginal discs. Furthermore, a dominant allele of hopscotch has been identified called Tumorour;-lethal (Tum~)s2,ss. This allele acts as an ‘oncogene’, causing a leukaemia-like disease in which there is abnormal proliferation and differentiation of larval haematopoietic cells. There is a single amino acid difference between the proteins encoded by Turn1and wild-type hopscotch, which results in a hyperphosphorylated protein. Interestingly, the removal of a single copy of mofe//efrom TumLflies decreases the lethalty of Tell, suggesting that mar&e is involved in celt proliferation and confirming that mar&? acts downstream of hopsc0tch5~. These data establsh the existence of the JAK-STAT pathway in Drosophila. How and where this pathway is activated is not known. Furthermore, it will be interesting to determine whether DrosoplGluhave genes encoding cytokines or cytokine receptor-like proteins.

337

Cytokine or growth factor

A

I\ n

Ligand binding and receptor aggregation

F

of apoptosis by erythropoietinz4 and the induction of an antiviral state by IFN-y (Ref. 25), respectively. STAT activation

There is a great deal of evidence that the JAKs phosphorylate and activate STATS. As discussed above, cytokine receptors unable to activate JAKs are defective in STAT activation. Overexpression of JAKs in Cos cells is sufficient to activate STATSindependently of cytokine stimulation, and purified JAKs directly phosphorylate STATS in in vitro STAT dimerization studieP. An interaction between JAKs and translocation and STATS has been observed in vitroz7. This requires the SH2 domain of the N STATS and suggests that a STAT, once recruited to an activated receptor, may bind to the associated JAK to facilitate activation, or vice versa. Other tyrosine kinases also phosphorylate and activate STATS. A number of STATSbinding to specific DNA elements growth factor receptors that have inFIGURE 1 trinsic tyrosine kinase activity [e.g. receptors for epidermal growth factor (EGF), A model for STAT activation by cytokine receptors. Ligand-induced receptor aggregation results platelet-derived growth factor (PDGF) in the juxtapositioning of associated JAKs. This is thought to facilitate the transphosphorylation of and colony-stimulating factor 1 (CSF-l)] JAKs, resulting in an increase of their kinase activity. The activated jAKs phosphorylate phosphorylate members of the STAT receptor-tyrosines, providing docking sites for the STATS. The STATS are recruited to the receptor family. Treatment of cells with EGF acthrough the interaction of their SH2 domains with the phosphorylated tyrosines. After tivates STATl, STAT3 and JAKl (Ref. 1). phosphorylation of the STATS on a tyrosine residue C-terminal of their SH2 domain, they The absence of JAKl, however, does not dissociate from the receptor complex and form homo- or heterodimers through reciprocal seem to affect the ability of EGF to ininteractions between their SH2 domain and the phosphorylated tyrosine residue. The STAT duce STAT phosphorylationz8. Additiondimers translocate to the nucleus, bind response elements and activate transcription of genes. ally, STAT1 has been shown to associate JAK, Janus kinase; P, phosphate; STAT, signal transducer and activator of transcription. directly with the EGF receptorzy, and the EGF receptor can phosphorylate STAT1 number of studies have found such interactions. For irz vitroz6. Similarly, PDGF-induced STAT activation is example, IRS-l (Ref. 16), SHC (Ref. 17), Grb2 (Ref. 18), not dependent on any particular JAK family memHCP (Ref. 19), Syp (Ref. 20) and Vav (Ref. 21) have all ber30. Together, these results suggest that STATS can been shown to interact either in v&o or in vivo with be phosphorylated directly by receptor tyrosine kivarious JAK family members in response to different nases. Further work is required to establish the roles cytokines. A consistent pattern showing which proof JAK and STAT activation by these growth factor reteins are directly associated with and activated by JAKs ceptors. For example, what is the role of JAK actiand which are activated by receptor recruitment has vation if it is not required for STAT phosphorylation? yet to emerge. There is no reason why both mechaSome reports have suggested that STAT activation nisms should not be used for a given JAK-signallingmakes only a minor contribution to induction of, for protein combination in different receptor complexes. example, the c-fos geneZ8,but others have shown a The direct activation of signalling pathways by stronger, additive, effect31-33. JAKs has functional significance. In a number of The non-receptor tyrosine kinases Src and Abl may cases, cytokine receptors lacking phosphorylatable also activate members of the STAT family3P37. Srctyrosines have been shown to be sufficient to induce transformed cells show constitutively activated a mitogenic response. For example, a truncated verSTAT3, and this activation correlates with the insion of gp130 (the IL-6 receptor), lacking cytosolic duction of src expression in cells containing an intyrosines but retaining the ability to activate JAK2, ducible form of src35.In addition, STAT3 associates induces mitogenesis in an IL-6-dependent manner with Src in vivo and in vitro, and Src phosphorylates in Ba/F3 cellP. Similar results have been obtained STAT3 in vitro. Similarly, cells transformed with oncowith erythropoietin; Quelle et al. have shown that genie versions of the tyrosine kinase Abl contain conerythropoietin-induced mitogenesis requires JAK2 stitutively active STATS~~,~~. It is not known whether activation but not STAT activationz3, indicating that Abl phosphorylates STATS directly, or indirectly mitogenesis can be separated from STAT activation, through the activation of JAKs. v-Abl was shown to at least in some cases.JAK-dependent functions that associate with JAKl and JAK3, leading to the specuare separate from receptor phosphorylation or STAT lation that v-Abl activates JAKs, which in turn actiactivation have also been implicated in the inhibition vate STATS~~.However, a separate study using Bcr-Abl Recruitment and phosphorylation

338

trends in CELL BIOLOGY (Vol. 6) September 1996

Tyrosine kinases e.g. EGF-R, Src

JAKs 4

and pZlO-Abl failed to find a correlation between JAK activation and STAT activation, leading the authors to suggest that, in these cases, v-Abl directly phosphorylates STATS37. The functional significance of these observations is unclear and it will be interesting to determine whether STAT activation is required for transformation by Src or Abl. STAT activity is modulated phosphorylation

p70Sfikinase

I Transcriotion factors

by swine

Serine phosphorylation of STATScan modulate the DNA-binding and/or transcriptional activity of STAT dimers. STAT3 DNA-binding activity to particular DNA elements in certain cell types is reduced by the inhibition of serine/threonine kinases38. More directly, Wen et al. showed that STAT1 and STAT3 are phosphorylated on a serine in the C-terminal transactivation domain3g. Mutation of this serine greatly reduced the ability of STAT1 and STAT3 to induce transcription but did not affect their DNA-binding activities. Furthermore, Eilers et al. have shown that the amount of serine/threonine phosphorylation of STAT1 increases when a monocyte cell line is induced to differentiate, and this correlates with an increase in IFN-y-induced gene expression40. Together, these studies suggest that STATSmay be phosphorylated on multiple serine/threonine residues and that this can modulate their DNA-binding and/or transcriptionactivation activity. The kinases responsible for the serine phosphorylation of STATS are not known. STAT serine phosphorylation is induced by cytokines in some cell types but is constitutive in others. The phosphorylated serine identified in the C-terminus of STAT1 and STAT3 lies within a consensus site for MAP kinases and is phosphorylated by MAP kinase in vitro39. In support of a role for MAP kinases in STAT serine phosphorylation (for review, see Refs 10 and ll), a recent report has suggested that a MAP kinase (ERK 2) is associated with the IFN-a receptor; in this system, a dominant-negative MAP kinase inhibits STAT1 transcriptional activation4i. Similarly, in monocytes, IFN-a but not IFN-)I transiently induces MAP kinase activity (T. Decker, pers. commun.), whereas, in other cell types, IFN-y has been observed to induce MAP kinase activation4z. Other studies have failed to find evidence for MAP kinase activation by IFN (Ref. 33). It seems unlikely that MAP kinase is the only serine kinase that phosphorylates STATS.For example, the transcriptional activity of STAT3 induced by IL-6 is not inhibited by a dominant-negative Ras protein43, and an inhibitor of MAP kinase kinase does not affect IL-a-induced STATS serine phosphorylation (D. Cantrell, pers. commun.). It may be that different STATS and different cell types utilize different pathways to effect different serine phosphorylations. Concluding

1 Signalling proteins e.g. SHC, IRS-I,

remarks

JAK-STAT pathways have been implicated in the signal transduction of an increasing number of ligands, including cytokines, which bind to type I and II CJ@Okine receptors, growth factors such as EGF, PDGF and CSF-1, and some ligands that utilize serpentine receptors coupled to heterotrimeric G proteins44. The trends in CELL BIOLOGY (Vol. 6) September 1996

\

Transcriptional activation

FIGURE 2 Connections between )AKs and STATSand other signalling pathways. (a) STATS can be tyrosine phosphorylated by kinases other than JAKs, and other tyrosine kinases may activate JAKs. (b) JAKs phosphorylate a range of signalling molecules, resulting in the activation of a number of signalling pathways. Among these may be the serine kinases that phosphorylate STATS. (c) Serine phosphorylation of STATS modulates STAT activity. IRS-l, insulin receptor substrate-l; JAK, Janus kinase; STAT, signal transducer and activator of transcription.

major challenges are to determine the mechanisms and relative importance of JAKs and STATS in signalling by these ligands. This has already been partially accomplished for IFN; mutant cell lines unresponsive to IFN have been isolated and shown to lack individual JAK and STAT family members13,25.Transgenie mice are beginning to provide answers for other cytokines. Experiments with mice deficient in STAT1 have confirmed the importance of this STAT in IFN responses5,6.Mice lacking JAK3 are defective in responses to the IL-2/IL-4 family of cytokines and have a SCID (severe combined immunodeficiency) phenotype45,46. This establishes the central role of JAK3 in immune responses. STATG-deficient mice have defects in their responses to IL-4, indicating an important role for STAT6 in these responses47,48.The recent developments that connect JAKs and STATSto other signalling pathways raise questions concerning the mechanisms and functions of these interactions and re-emphasize the importance of crosstalk between pathways in determining the responses of cells to cytokine stimulation. References 1 SCHINDLER,C. and DARNELL,1. E., Jr (1995) Annu. Rev. Biochem.64,621-651 2 IHLE, J. N. (1995) Nature 377, 591-594 3 IVASHKIV,L. B. (1995) immunity 3, 14 4 IHLE,J. N. (1996) Cell 84, 331-334 5 DURBIN, J. E., HACKENMILLER,R., SIMON, M. C. and LEVY,D. E. (1996) Cell 84, 443-450 6 MERAZ,M. A. et al. (1996) Cell 84,431442 7 HEIM, M. H., KERR,I. M., STARK,C. R. and DARNELL,1. E., jr (1995) Science267, 1347-I 349 8 STAHL,N., FARRUCCELLA, T. J., BOULTON, T. C., ZHONC, Z., DARNELL,J. E., Jr and YANCOPOULOS,G. D. (1995) Science 267,1349-l 353 9 SEIDEL,H. M., MILOCCO, L. H., LAMB, P., DARNELL,1. E.,

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10 11 12 13 14 15 16 17

18 19 20 21 22

Acknowledgements We apologize to our colleagues whose work we have been unable to cite owing to space limitations. We thank I. M. Kerr and T. Decker for helpful comments and suggestions.

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23 24 25 26

27 28 29 30

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trends in CELL BIOLOGY (Vol. 6) September 1996