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Janus kinases and their role in growth and disease
life Sciences, Vol. 64. No. 24. pp. 2173-2186. 1999 Published by Elsevier Science Inc. Printedin the USA. All rights reserved OcQ4-3205/99/$-smfront matter ELSEVIER
MINIREVIEW JANUS KINASES AND THEIR ROLE IN GROWTH AND DISEASE
M. Aringer, A. Cheng*, J.W. Nelson, M. Chen, C. Sudarshan,Y.-J. Zhou, and J.J. O’Shea
Lymphocyte Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, *Howard Hughes Medical Institute - National Institutes of Health Research Scholars Program, National Institutes of Health, Bethesda, MD 20892-l 820 (Received in final form February 23, 1999)
Janus kinases (JAR) play a crucial role in the initial steps of cytokine signaling. Each of the four members (JAKl, JAK2, JAK3, TYK2) of this non-receptor tyrosine kinase family is indispensable for the effects of distinct cytokines. Moreover, recent reports have added to our knowledge on their highly specific functions: JAK3 knockout mice and JAK3 deficient patients cannot signal through the interleukin-2,4,7,9, or 15 receptors and suffer from severe combined immunodeficiency (SCID). JAKl and JAK2 knockout mice do not survive, their cells again showing distinct patterns of cytokine signaling deficits. At the other end of the spectrum, JAK fusion proteins have been shown to play a role in leukemias. In addition, a new class of JAK-specific inhibitors was described by several groups, the CIS/SOCS/Jab family. This review on the rapidly growing field focuses on JAK function and regulation, and on their emerging role in development and human disease. Key Words: interferons,
interleukins,
cytokine receptors, signal transduction,
Janus kinases
Cytokines represent a family of extracellular polypeptides capable of mediating intercellular communication through an array of diverse cellular responses (reviewed in 1) via binding to their cognate receptors found on the surfaces of their target cells. Once bound, these molecules transmit signals via phosphorylation of a number of downstream proteins. Unlike many other receptors (e.g., the growth factor receptors), type I (colony stimulating factors, hematopoietic cytokines, most
Corresponding Author: Dr. John J. O’Shea, Chief, Lymphocyte Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 10 Center Drive MSC 1820. Bethesda, MD 20892-1820, USA. Phone: 301 496-6026, Fax: 301 402-0012. email: [email protected]
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interleukins (IL), neuropoietic cytokines, GH, PRL, and Leptin) and type II (IFN a,P,y; IL-lo) cytokine receptors lack intrinsic kinase activity. These receptors therefore recruit and / or activate nonreceptor protein tyrosine kinases (reviewed in 2). Through a series of extensive studies since the 198Os, 11 mammalian nonreceptor tyrosine kinase families have been identified, each with the ability to bind to cytoplasmic motifs on receptors and to direct signaling. Of these, the family of Janus kinases (JAKs) are essential for the first steps of cytokine-mediated signaling (reviewed in 3). The Janus kinases contain both a kinase and a pseudokinase domain, and thus were named after the two-faced Roman God of Gates and Passages. TYK2 was discovered by screening a T-cell library by low stringency hybridization with a probe representing a known tyrosine kinase catalytic domain (4). Subsequently, JAKl, JAK2, and JAK3 were all identified utilizing polymerase chain reactions with degenerate primers corresponding to conserved motifs within tyrosine kinase catalytic domains (5,6,7,8,9,10). It is not known whether additional JAK family members exist. EST databases, however, have so far not provided evidence of additional mammalian JAK family members. Recent advances have been made toward a better understanding of cytokine signaling and human disease, thus providing a wealth of information related to JAK function, JAK regulation and counterregulation, and their role in pathogenesis. In this review, we will focus on the mechanisms of JAK regulation with emphasis on development and human disease.
Structure and
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The JAK family of kinases consists of four known members (TYK2, JAKl, JAK2, JAK3) ranging in size from 110 kDa to 140 kDa that localize to three chromosomal clusters. The gene encoding JAKl maps to a region in human chromosome lp3 1.3 (11). JAK2 was mapped to chromosome 9~24 (1 I), andJAK3andTYK2arelocatedonchromosome 19(19pl3.1 (12,13)and 19pl3.2(14),respectively). The murine counterparts map to murine chromosome 4 (JAKI), 8 (JAK3), and 19 (JAK2); the region for murine TYK2 has not yet been identified. TYK2, JAKl and JAK2 appear to be ubiquitously expressed. JAK3 expression, however, is predominantly restricted to cells of the hematopoietic lineage (8,15). Interestingly, three splice variants of JAK3 have been reported in hematopoietic and epithelial cancer cells, which contain identical amino-terminal regions but diverge at the C-terminus (16). The significance of this is unclear, nor is it known whether these alternatively spliced variants have physiological or pathological consequences. Also unique to JAK3 is significant enhancement of expression following T cell, B cell, or monocyte activation or myeloid cellular differentiation, illustrating a pivotal role of JAK3 in immunoregulation (8, 13, 15, 17). Amino acid sequence alignment of the JAKs revealed that they possess seven highly conserved domains (JHl-JH7; Figure 1); with the exception of the catalytic domain their respective and cooperative functions remain largely unknown. Interestingly, the JAK family members possess two unique features. First, the C-terminal region consists of tandem kinase and kinase-like modules (JHl, JH2), only one of which (JHl) is catalytically active; these two regions make up approximately 50% of the entire molecule. JHl contains the typical conserved residues identified in tyrosine specific kinases. The activation loops of all JHl domains contain a KE/DYY motif which appears to be a site of autophosphorylation; phosphorylation of the first of these tandem tyrosine residues is essential for full kinase activity (18,19,20,21). The function of the JH2 domain is not yet completely clear. This domain has many features of a tyrosine kinase catalytic domain; however, many residues that are essential for phosphotransferase activity are altered from the canonical motifs (5). Moreover, recent studies in patients with JAK3 mutations in the JH2 domain (22) as well as TYK2, JAK2 and Drosophila JAK (Hopscotch) studies suggest a role in intrinsic regulation, although the data are to some degree contradictory. For TYK2, a mutant lacking the JH2 domain lacked catalytic activity in vitro and was unable to transmit
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JH5
FIG. 1. The domain structure of the JAK kinases.
interferon-dependent signals (23). In contrast, a similar JAK2 mutant was able to transmit growth hormone dependent signals (24). A chimeric receptor CD16-JAK2 mutant lacking the JH2 domain had increased catalytic activity compared to the construct containing JH2 (25). This finding was interpreted as JH2 providing a tonic inhibitory function on the catalytic domain. Consistent with this notion is the identification of a gain-of-function mutation in the Drosophila JAK Hopscotch, which leads to a hyperactive kinase and subsequent leukemia in the flies (26). In JAK2, the same mutation also creates a kinase with increased activity to phosphorylate coexpressed STATS. Thus, the JH2 domain appears to have a complex regulatory function in the context of different JAKs. Our own studies on a subset of JAK3-SCID patients with mutations in the JH2 domain provide evidence for the functional importance of the domain in regulating JAK3 activity. Our results suggest that the JH2 domain is required for full catalytic activity of JAK3, but also appears to inhibit kinase activity. Interestingly, it may also affect the ability of JAK3 to phosphorylate certain substrates (M. CHEN et al, manuscript in preparation). As a second significant feature, the JAK N-terminal regions (JH7-JH3) do not show extensive homology with the members of the other 10 nonreceptor tyrosine kinase families. Similarities between the JH3 domain and SH2 domains have been reported; it is, however, not clear if the JH3 region has the functional characteristics of SH2 domains, namely the ability to bind phosphotyrosine (27,28). Experimental evidence has shown that the N-terminal regions of JAKs play a role in coordinating JAK functions and are required to interact with their cognate cellular receptors (24,29,30,3 1,32,33,34,35). For example, a 293 amino acid N-terminal segment (JH7-6) of JAK3 was necessary and sufficient for its interaction with the IL-2R subunit yc (29). This relatively small fragment also inhibited full length JAK3 from binding to the receptor chain when used in a competition experiment (29). In contrast, the respective N-terminal TYK2 fragment needed for full binding of the IFNa-receptor 1 (IFNaRl) was considerably larger (601 amino acids)(33). The functional importance of the JAKs was first made clear by studies in cell lines defective in interferon signaling. In these cell lines, the interferon signaling pathways could be restored by transfection with cDNA encoding TYK2, JAKl and/or JAK2 (36,37,38,39). Subsequent studies done with other families of receptors offered additional evidence that pointed to the importance of the JAKs in cytokine signaling (40). The findings of these studies performed in cell lines can now be confirmed by the development of knockout mice (see below). By now, many reports have documented activation of various JAKs by most, if not all cytokines (Table 1: JAKs activated by cytokines) (41,42). JAKl, JAK2, and TYK2 can be activated by a broad range of cytokines. Again in contrast, JAK3 specifically associates with the common gamma chain (yc), and hence restricts its activation to cytokines that bind yc (i.e. the Interleukins 2,4,7,9, and 15; the IL-13 receptor may include yc (43), but there are conflicting data on whether IL-13 activates JAK3 (43,44,45,46). The association of JAKs with their cognate receptor chains is obligatory to subsequent JAK phosphorylation and activation. Growing evidence suggested that the JAKs are the primary means of cytokine signaling, thus great interest has focused on mapping the regions involved in the interaction between receptors and JAKs. Initial studies done on cytokine receptor chains revealed the presence of two membrane-proximal regions termed Box 1 and Box 2 (47). Since then, subsequent in vivo and
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TABLE 1 JAK Activation
by Various Cytokines
TYK2
JAK2
+
+
+ +
IL-3, IL-5, GM-CSF IL-6, IL- 11, OSM, CNTF, LIF
+
IL-12
+
+
+ +
Leptin
+
hGI-1, PRL, Epo, Thrombopoietin
+
IFNa, IFNy
IFNP, IL- 10
JAK3 +
+
IL-2, IL-4, IL-7, IL-9, IL-15 IL-13
JAKl
+
+ +
+
in v&-o studies have demonstrated that both these regions are necessary for receptor-JAK association (48,49,50,5 152). The exact regions of the IL-2 (and IL- 15) receptor p chain binding JAKl and JAK3, respectively, have very recently been characterized (53). The sequence of events leading to JAK activation and signal propagation begins with extracellular cytokine binding to its cognate receptor and occurs in three steps: First, ligand-mediated receptor dimer-/oligomerization occurs, resulting in transphosphorylation and activation of the JAKs at particular tyrosine residues within the kinase domain (18,19). Second, activated JAKs phosphorylate particular tyrosine residues in the cytoplasmic portion of the receptor chain. These phosphorylated sites serve as docking sites for various transcription factors including the STATS (signal transducers and activators of transcription). Finally, JAKs phosphorylate the bound STAT molecules, which subsequently dissociate from the receptor, dimerize and translocate to the nucleus to modulate gene transcription (reviewed in 54 and 55). While JAK3 is exclusively activated by the yc cytokines, and thus is relatively specific, JAKl and JAK2 appear to be activated by a number of different receptor chains. Hence, cytokine signaling specificity appears not to be conferred by the JAKs, but rather, is probably due to different STAT homo- and heterodimers formed as a result of receptor activation. In addition to phosphorylating various STATS, the JAKs also interact with several other proteins: JAKl and JAK3 associate with and signal through the Insulin Receptor Substrates IRS- 1 and IRS-2, which in turn can activate PI3-kinase (56). JAK2 associates with Grb2 and SOS (57), as well as with Vav, in the regulation of which it may play a role (58). JAKI and JAK2 associate with and phosphorylate STAM (59), leading to myc induction, and SHP-2, which can then associate with Grb2 (60,61). JAK3 also employs this pathway upon IL-2 stimulation and appears to activate the Ras/MAPK pathway via SHP-2 (62). And this list is likely to grow.
There is also considerable evidence for negative regulation of the JAK-STAT pathway. Two different types of inhibitors have been described recently. While the PIAS proteins bind and inhibit at the level
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of the STATS (63), different groups have described members of an SH2 containing family of proteins which are able to bind to JAKs and/or receptors and block signaling (64,65,66,67,68,69,70, reviewed in 7 1 and 72). The family members are immediate early genes, which fits their role as classic negative feedback regulators. For the moment, the proteins bear several different names, reflecting the fact that they were cloned and characterized by several different groups. A general agreement on the nomenclature of the JAK-inhibitory proteins is to be hoped for, as their numbers appear to be increasing. The first identified inhibitor of STAT signaling was named CIS, for Cytokine-inducible src homology 2-containing protein (64). CIS binds to phosphotyrosine residues in the EpoR and IL-3 PC and prevents the activation of STAT5 (65). The mechanism by which CIS inhibits STAT5 activation remains unknown but might include the ability to directly prevent STAT dimerization thereby inhibiting nuclear” translocation, or by sequestration of specific phosphotyrosines on cytokine receptors, preventing the binding of positive growth regulatory signaling proteins. Two years later, new CIS-related proteins were identified and characterized, which unlike CIS (which bound cytokine receptors) directly bound to Janus kinases (66,67,68). The suppressor Qf wokine signaling-l protein (SOCS-1, also known as JAR or SSI-1) which possesses distant sequence identity to CIS was first identified by its ability to inhibit the IL-6 -mediated differentiation of Ml cells. SOCS1 can evidently associate with all the JAKs and blocks the downstream activation of STAT3. In addition, SOCS-1 can also bind to Tee, another nonreceptor tyrosine kinase, again suppressing kinase activity (73). This action is thought to be mediated by pseudosubstrate interference (66). A conserved motif within the C-terminus of SOCS-1, RDY, may mimic the activation loop of the Janus kinases (KEYY, as found in Jak2) thus sabotaging JAK activation. However, it is worth noting that the residues flanking this motif in Jaks and SOCS-1 are not highly similar. Nonetheless, the mechanism of pseudosubstrate interference has been well documented previously in the functional regulation of protein kinase C and myosin light chain kinase (74,75). Taking advantage of homology searches, Hilton et al. (76) discovered several genes coding for proteins similar to SOCS-1. Of twenty genes identified, five structural classes emerged based on the presence of structural domains. All SOCS superfamily members possess a similar C-terminal region referred to as the SOCS box that is approximately 40 amino acids in length and contains a number of highly conserved residues (66,76). The SOCS class I family consists of eight proteins (CIS and SOCS l-7) which contain unique N-terminal regions, an SH2 domain and the SOCS box. The SOCS proteins 4-7 are further characterized by the presence of a very large N-terminal region (Figure 2). Different SOCS proteins appear to regulate JAKs singly or in toto. SOCS-1 can inhibit all JAKs, Tee and possibly other kinases. SOCS-3, on the other hand, may be more specific: It was implicated in the inhibition of leptin signaling (77) and also shown to be upregulated by growth hormone and to block
Ia. CIS, SOCS-1, SOCS-2, SOCS-3
Ib. SOCS-4, SOCS-5, SOCS-6,
SOCS-7
FIG. 2. The structure of the SOCS class I proteins.
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(78). Thus, this may implicate certain SOCS as general inhibitors regulate the activity of one JAK.
The other proteins containing SOCS boxes may turn out to have other functions: The SOCS class II family consists of two members, WSB-1 and WSB-2, each of which possesses a C-terminal SOCS box and eight N-terminal WD40 motifs known to bind specifically to PH domains. Class III family members (SSB-1, SSB-2 and SSB-3) each possess a C-terminal SOCS box and a SPRY domain that is thought to mediate specific protein-protein interactions. ASB-1, ASB-2 and ASB-3 comprise class IV and contain from five (ASB-1 and ASB-3) to nine (ASB-2) ankyrin repeats in addition to the Cterminal SOCS box. The SOCS class V family consists of two small GTPase proteins that contain an N-terminal catalytic GTPase domain and a SOCS box at the C-terminal. The function of the SOCS box remains unknown, but each SOCS protein possesses other domains or motifs known to interact with other proteins/substrates. Therefore it is possible that the other domains function to localize SOCS proteins to specific intracellular compartments, while the SOCS box either serves to inhibit particular protein-protein associations or may act as a docking site for other proteins to bind and carryout inhibitory functions (76). Clearly, much work remains to be done to elucidate the function of each SOCS class, whether other members (or classes) exist and whether functional redundancy exists between various SOCS proteins.
JAKs inDevelonment The role of JAKs in development has begun to be uncovered. Work done in Drosophila melanogaster demonstrates an essential role of JAKs in development and growth (79). Mutation of the Drosophila JAK (Hopscotch (Hop)) gene results in severe developmental abnormalities. These include profound segmentation defects, stripe-specific defects in the expression of pair-rule genes (even-skipped, runt, and&shi tarazu ) and in the expression of segment-polarity genes (such as engrailed and wingless ) (80). Clearly, regulation of JAK activity is important for the maintenance of normal growth and development in flies. Studies with JAK knockout mice have enhanced our knowledge of JAKs function in mammalian development and growth. JAK3-deficient mice, the first to be generated (81,82,83), exhibit severe defects in the development of lymphoid cells. These mice have smaller thymi , spleens and lymph nodes as compared to wild type mice. They also have greatly reduced numbers of thymocytes, and TABLE 2 JAK Knockout Mice and Their Cytokine Signaling
Defects
JAK2 -/JAKI-/embryonically lethal perinatally lelhal no blood formation unnblc to nurse (neuronal Lipoptosis) IFNo& IFNv
IL- 10
IL-2, IL-4, IL-7 IL-3 IL-5, Epo, Tpo, GM-CSF IL-6, LlF CNTF, CT- 1, Oncostatin M
D D D PD
JAK3 -/viable immunodeficient
D D D D
PD D
D denotes deficient, PD partially deficient signaling upon stimulation
with the cytokine.
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defective B cell development; furthermore, the residual T cells are functionally deficient. The severe lymphopenia exhibited by these mice was reminiscent of the severe combined immunodeficiency (SCID) phenotype found in human patients with mutations of JAK3 or yc (84,85,86,87). And indeed, the phenotype of JAK3-deficient mice is also similar to that of mice lacking the yc chain (88,89). Additional studies on the consequences of murine JAK3 deficiency have found an impairment in T cell negative selection (90) and have demonstrated the importance of JAK3 for peripheral T cell function (91). In contrast to JAK3, which is expressed predominantly in hematopoietic cells, JAKl, JAK2 and TYK2 are ubiquitously expressed. Therefore, the loss of these kinases was expected to result in a more profound phenotype. Accordingly, JAK2 deficiency is embryonically lethal (92,93), and JAKl deficiency (94) is perinatally lethal. In addition, studies in zebrafish have pointed to an essential role of JAKl in zebrafish embryogenesis and thus early vertebrate development (95). Zebrafish JAKl is maternally encoded, stored in unfertilized eggs and is expressed throughout the midblastula stage. Injection of RNA encoding dominant-negative JAKl results in inhibition of specific cellular migration and reduction of anterior structure formation. Mouse embryos deficient of JAKl or JAK2, however, do not demonstrate any obvious structural problems as described in the zebrafish studies. The findings in zebra fish embryos may thus be due to redundant effects, as the effects of the dominant negative form might also block other than pure JAKl functions. Double knockout mice may eventually shed light on this problem. Embryonic fibroblasts from JAKl knockout mice do not respond to the class II cytokine receptors ligands IFNy and IFNc(. Similarly, neurons from JAKl deficient mice fail to respond to the ligands of gp130 receptor family members - leukemia inhibitory factor (LIF), Ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-l), and oncostatin M (OSM). Like the JAK3 deficient mice, murine JAKl deficiency leads to reduced numbers of T and B lymphocytes. This has been attributed to impaired IL-7 signal transduction in the absence of JAKl. Thus it is clear that JAKl plays an indispensable role in mediating biologic responses induced by a specific subset of cytokine receptors. The cause of perinatal death of JAKl deficient mice is not clear. Studies indicate that neurons from JAKl deficient mice are incapable of responding to LIF, CT-l, and CNTF and die due to apoptosis. Accordingly, these mice have reduced numbers of sensory neurons and do not nurse. Thus, neuronal defects caused by lack of JAKl could explain the perinatal death observed in JAKl deficient mice. JAK2 deficient mice have been shown to lack definitive erythropoiesis. Analysis of day 11 embryos showed lack of hematopoietic progenitor cells. The embryonic lethality of the JAK2 deficient mice has thus been attributed to the lack of formation of blood cells. The similarity of the phenotype of JAK2 deficient mice and erythryopoietin (Epo) and EpoR deficient mice that also exhibit lack of definitive erythropoiesis, suggest a specific role for Jak2 in Epo signal transduction. Fetal cells from JAK2 deficient mice also fail to respond to thrombopoietin (Tpo), GM-CSF, IL-3 and IL-5. Similarly, IFNy fails to elicit an antiviral response from fibroblasts of JAK2 deficient mice. Thus, like JAKl , JAK2 also plays a critical and specific role in the function of a specific subset of cytokine receptors. Observations on TYK2 knockout mice have not yet been reported. Based on the studies with other JAK knockout mice it is likely that TYK2 also plays a specific role in mediating biologic responses of specific cytokines. These studies with knockout mice have led us to better appreciate the central role the JAKs play in several aspects of development. We can expect to hear more shortly (such as TYK2and double knock-out mice).
JAK3 in SCID Whereas human deficiencies of the ubiquitous JAKs have not been observed (and are not likely to occur given the knockout mouse findings), JAK3 deficient patients have been described. Given that
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JAK3 is predominantly expressed in leukocytes, affected patients not unexpected.
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this was more likely and the finding of SCID in the
SCIDs are a rare, heterogeneous group of pediatric disorders characterized by marked hypoplastic lymphoid development, failure to thrive and profound lymphopenia (96,97). Attempts to identify the molecular pathogenesis of this syndrome resulted in the identification of a number of genetic mutations, of which the most common are deficiencies of the X-chromosomal common gamma chain (yc) gene, encoding a shared component of the interleukin-2,4, 7,9, and 15 receptors (85,86,98). Work on the pathogenesis of X-linked SCID (XSCID) showed the importance of the yc-JAK3 association in generating disease (99): Mutations in yc that completely disrupted its JAK3 association resulted in severe forms of immunodeficiency, while mutations that minimally diminished this association resulted in attenuated immunodeficiency. These observations led to the hypothesis that patients with JAK3 mutations would suffer from an autosomal recessive form of SCID. Indeed, except for the pattern of inheritance patients with JAK3 deficiency (MIM600173) displayed an identical SCID phenotype with a severity comparable to XSCID (87,88). As expected, reconstituting JAK3 in the B cells of these patients restored cellular proliferation in response to mitogenic agents (100). Interestingly, whereas in murine SCID B-cells are absent and some T-cells present, but not functional (see above), human SCID patients completely lack T cells, and their B cells are not functional. Why this occurs is not clear so far, but the finding suggests species-specific pathways that rescue the development of lymphocyte subsets. Most of the described mutations in JAK3 (Figure 3) lead to unstable and/or truncated JAK3 proteins. However, two point mutations (LEl and LP2) are of particular interest: The LEl (E481G) mutation
1. Premature termination
2. Deletions ,--c-------- JH5_________
_________________r_ I I _ _ __’ ‘bt; __-_____‘i---__-__-___
3. Other Mutations LE1 (E481 G)
LP2 (C759R)
FIG. 3. Known JAK3 mutations in patients with SCID.
, ) #1 , 1 !Ii’
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T ALL-case
Pre-B ALL-case
CML-case
FIG. 4. TEL-JAK2 fusion proteins found in leukemic cells. in the JH3 region led to a somewhat leaky phenotype with the patient progressively developing T lymphocytes, although functionally defective (101). The LP2 (C759R) mutation was found in the JH2 (pseudokinase) domain producing a constitutively autophosphorylated, but non-functional protein (22). This mutation underscores the importance of the JH2 domain with respect to regulation of JAK catalytic activity. A compendium of JAK3 mutations can be found at http://www.uta.ftiaitokset/imt/ bioinfolJAK3basel. JAKs in Malignancies In Drosophila, activating mutations of their Janus kinase Hop (designated as turn-l (tumorous lethal)) are known to result in leukemia-like hematopoietic defects (26,102,103,104). In human leukemic cells recently translocations which constitutively activate JAK2 have been found (105,106). In combination, the two reports on a total of three patients (two with childhood lymphoblastic, one with atypical adult CML)(Figure 4) make a causal role of JAK2 overexpression indeed likely. Patients with leukemias associated with putative Tel-JAK3 (n=l) and Tel-TYK2 (n=2) fusion proteins have also been reported (107). In addition to these newly found fusion-proteins, a number of other studies have implicated JAK hyperactivation to play a role in human T cell leukemia virus Itransformed T cells (log), Sezary’s syndrome (log), v-abl-transformed cells (110) and acute lymphoblastic leukemia (111).
Since their initial discovery in 1990, the JAKs have emerged as an integral aspect of cytokine signaling and cellular growth. Though the advances made thus far have added much to our understanding of immunoregulation, several issues remain unresolved. One major issue lies in the regulation of JAK activity: Previous studies done in JAK2 and JAK3 have demonstrated that tyrosine residues regulate intrinsic kinase activity (18,19). It is likely that more phosphorylation residues of functional importance exist, which may contribute to catalytic regulation and/or binding to other proteins. A second issue is to resolve the three-dimensional structure of the JAK protein. This
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information will offer a better understanding of the function of each individual domain upon the protein as a whole. Finally, the complex system of inhibitors needs to be thoroughly uncovered and understood. Many of the involved inhibitory proteins are still unknown and there is also a profound lack of information on the structural motifs relevant for their mode of function. Once we obtain answers to these queries, we may begin to understand the complex molecular pathways involved in immune regulation and predict how hematopoietic abnormalities may be treated.
Martin Aringer is a recipient of a Max Kade Foundation
1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22.
Postdoctoral
Research Exchange
Grant.
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MINIREVIEW JANUS KINASES AND THEIR ROLE IN GROWTH AND DISEASE
M. Aringer, A. Cheng*, J.W. Nelson, M. Chen, C. Sudarshan,Y.-J. Zhou, and J.J. O’Shea
Lymphocyte Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, *Howard Hughes Medical Institute - National Institutes of Health Research Scholars Program, National Institutes of Health, Bethesda, MD 20892-l 820 (Received in final form February 23, 1999)
Janus kinases (JAR) play a crucial role in the initial steps of cytokine signaling. Each of the four members (JAKl, JAK2, JAK3, TYK2) of this non-receptor tyrosine kinase family is indispensable for the effects of distinct cytokines. Moreover, recent reports have added to our knowledge on their highly specific functions: JAK3 knockout mice and JAK3 deficient patients cannot signal through the interleukin-2,4,7,9, or 15 receptors and suffer from severe combined immunodeficiency (SCID). JAKl and JAK2 knockout mice do not survive, their cells again showing distinct patterns of cytokine signaling deficits. At the other end of the spectrum, JAK fusion proteins have been shown to play a role in leukemias. In addition, a new class of JAK-specific inhibitors was described by several groups, the CIS/SOCS/Jab family. This review on the rapidly growing field focuses on JAK function and regulation, and on their emerging role in development and human disease. Key Words: interferons,
interleukins,
cytokine receptors, signal transduction,
Janus kinases
Cytokines represent a family of extracellular polypeptides capable of mediating intercellular communication through an array of diverse cellular responses (reviewed in 1) via binding to their cognate receptors found on the surfaces of their target cells. Once bound, these molecules transmit signals via phosphorylation of a number of downstream proteins. Unlike many other receptors (e.g., the growth factor receptors), type I (colony stimulating factors, hematopoietic cytokines, most
Corresponding Author: Dr. John J. O’Shea, Chief, Lymphocyte Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 10 Center Drive MSC 1820. Bethesda, MD 20892-1820, USA. Phone: 301 496-6026, Fax: 301 402-0012. email: [email protected]
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interleukins (IL), neuropoietic cytokines, GH, PRL, and Leptin) and type II (IFN a,P,y; IL-lo) cytokine receptors lack intrinsic kinase activity. These receptors therefore recruit and / or activate nonreceptor protein tyrosine kinases (reviewed in 2). Through a series of extensive studies since the 198Os, 11 mammalian nonreceptor tyrosine kinase families have been identified, each with the ability to bind to cytoplasmic motifs on receptors and to direct signaling. Of these, the family of Janus kinases (JAKs) are essential for the first steps of cytokine-mediated signaling (reviewed in 3). The Janus kinases contain both a kinase and a pseudokinase domain, and thus were named after the two-faced Roman God of Gates and Passages. TYK2 was discovered by screening a T-cell library by low stringency hybridization with a probe representing a known tyrosine kinase catalytic domain (4). Subsequently, JAKl, JAK2, and JAK3 were all identified utilizing polymerase chain reactions with degenerate primers corresponding to conserved motifs within tyrosine kinase catalytic domains (5,6,7,8,9,10). It is not known whether additional JAK family members exist. EST databases, however, have so far not provided evidence of additional mammalian JAK family members. Recent advances have been made toward a better understanding of cytokine signaling and human disease, thus providing a wealth of information related to JAK function, JAK regulation and counterregulation, and their role in pathogenesis. In this review, we will focus on the mechanisms of JAK regulation with emphasis on development and human disease.
Structure and
. . of t@watlog
The JAK family of kinases consists of four known members (TYK2, JAKl, JAK2, JAK3) ranging in size from 110 kDa to 140 kDa that localize to three chromosomal clusters. The gene encoding JAKl maps to a region in human chromosome lp3 1.3 (11). JAK2 was mapped to chromosome 9~24 (1 I), andJAK3andTYK2arelocatedonchromosome 19(19pl3.1 (12,13)and 19pl3.2(14),respectively). The murine counterparts map to murine chromosome 4 (JAKI), 8 (JAK3), and 19 (JAK2); the region for murine TYK2 has not yet been identified. TYK2, JAKl and JAK2 appear to be ubiquitously expressed. JAK3 expression, however, is predominantly restricted to cells of the hematopoietic lineage (8,15). Interestingly, three splice variants of JAK3 have been reported in hematopoietic and epithelial cancer cells, which contain identical amino-terminal regions but diverge at the C-terminus (16). The significance of this is unclear, nor is it known whether these alternatively spliced variants have physiological or pathological consequences. Also unique to JAK3 is significant enhancement of expression following T cell, B cell, or monocyte activation or myeloid cellular differentiation, illustrating a pivotal role of JAK3 in immunoregulation (8, 13, 15, 17). Amino acid sequence alignment of the JAKs revealed that they possess seven highly conserved domains (JHl-JH7; Figure 1); with the exception of the catalytic domain their respective and cooperative functions remain largely unknown. Interestingly, the JAK family members possess two unique features. First, the C-terminal region consists of tandem kinase and kinase-like modules (JHl, JH2), only one of which (JHl) is catalytically active; these two regions make up approximately 50% of the entire molecule. JHl contains the typical conserved residues identified in tyrosine specific kinases. The activation loops of all JHl domains contain a KE/DYY motif which appears to be a site of autophosphorylation; phosphorylation of the first of these tandem tyrosine residues is essential for full kinase activity (18,19,20,21). The function of the JH2 domain is not yet completely clear. This domain has many features of a tyrosine kinase catalytic domain; however, many residues that are essential for phosphotransferase activity are altered from the canonical motifs (5). Moreover, recent studies in patients with JAK3 mutations in the JH2 domain (22) as well as TYK2, JAK2 and Drosophila JAK (Hopscotch) studies suggest a role in intrinsic regulation, although the data are to some degree contradictory. For TYK2, a mutant lacking the JH2 domain lacked catalytic activity in vitro and was unable to transmit
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JH5
FIG. 1. The domain structure of the JAK kinases.
interferon-dependent signals (23). In contrast, a similar JAK2 mutant was able to transmit growth hormone dependent signals (24). A chimeric receptor CD16-JAK2 mutant lacking the JH2 domain had increased catalytic activity compared to the construct containing JH2 (25). This finding was interpreted as JH2 providing a tonic inhibitory function on the catalytic domain. Consistent with this notion is the identification of a gain-of-function mutation in the Drosophila JAK Hopscotch, which leads to a hyperactive kinase and subsequent leukemia in the flies (26). In JAK2, the same mutation also creates a kinase with increased activity to phosphorylate coexpressed STATS. Thus, the JH2 domain appears to have a complex regulatory function in the context of different JAKs. Our own studies on a subset of JAK3-SCID patients with mutations in the JH2 domain provide evidence for the functional importance of the domain in regulating JAK3 activity. Our results suggest that the JH2 domain is required for full catalytic activity of JAK3, but also appears to inhibit kinase activity. Interestingly, it may also affect the ability of JAK3 to phosphorylate certain substrates (M. CHEN et al, manuscript in preparation). As a second significant feature, the JAK N-terminal regions (JH7-JH3) do not show extensive homology with the members of the other 10 nonreceptor tyrosine kinase families. Similarities between the JH3 domain and SH2 domains have been reported; it is, however, not clear if the JH3 region has the functional characteristics of SH2 domains, namely the ability to bind phosphotyrosine (27,28). Experimental evidence has shown that the N-terminal regions of JAKs play a role in coordinating JAK functions and are required to interact with their cognate cellular receptors (24,29,30,3 1,32,33,34,35). For example, a 293 amino acid N-terminal segment (JH7-6) of JAK3 was necessary and sufficient for its interaction with the IL-2R subunit yc (29). This relatively small fragment also inhibited full length JAK3 from binding to the receptor chain when used in a competition experiment (29). In contrast, the respective N-terminal TYK2 fragment needed for full binding of the IFNa-receptor 1 (IFNaRl) was considerably larger (601 amino acids)(33). The functional importance of the JAKs was first made clear by studies in cell lines defective in interferon signaling. In these cell lines, the interferon signaling pathways could be restored by transfection with cDNA encoding TYK2, JAKl and/or JAK2 (36,37,38,39). Subsequent studies done with other families of receptors offered additional evidence that pointed to the importance of the JAKs in cytokine signaling (40). The findings of these studies performed in cell lines can now be confirmed by the development of knockout mice (see below). By now, many reports have documented activation of various JAKs by most, if not all cytokines (Table 1: JAKs activated by cytokines) (41,42). JAKl, JAK2, and TYK2 can be activated by a broad range of cytokines. Again in contrast, JAK3 specifically associates with the common gamma chain (yc), and hence restricts its activation to cytokines that bind yc (i.e. the Interleukins 2,4,7,9, and 15; the IL-13 receptor may include yc (43), but there are conflicting data on whether IL-13 activates JAK3 (43,44,45,46). The association of JAKs with their cognate receptor chains is obligatory to subsequent JAK phosphorylation and activation. Growing evidence suggested that the JAKs are the primary means of cytokine signaling, thus great interest has focused on mapping the regions involved in the interaction between receptors and JAKs. Initial studies done on cytokine receptor chains revealed the presence of two membrane-proximal regions termed Box 1 and Box 2 (47). Since then, subsequent in vivo and
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TABLE 1 JAK Activation
by Various Cytokines
TYK2
JAK2
+
+
+ +
IL-3, IL-5, GM-CSF IL-6, IL- 11, OSM, CNTF, LIF
+
IL-12
+
+
+ +
Leptin
+
hGI-1, PRL, Epo, Thrombopoietin
+
IFNa, IFNy
IFNP, IL- 10
JAK3 +
+
IL-2, IL-4, IL-7, IL-9, IL-15 IL-13
JAKl
+
+ +
+
in v&-o studies have demonstrated that both these regions are necessary for receptor-JAK association (48,49,50,5 152). The exact regions of the IL-2 (and IL- 15) receptor p chain binding JAKl and JAK3, respectively, have very recently been characterized (53). The sequence of events leading to JAK activation and signal propagation begins with extracellular cytokine binding to its cognate receptor and occurs in three steps: First, ligand-mediated receptor dimer-/oligomerization occurs, resulting in transphosphorylation and activation of the JAKs at particular tyrosine residues within the kinase domain (18,19). Second, activated JAKs phosphorylate particular tyrosine residues in the cytoplasmic portion of the receptor chain. These phosphorylated sites serve as docking sites for various transcription factors including the STATS (signal transducers and activators of transcription). Finally, JAKs phosphorylate the bound STAT molecules, which subsequently dissociate from the receptor, dimerize and translocate to the nucleus to modulate gene transcription (reviewed in 54 and 55). While JAK3 is exclusively activated by the yc cytokines, and thus is relatively specific, JAKl and JAK2 appear to be activated by a number of different receptor chains. Hence, cytokine signaling specificity appears not to be conferred by the JAKs, but rather, is probably due to different STAT homo- and heterodimers formed as a result of receptor activation. In addition to phosphorylating various STATS, the JAKs also interact with several other proteins: JAKl and JAK3 associate with and signal through the Insulin Receptor Substrates IRS- 1 and IRS-2, which in turn can activate PI3-kinase (56). JAK2 associates with Grb2 and SOS (57), as well as with Vav, in the regulation of which it may play a role (58). JAKI and JAK2 associate with and phosphorylate STAM (59), leading to myc induction, and SHP-2, which can then associate with Grb2 (60,61). JAK3 also employs this pathway upon IL-2 stimulation and appears to activate the Ras/MAPK pathway via SHP-2 (62). And this list is likely to grow.
There is also considerable evidence for negative regulation of the JAK-STAT pathway. Two different types of inhibitors have been described recently. While the PIAS proteins bind and inhibit at the level
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of the STATS (63), different groups have described members of an SH2 containing family of proteins which are able to bind to JAKs and/or receptors and block signaling (64,65,66,67,68,69,70, reviewed in 7 1 and 72). The family members are immediate early genes, which fits their role as classic negative feedback regulators. For the moment, the proteins bear several different names, reflecting the fact that they were cloned and characterized by several different groups. A general agreement on the nomenclature of the JAK-inhibitory proteins is to be hoped for, as their numbers appear to be increasing. The first identified inhibitor of STAT signaling was named CIS, for Cytokine-inducible src homology 2-containing protein (64). CIS binds to phosphotyrosine residues in the EpoR and IL-3 PC and prevents the activation of STAT5 (65). The mechanism by which CIS inhibits STAT5 activation remains unknown but might include the ability to directly prevent STAT dimerization thereby inhibiting nuclear” translocation, or by sequestration of specific phosphotyrosines on cytokine receptors, preventing the binding of positive growth regulatory signaling proteins. Two years later, new CIS-related proteins were identified and characterized, which unlike CIS (which bound cytokine receptors) directly bound to Janus kinases (66,67,68). The suppressor Qf wokine signaling-l protein (SOCS-1, also known as JAR or SSI-1) which possesses distant sequence identity to CIS was first identified by its ability to inhibit the IL-6 -mediated differentiation of Ml cells. SOCS1 can evidently associate with all the JAKs and blocks the downstream activation of STAT3. In addition, SOCS-1 can also bind to Tee, another nonreceptor tyrosine kinase, again suppressing kinase activity (73). This action is thought to be mediated by pseudosubstrate interference (66). A conserved motif within the C-terminus of SOCS-1, RDY, may mimic the activation loop of the Janus kinases (KEYY, as found in Jak2) thus sabotaging JAK activation. However, it is worth noting that the residues flanking this motif in Jaks and SOCS-1 are not highly similar. Nonetheless, the mechanism of pseudosubstrate interference has been well documented previously in the functional regulation of protein kinase C and myosin light chain kinase (74,75). Taking advantage of homology searches, Hilton et al. (76) discovered several genes coding for proteins similar to SOCS-1. Of twenty genes identified, five structural classes emerged based on the presence of structural domains. All SOCS superfamily members possess a similar C-terminal region referred to as the SOCS box that is approximately 40 amino acids in length and contains a number of highly conserved residues (66,76). The SOCS class I family consists of eight proteins (CIS and SOCS l-7) which contain unique N-terminal regions, an SH2 domain and the SOCS box. The SOCS proteins 4-7 are further characterized by the presence of a very large N-terminal region (Figure 2). Different SOCS proteins appear to regulate JAKs singly or in toto. SOCS-1 can inhibit all JAKs, Tee and possibly other kinases. SOCS-3, on the other hand, may be more specific: It was implicated in the inhibition of leptin signaling (77) and also shown to be upregulated by growth hormone and to block
Ia. CIS, SOCS-1, SOCS-2, SOCS-3
Ib. SOCS-4, SOCS-5, SOCS-6,
SOCS-7
FIG. 2. The structure of the SOCS class I proteins.
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(78). Thus, this may implicate certain SOCS as general inhibitors regulate the activity of one JAK.
The other proteins containing SOCS boxes may turn out to have other functions: The SOCS class II family consists of two members, WSB-1 and WSB-2, each of which possesses a C-terminal SOCS box and eight N-terminal WD40 motifs known to bind specifically to PH domains. Class III family members (SSB-1, SSB-2 and SSB-3) each possess a C-terminal SOCS box and a SPRY domain that is thought to mediate specific protein-protein interactions. ASB-1, ASB-2 and ASB-3 comprise class IV and contain from five (ASB-1 and ASB-3) to nine (ASB-2) ankyrin repeats in addition to the Cterminal SOCS box. The SOCS class V family consists of two small GTPase proteins that contain an N-terminal catalytic GTPase domain and a SOCS box at the C-terminal. The function of the SOCS box remains unknown, but each SOCS protein possesses other domains or motifs known to interact with other proteins/substrates. Therefore it is possible that the other domains function to localize SOCS proteins to specific intracellular compartments, while the SOCS box either serves to inhibit particular protein-protein associations or may act as a docking site for other proteins to bind and carryout inhibitory functions (76). Clearly, much work remains to be done to elucidate the function of each SOCS class, whether other members (or classes) exist and whether functional redundancy exists between various SOCS proteins.
JAKs inDevelonment The role of JAKs in development has begun to be uncovered. Work done in Drosophila melanogaster demonstrates an essential role of JAKs in development and growth (79). Mutation of the Drosophila JAK (Hopscotch (Hop)) gene results in severe developmental abnormalities. These include profound segmentation defects, stripe-specific defects in the expression of pair-rule genes (even-skipped, runt, and&shi tarazu ) and in the expression of segment-polarity genes (such as engrailed and wingless ) (80). Clearly, regulation of JAK activity is important for the maintenance of normal growth and development in flies. Studies with JAK knockout mice have enhanced our knowledge of JAKs function in mammalian development and growth. JAK3-deficient mice, the first to be generated (81,82,83), exhibit severe defects in the development of lymphoid cells. These mice have smaller thymi , spleens and lymph nodes as compared to wild type mice. They also have greatly reduced numbers of thymocytes, and TABLE 2 JAK Knockout Mice and Their Cytokine Signaling
Defects
JAK2 -/JAKI-/embryonically lethal perinatally lelhal no blood formation unnblc to nurse (neuronal Lipoptosis) IFNo& IFNv
IL- 10
IL-2, IL-4, IL-7 IL-3 IL-5, Epo, Tpo, GM-CSF IL-6, LlF CNTF, CT- 1, Oncostatin M
D D D PD
JAK3 -/viable immunodeficient
D D D D
PD D
D denotes deficient, PD partially deficient signaling upon stimulation
with the cytokine.
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defective B cell development; furthermore, the residual T cells are functionally deficient. The severe lymphopenia exhibited by these mice was reminiscent of the severe combined immunodeficiency (SCID) phenotype found in human patients with mutations of JAK3 or yc (84,85,86,87). And indeed, the phenotype of JAK3-deficient mice is also similar to that of mice lacking the yc chain (88,89). Additional studies on the consequences of murine JAK3 deficiency have found an impairment in T cell negative selection (90) and have demonstrated the importance of JAK3 for peripheral T cell function (91). In contrast to JAK3, which is expressed predominantly in hematopoietic cells, JAKl, JAK2 and TYK2 are ubiquitously expressed. Therefore, the loss of these kinases was expected to result in a more profound phenotype. Accordingly, JAK2 deficiency is embryonically lethal (92,93), and JAKl deficiency (94) is perinatally lethal. In addition, studies in zebrafish have pointed to an essential role of JAKl in zebrafish embryogenesis and thus early vertebrate development (95). Zebrafish JAKl is maternally encoded, stored in unfertilized eggs and is expressed throughout the midblastula stage. Injection of RNA encoding dominant-negative JAKl results in inhibition of specific cellular migration and reduction of anterior structure formation. Mouse embryos deficient of JAKl or JAK2, however, do not demonstrate any obvious structural problems as described in the zebrafish studies. The findings in zebra fish embryos may thus be due to redundant effects, as the effects of the dominant negative form might also block other than pure JAKl functions. Double knockout mice may eventually shed light on this problem. Embryonic fibroblasts from JAKl knockout mice do not respond to the class II cytokine receptors ligands IFNy and IFNc(. Similarly, neurons from JAKl deficient mice fail to respond to the ligands of gp130 receptor family members - leukemia inhibitory factor (LIF), Ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-l), and oncostatin M (OSM). Like the JAK3 deficient mice, murine JAKl deficiency leads to reduced numbers of T and B lymphocytes. This has been attributed to impaired IL-7 signal transduction in the absence of JAKl. Thus it is clear that JAKl plays an indispensable role in mediating biologic responses induced by a specific subset of cytokine receptors. The cause of perinatal death of JAKl deficient mice is not clear. Studies indicate that neurons from JAKl deficient mice are incapable of responding to LIF, CT-l, and CNTF and die due to apoptosis. Accordingly, these mice have reduced numbers of sensory neurons and do not nurse. Thus, neuronal defects caused by lack of JAKl could explain the perinatal death observed in JAKl deficient mice. JAK2 deficient mice have been shown to lack definitive erythropoiesis. Analysis of day 11 embryos showed lack of hematopoietic progenitor cells. The embryonic lethality of the JAK2 deficient mice has thus been attributed to the lack of formation of blood cells. The similarity of the phenotype of JAK2 deficient mice and erythryopoietin (Epo) and EpoR deficient mice that also exhibit lack of definitive erythropoiesis, suggest a specific role for Jak2 in Epo signal transduction. Fetal cells from JAK2 deficient mice also fail to respond to thrombopoietin (Tpo), GM-CSF, IL-3 and IL-5. Similarly, IFNy fails to elicit an antiviral response from fibroblasts of JAK2 deficient mice. Thus, like JAKl , JAK2 also plays a critical and specific role in the function of a specific subset of cytokine receptors. Observations on TYK2 knockout mice have not yet been reported. Based on the studies with other JAK knockout mice it is likely that TYK2 also plays a specific role in mediating biologic responses of specific cytokines. These studies with knockout mice have led us to better appreciate the central role the JAKs play in several aspects of development. We can expect to hear more shortly (such as TYK2and double knock-out mice).
JAK3 in SCID Whereas human deficiencies of the ubiquitous JAKs have not been observed (and are not likely to occur given the knockout mouse findings), JAK3 deficient patients have been described. Given that
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this was more likely and the finding of SCID in the
SCIDs are a rare, heterogeneous group of pediatric disorders characterized by marked hypoplastic lymphoid development, failure to thrive and profound lymphopenia (96,97). Attempts to identify the molecular pathogenesis of this syndrome resulted in the identification of a number of genetic mutations, of which the most common are deficiencies of the X-chromosomal common gamma chain (yc) gene, encoding a shared component of the interleukin-2,4, 7,9, and 15 receptors (85,86,98). Work on the pathogenesis of X-linked SCID (XSCID) showed the importance of the yc-JAK3 association in generating disease (99): Mutations in yc that completely disrupted its JAK3 association resulted in severe forms of immunodeficiency, while mutations that minimally diminished this association resulted in attenuated immunodeficiency. These observations led to the hypothesis that patients with JAK3 mutations would suffer from an autosomal recessive form of SCID. Indeed, except for the pattern of inheritance patients with JAK3 deficiency (MIM600173) displayed an identical SCID phenotype with a severity comparable to XSCID (87,88). As expected, reconstituting JAK3 in the B cells of these patients restored cellular proliferation in response to mitogenic agents (100). Interestingly, whereas in murine SCID B-cells are absent and some T-cells present, but not functional (see above), human SCID patients completely lack T cells, and their B cells are not functional. Why this occurs is not clear so far, but the finding suggests species-specific pathways that rescue the development of lymphocyte subsets. Most of the described mutations in JAK3 (Figure 3) lead to unstable and/or truncated JAK3 proteins. However, two point mutations (LEl and LP2) are of particular interest: The LEl (E481G) mutation
1. Premature termination
2. Deletions ,--c-------- JH5_________
_________________r_ I I _ _ __’ ‘bt; __-_____‘i---__-__-___
3. Other Mutations LE1 (E481 G)
LP2 (C759R)
FIG. 3. Known JAK3 mutations in patients with SCID.
, ) #1 , 1 !Ii’
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T ALL-case
Pre-B ALL-case
CML-case
FIG. 4. TEL-JAK2 fusion proteins found in leukemic cells. in the JH3 region led to a somewhat leaky phenotype with the patient progressively developing T lymphocytes, although functionally defective (101). The LP2 (C759R) mutation was found in the JH2 (pseudokinase) domain producing a constitutively autophosphorylated, but non-functional protein (22). This mutation underscores the importance of the JH2 domain with respect to regulation of JAK catalytic activity. A compendium of JAK3 mutations can be found at http://www.uta.ftiaitokset/imt/ bioinfolJAK3basel. JAKs in Malignancies In Drosophila, activating mutations of their Janus kinase Hop (designated as turn-l (tumorous lethal)) are known to result in leukemia-like hematopoietic defects (26,102,103,104). In human leukemic cells recently translocations which constitutively activate JAK2 have been found (105,106). In combination, the two reports on a total of three patients (two with childhood lymphoblastic, one with atypical adult CML)(Figure 4) make a causal role of JAK2 overexpression indeed likely. Patients with leukemias associated with putative Tel-JAK3 (n=l) and Tel-TYK2 (n=2) fusion proteins have also been reported (107). In addition to these newly found fusion-proteins, a number of other studies have implicated JAK hyperactivation to play a role in human T cell leukemia virus Itransformed T cells (log), Sezary’s syndrome (log), v-abl-transformed cells (110) and acute lymphoblastic leukemia (111).
Since their initial discovery in 1990, the JAKs have emerged as an integral aspect of cytokine signaling and cellular growth. Though the advances made thus far have added much to our understanding of immunoregulation, several issues remain unresolved. One major issue lies in the regulation of JAK activity: Previous studies done in JAK2 and JAK3 have demonstrated that tyrosine residues regulate intrinsic kinase activity (18,19). It is likely that more phosphorylation residues of functional importance exist, which may contribute to catalytic regulation and/or binding to other proteins. A second issue is to resolve the three-dimensional structure of the JAK protein. This
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information will offer a better understanding of the function of each individual domain upon the protein as a whole. Finally, the complex system of inhibitors needs to be thoroughly uncovered and understood. Many of the involved inhibitory proteins are still unknown and there is also a profound lack of information on the structural motifs relevant for their mode of function. Once we obtain answers to these queries, we may begin to understand the complex molecular pathways involved in immune regulation and predict how hematopoietic abnormalities may be treated.
Martin Aringer is a recipient of a Max Kade Foundation
1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22.
Postdoctoral
Research Exchange
Grant.
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