Oncogene (2000) 19, 5662 ± 5679 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Janus kinases: components of multiple signaling pathways

Sushil G Rane1 and E Premkumar Reddy*,1

1Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, Pennsylvania, PA 19140, USA

Cytoplasmic Janus protein tyrosine kinases (JAKs) are rapidly induce tyrosine phosphorylation of the recep- crucial components of diverse signal transduction path- tors and a variety of cellular proteins suggesting that ways that govern cellular survival, proliferation, di€er- these receptors transmit their signals through cellular entiation and apoptosis. Evidence to date, indicates that tyrosine kinases (Kishimoto et al., 1994). During the JAK kinase function may integrate components of past decade, new evidence has emerged to indicate that diverse signaling cascades. While it is likely that most cytokines transmit their signals via a new family activation of STAT proteins may be an important of tyrosine kinases termed JAK kinases (Ihle et al., function attributed to the JAK kinases, it is certainly 1995, 1997; Darnell, 1998; Darnell et al., 1994; not the only function performed by this key family of Schindler, 1999; Schindler and Darnell, 1995; Ward et cytoplasmic tyrosine kinases. Emerging evidence indi- al., 2000; Pellegrini and Dusanter-Fourt, 1997; Leo- cates that phosphorylation of cytokine and growth factor nard and O'Shea, 1998; Leonard and Lin, 2000; Heim, receptors may be the primary functional attribute of 1999). JAK kinases. The JAK-triggered receptor phosphoryla- Conventional protein tyrosine kinases (PTKs) pos- tion can potentially be a rate-limiting event for a sess catalytic domains ranging from 250 to 300 amino successful culmination of downstream signaling events. acids, corresponding to about 30 kDa (Hanks et al., In support of this hypothesis, it has been found that JAK 1988). The location of the kinase catalytic domain, in kinase function is required for optimal activation of the most enzymes, lies near the carboxy terminus of the Src-kinase cascade, the Ras-MAP kinase pathway, the molecule, whereas the amino terminus performs a PI3K-AKT pathway and STAT signaling following the regulatory role (Figure 1). The catalytic domains interaction of cytokine/interferon receptors with their comprise of a characteristic signature of conserved ligands. Aberrations in JAK kinase activity, that may amino acid residues. PTKs can be further grouped as lead to derailment of one or more of the above mentioned members of either the Src subfamily, Tec subfamily or pathways could disrupt normal cellular responses and one of the three di€erent growth factor receptor result in disease states. Thus, over-activation of JAK subfamilies, the EGF-receptor subfamily, the insulin kinases has been implicated in tumorigenesis. In contrast, receptor subfamily or the PDGF-receptor subfamily. loss of JAK kinase function has been found to result in The protein tyrosine kinases encoded by the c-abl and disease states such as severe-combined immunode®ciency. c-fes/fps genes are distantly related to the Src In summary, optimal JAK kinase activity is a critical subfamily. At least nine of the protein tyrosine kinases determinant of normal transmission of cytokine and including c-fgr,c-src,c-abl and c-fes have been growth factor signals. Oncogene (2000) 19, 5662 ± 5679. transduced by retroviruses, where they perform a transforming function (Pierce, 1989). Apart from the Keywords: JAK kinases; STATs; ras; PI3K; CIS/JAB/ kinase catalytic domains, PTKs possess certain other SOCS/SIS protein family; signal transduction characteristic motifs that enable the kinases to interact with diverse signaling intermediates (Figure 1). These domains are represented by the (a) Src-homology 2 (SH2), (b) SH3 domains, (c) the pleckstrin homology Tyrosine kinases and signal transduction domain (PH), (d) a negative regulatory tyrosine in the carboxy terminus and (e) myristylation or palmitoyla- Signal transduction involves multiple sets of proteins tion lipid modi®cation sites at the N-terminus (Cooper interacting in a harmonious fashion to integrate diverse and Howell, 1993; Pawson, 1995). The PH and the cues emanating from the extracellular milieu resulting lipid modi®cation sites are considered to be important in changes in gene expression. Majority of the for attachment of the kinases to membranes. The PH mammalian signal transduction processes are initiated domain may also facilitate association of other as a result of ligand receptor interactions (Kishimoto et signaling proteins to membranes thereby bringing them al., 1994). These interactions result in biochemical in close proximity to the kinases. The JAK kinase changes, which are processed and delivered to the family, which constitutes a distinct family of tyrosine nucleus to bring about changes in gene expression. kinases is a relatively new member of tyrosine gene Molecular cloning of cytokine receptors and subse- families and has been the focus of intensive investiga- quent structure-function studies has revealed that tion during the past few years. While it was originally unlike growth-factor receptors, cytokine receptors lack thought that this family of kinases is mainly involved a cytoplasmic kinase domain. Nevertheless, interaction in interferon and cytokine mediated signal transduction of a cytokine with its receptor has been found to pathways, recent evidence suggests that these kinases act as mediators of multiple signaling pathways and are essential for the normal function of the mammalian *Correspondence: EP Reddy organism. Signal transduction by JAK kinases SG Rane and EP Reddy 5663

Figure 1 Structural organization of the Janus kinases. The structural domains featured in the JAK kinase family are referred to as the JAK homology regions (JH1-JH7). JAK kinases, apart from featuring the functional kinase domain (JH1) at the carboxy terminus, also possess a pseudo-kinase domain (JH2). This kinase like domain in spite of harboring most of the conserved amino acid residues, which are a characteristic of the kinase domain, lacks any observable tyrosine kinase activity. Sequences amino terminal to the kinase and kinase-like domains bear no resemblance to any characterized protein motif. However, there are blocks of homology which are shared among the members of the Janus Kinase family. Also shown are the structures of CIS/SOCS family of proteins that negatively regulate JAK kinase activity

Discovery and structure-function analysis of the Janus The chromosomal locations of the murine and kinases human JAKs have been determined. In humans, the JAK1, JAK2 and TYK2 genes have been mapped to Discovery of the Janus kinases (JAKs) occurred at a chromosomes 1p13.3 (Pritchard et al., 1992), 9p24 time when a variety of approaches were being tested in (Pritchard et al., 1992) and 19p13.2 (Firmback-Kraft attempts to identify novel protein tyrosine kinases. The et al., 1990) respectively. However, using interspeci®c ®rst JAK kinase, which was named TYK2 (tyrosine hybrids JAK2 has been reportedly localized to murine kinase 2), was obtained upon screening a T-cell library chromosome 19 at a region which corresponds to the using low stringency hybridization techniques (Kro- human chromosome 10q23-q24.1 and not to 9p24 as lewski et al., 1990; Firmbach-Kraft et al., 1990). The previously reported (Ihle et al., 1995). JAK3 has been unique structure and function of TYK2 became obvious mapped to human chromosome 19p13.1 (Kumar et only after other members of the JAK family were al., 1996; Riedy et al., 1996). The corresponding identi®ed and characterized. Polymerase chain reaction murine genes have also been mapped with JAK1 on (PCR) using degenerate oligonucleotides spanning the mouse chromosome 4, JAK2 on mouse chromo- heavily conserved kinase domains of members of the some 19 and JAK3 on mouse chromosome 8 (Kono Src family of protein tyrosine kinases resulted in et al., 1996). identi®cation of partial cDNA clones of JAK1 and The unique structure of the JAK kinases clearly JAK2 (Wilks, 1989). Full length cDNA clones of JAK1 distinguishes them from other members of the and JAK2 were subsequently cloned using the partial protein tyrosine kinase family (Figure 1). The most cDNA fragments as probes (Wilks, 1991; Harpur et al., intriguing feature of these proteins is the presence 1992). Their discovery, along with several other of two domains (JH1 and JH2), with extensive members of the tyrosine kinase family resulted in the homology to the tyrosine kinase domains. A second usage of the acronym JAK (Just Another Kinase). interesting feature is the absence of any SH2 or Subsequent sequencing studies revealed that the JAK SH3 domains. Instead, these proteins encode a family of protein tyrosine kinases (PTKs) di€ers group of well-conserved domains termed as JAK markedly from other classes of PTKs by the presence homology (JH1-JH7) domains that follow a non- of an additional kinase domain. To denote this unique conserved amino terminus of about 30 ± 50 amino structural feature, these kinases were renamed as `Janus acids. Of the dual kinase domains identi®ed only kinases' in reference to an ancient two-faced Roman the JH1 domain appears to be functional. The JH2 God of gates and doorways (Darnell, 1998; Darnell et domain, which harbors considerable homology to al., 1994; Ihle et al., 1995, 1997; Pellegrini and the tyrosine kinase domains lacks certain critical Dusanter-Fourt, 1997; Schindler and Darnell, 1995; amino acids required for a functional kinase and Heim, 1999; Leonard and O'Shea, 1998; Schindler, does not appear to be associated with a kinase 1999; Leonard and Lin, 2000; Ward et al., 2000). activity. Both the tyrosine kinase domain (JH1) and Expression studies indicated that JAK1, JAK2 and the pseudo-kinase domain (JH2) are housed at the TYK2 are ubiquitously expressed and are encoded by carboxy terminus of the protein (Figure 1). The transcripts of 5.4 kb (JAK1), two transcripts of 5.3 and other conserved blocks of sequences (JH3-JH7) that 5.0 kb (JAK2), and a 4.4 kb transcript (TYK2), are characteristic to members of the JAK family, respectively. Several independent groups identi®ed comprise approximately 600 N-terminal amino-acids and cloned a fourth member of the JAK family, residues. The precise functions of the JH3-JH7 JAK3 (Johnston et al., 1994; Rane et al., 1994; domains as well as the pseudokinase domain Kawamura et al., 1994; Takahashi and Shirasawa, (JH2) are currently under investigation. The se- 1994; Witthuhn et al., 1994). JAK3 is encoded by a quences of the JH3-JH7 domains bear no resem- 4.2 kb transcript that codes for a 120 kD protein that blance to any characterized protein motif. is expressed predominantly in cells of hematopoietic Considering the variety of interactions and functions origin. Recently, reports describing JAK3 expression in performed by the JAK kinase family members it normal and transformed human cell lines of various seems plausible that these domains facilitate some origins have been published (Lai et al., 1995; Verbsky key functions like protein-protein interactions, et al., 1996). recruitment of substrates, etc.

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5664 N-terminal domain of JAKs mediate their interaction substrates to gain access to the active site (Duhe and with cytokine/interferon/growth hormone receptors Farrar, 1995). Zhou et al. (1997) who engineered point mutations in the activation loop of the JAK3 kinase Recent studies indicate that the amino terminal regions domain studied the importance of tyrosine phosphor- may be involved in receptor binding (Chen et al., 1997; ylation on critical tyrosine residues in modulating JAK Gauzzi et al., 1997). For example, Cacalano et al. kinase catalytic activities. The authors detected multi- (1999) recently mapped the region on JAK3 that is ple auto-phosphorylation sites on JAK3 and mutated obligatory for its functional interaction with the IL-2 two tyrosine residues to phenylalanine (Y980F and/or receptor. These authors identi®ed a single amino acid Y981F). The Y980F mutant exhibited decreased kinase substitution (Y100C) in the N-terminus of the JH7 activity whereas the Y981F mutant exhibited signi®- domain of in a patient with autosomal severe combined cantly increased kinase activity and the double mutant immunode®ciency (SCID). As a result of this mutation, Y980F/Y981F displayed intermediate kinase activity. IL-2 responsive signaling was compromised in B-cell Furthermore, the authors also demonstrated that lines derived from the patient cells. Furthermore, the optimal phosphorylation of JAK3 on other phosphor- authors demonstrated that a region encompassing the ylation sites as well as substrate phosphorylation was JH6 and JH7 domains of JAK3 was sucient for dependent on Y980 phosphorylation. Similarly, Gauzzi interaction of the kinase with the proline-rich Box1 et al. (1996) showed that substitution of two adjacent region of the IL-2 receptor and was sucient in tyrosine residues in the Tyk2 activation loop with reconstituting the IL-2 dependent response. Zhao et al. phenylalanine resulted in the abrogation of TYK2 (1995) using a mutational analysis approach demon- activity in response to IFNa/b. strated that truncation of 239 amino terminal residues of JAK2, but not the JH1 and JH2 domains, resulted in abrogation of the interaction of JAK2 with the Activation of JAK kinases by cytokines/interferons and membrane proximal region of the beta-chain of the growth hormones GM-CSF receptor. A similar N-terminal truncation of JAK2 also abolished its interaction with the growth Janus kinases are generally believed to be present in hormone receptor (Frank et al., 1995). Several other unstimulated cells in an inactive form. Ligand-induced studies that utilized diverse receptor systems have receptor oligomerization, such as cytokine interaction further validated the role of the N-terminus of JAK with its speci®c receptor, serves as a trigger to signal kinases as modular domains that dictate interactions of the recruitment of JAKs to close proximity of the the kinases with receptors. Thus, the amino terminal receptors. Local aggregation of the JAK kinases and region of JAK1 and JAK2 was required for association their subsequent activation by either auto or trans with the IFN gamma receptor chains and downstream phosphorylation is an event which either involves other signaling (Kohlhuber et al., 1997). members of the JAK kinase family, Src family of kinases and/or receptor tyrosine kinases. The JAK kinases either alone as monomers or as homo or JH1 and JH2 domains heterodimers have been implicated in signal transduc- tion processes initiated by a variety of growth factors The kinase like JH2 domain, despite harboring most of and cytokines (Table 1). A number of studies show the conserved amino acid residues that are a char- that JAK kinases are involved in signaling by acteristic of the kinase domain, lacks any observable interleukins such as IL-2, IL-4, IL-7, IL-9 and IL-15 tyrosine kinase activity. It is now clear that this kinase- (Witthuhn et al., 1994; Zeng et al., 1994; Johnston et like domain plays a regulatory role (Luo et al., 1997). al., 1994; Kirken et al., 1994; Tanaka et al., 1994; Yin Interestingly, Saharinen et al. (2000) recently ascribed a et al., 1994). JAK2 is activated following erythropoie- regulatory role to the JAK2 pseudo-kinase domain in tin stimulation of its receptor (Witthuhn et al., 1993) modulation of JAK2 kinase activity. In this study, the while JAK1 and JAK2 have been implicated in authors studied the functional signi®cance of the signaling by IL-3, GM-CSF and IL-5 (Silvennoinen various JAK2 protein domains by mutational analysis. et al., 1993a; Quelle et al., 1994; Lutticken et al., 1994). Deletion of the JAK2 pseudo-kinase domain, but not Stimulation by the cytokines IL-6, CNTF, and LIF of JH3-JH7 domains, negatively regulated JAK2 results in the activation of JAK1, JAK2 and TYK2 catalytic activity as well as STAT5 activation by kinases (Stahl et al., 1994; Narazaki et al., 1994; JAK2. Furthermore, this study indicated that JAK2 Guschin et al., 1995). Also, JAK2 and TYK2 are kinase inhibition was mediated by an interaction activated upon IL-12 stimulation and are proposed to between the JAK2 kinase and the psuedo-kinase play a critical role in IL-12 mediated T-cell di€erentia- domains. The above results were further substantiated tion (Bacon et al., 1995). Similarly, JAK kinases have when the authors detected an inhibition of JAK2 been shown to play a key role in the signal single-kinase domain activity upon trans expression of transduction pathways initiated in response to stimula- the pseudo-kinase domain whereas deletion of the tion by growth hormone (Argetsinger et al., 1993), pseudo-kinase domain resulted in de-regulation of prolactin (Rui et al., 1994; Dusanter-Fourt et al., 1994) JAK2 kinase activity in response to interferon gamma and G-CSF (Nicholson et al., 1994; Shimoda et al., resulting in an increase in STAT activity. 1994). The interferons also transduce their signals via Auto (trans) phosphorylation of conserved tyrosine the JAK kinases, JAK1, JAK2 and TYK2 (Muller et residues in the JAK kinase activation loop determines al., 1993; Velazquez et al., 1992, 1995; Watling et al., the levels of catalytic activation. Such a mode of 1993). JAK3 conveys signals emanating from IL-2, IL- activation is a conserved feature of tyrosine kinases in 4, IL-7, IL-15 and IL-19. In summary it appears that general and the tyrosine phosphorylation may allow speci®c JAK kinases, either alone or in combination

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5665 Table 1 Tyrosine kinases, CIS/SOCS proteins and STATs activated by cytokine/growth factor signal transduction pathways Cytokine JAK-kinase SOCS/CIS/JAB proteins Non-JAK kinase STAT

IL-2 Jak1, Jak2, Jak3 CIS1, CIS3, SOCS1 Fyn, Lck, Hck, Tec, Syk Stat5a, Stat5b, Stat3 IL-3 Jak2 CIS1, CIS2, CIS3, SOCS1 Lyn, Hck, Fyn Stat3, Stat5a, Stat5b IL-4 Jak1, Jak3 CIS1, CIS2, CIS3 Lck, Tec Stat6 IL-5 Jak2 ND Btk Stat1, Stat3, Stat5a, Stat5b IL-6 Jak1, Jak2, Tyk2 CIS1, CIS2, CIS3, SOCS1 Hck Stat1, Stat3 IL-7 Jak1, Jak3 CIS1, CIS3 Lyn Stat5a, Stat5b, Stat3 IL-9 Jak3 ND ND Stat5a, Stat5b, Stat3 IL-10 Jak1, Tyk2 CIS3 ND Stat1, Stat3 IL-11 Jak1, Jak2, Tyk2 CIS3 Src, Yes Stat3 IL-12 Jak2, Tyk2 CIS1, CIS3 Lck Stat4 IL-13 Jak1, Jak2, Tyk2 CIS1, CIS3, SOCS1 Lsk Stat6 IL-15 Jak1, Jak3 ND Lck Stat5a, Stab5b, Stat3 IFNa/b Jak1, Tyk2 ND Lck Stat1, Stat2 IFNg Jak1, Jak2 CIS1, CIS2, CIS3, SOCS1 Hck, Lyn Stat1 Epo Jak2 CIS1, CIS2, CIS3 Lyn Stat5a, Stat5b Tpo Tyk2, Jak2 CIS1, CIS3 ND Stat5a, Stat5b G-CSF Jak2, Jak3 CIS1, CIS2, CIS3 Lyn Stat3 GH Jak2 CIS1, CIS2, CIS3, SOCS1 Src kinases Stat5a, Stat5b, Stat3 PRL Jak2 CIS1, CIS2, CIS3, SOCS1 Src Stat5a, Stat5b Leptin Jak2 CIS1, CIS3 ND Stat3, Stat6, Stat5a, Stat5b GM-CSF Jak2 CIS1, CIS2, CIS3, SOCS1 Lyn, Hck Stat5a, Stat5b CNTF Jak1, Jak2, Tyk2 ND ND Stat3 CT-1 Jak1, Jak2, Tyk2 ND ND Stat3 LIF Jak1, Jak2, Tyk2 CIS1, CIS2, CIS3, SOCS1 Hck Stat3 OSM Jak1, Jak2, Tyk2 ND Src kinases Stat3 EGF Jak1 ND EGF-R, Src Stat1, Stat3, Stat5 PDGF Jak1 ND PDGF-R, Src Stat1, Stat3, Stat5 Insulin Jak2 ND IR, Src Stat1, Stat5B

ND=not determined

with other JAK kinases, may be preferentially and/or cross phosphorylation by other JAK kinases or activated depending on the type of the receptor that other tyrosine kinase family members. This activation is being activated. is presumed to result in an increased JAK kinase activity. The activated JAKs then phosphorylate receptors on target tyrosine sites. The phosphotyrosine Mode of action of JAKs: receptors are primary sites on the receptors can then serve as docking sites substrates of JAK activation that allow the binding of other SH2-domain containing signaling molecules such as STATs, Src-kinases, The importance of JAKs in mediating signals from a protein phosphatases and other adaptor signaling variety of cytokines/growth factors underscores their proteins such as Shc, Grb2 and Cbl. importance in signal transduction in general. However, This model is supported by several studies where the complexity associated with processing signals from investigators have demonstrated the ability of cyto- such diverse sources suggests a complicated mechanism kines such as Erythropoietin (EPO) to rapidly induce of action for the JAK kinases. Although the precise receptor oligomerization leading to JAK2 activation mechanism by which ligand binding results in the (Watowich et al., 1994). Furthermore, studies with activation of JAKs is not known, inferences from chimeric receptors, which combine the extracellular results of various studies has allowed the proposition domains of the EGF receptor with the cytoplasmic of a model to explain the mechanism of activation of domain of the Epo receptor, also provide evidence in JAK kinases (Figure 2). JAK activation is usually support of a role for receptor dimerization/oligomer- determined by an in vitro kinase assay that measures an ization in JAK kinase activation. EGF stimulation of increase in tyrosine phosphorylation of substrates. cells expressing the EGF-Epo chimeric receptors, leads JAKs, when expressed in the baculovirus system are to receptor oligomerization of the chimeras followed by enzymatically active and are phosphorylated on activation of the JAK2 kinase and subsequent tyrosine residues. Their overexpression in mammalian induction of mitogenesis (Ihle et al., 1995). Similar cells also leads to constitutive activation, most chimeric receptor based studies have elucidated the probably due to dimerization (Colamonici et al., importance of oligomerization of the bc-chain of the 1994a,b; Quelle et al., 1995; Duhe and Farrar, 1995; receptors for IL-3/IL-5/GM-CSF in signal transduction Yamamoto et al., 1994; Wang et al., 1995; Eilers et al., by their respective cytokines (Eder et al., 1994; 1996). On the other hand, a JAK kinase in complex Jubinsky et al., 1993). Also, studies with chimeric with a native un-liganded receptor is in a catalytically receptors for IL-2 suggest a similar role for receptor inactive latent state. Receptor dimerization/oligomer- oligomerization as a prerequisite for signal transduc- ization due to ligand binding results in the juxtaposi- tion by IL-2 (Watowich et al., 1994). In these cases, as tioning of the JAKs, which are in the vicinity either with the EGF-Epo chimeric receptors, the chimeras through homo- or heterodimeric interactions. This were functionally capable of induction of mitogenesis recruitment of the JAK kinases appears to result in supporting the receptor oligomerization concept for their phosphorylation either via autophosphorylation JAK kinase activation. Such a model is readily

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5666

Figure 2 Signal transduction pathways mediated by JAK kinases. Janus kinases are cytoplasmic tyrosine kinases that are generally believed to be present in unstimulated cells in an inactive form. Ligand-induced receptor oligomerization such as cytokine interaction with its speci®c receptor serves as a trigger to signal the recruitment of the JAKs to close proximity of the receptors. The local aggregation of the JAK kinases and their subsequent activation by either auto or trans phosphorylation results in the phosphorylation (indicated as -P) of the receptors as well as downstream substrates. The best characterized molecular event following the activation of JAK kinases is the tyrosine phosphorylation and activation of a family of latent cytoplasmic transcription factors termed the Signal Transducers and Activators of Transcription (STATs). The STATs upon activation oligomerize, translocate to the nucleus and bind speci®c sequence elements on the promoters of target genes to activate transcription. In addition to the activation of STATs, JAKs mediate the recruitment of other molecules involved in signal transduction such as the src-family kinases, protein tyrosine phosphatases, MAP kinases, PI3 kinase etc. These molecules process downstream signals via the Ras-Raf-MAP kinase and PI3 kinase pathways which results in the activation of additional transcription factors. The combined action of STATs and the other transcription factors (TF) activated by these pathways dictate the phenotype produced by a given cytokine/interferon stimulation

applicable for both single chain receptors such as those (1996) used mutational analysis to demonstrate that for Epo, prolactin, growth hormone and G-CSF as the Box 1 region of cytokine receptors speci®es the well as multi-chain receptors such as those for IL-3, region of interaction with JAK kinases. IL-2 stimula- IL-5 and GM-CSF. Cytokine binding results in the tion leads to the activation of JAK3 but not of JAK2 association of the JAKs with one of the subunits. The whereas erythropoietin conveys its signals by recruiting receptor associated JAK kinase can then either process and activating JAK2. These authors created chimeric the signal or subsequent to the receptor oligomeriza- receptor molecules by switching the Box 1 region of the tion can recruit other JAKs in the vicinity. Homo or EpoR with that of the beta chain of the IL-2R. The heterodimerization of the JAKs followed by their EpoR-IL-2R fusion receptor was sucient to induce activation upon phosphorylation ®nally results in the activation of JAK2 in response to stimulation with IL- propagation of the initial signal. 2 thereby con®rming the role of Box 1 region in While the importance of receptor oligomerization in determining the speci®city of JAK kinase activation. the activation of the JAK kinases was being Mutational analysis of several receptors corroborated documented, other studies focused on delineating the such a theory. Mutations in the membrane proximal regions of the receptors that play a critical role in regions of the Epo receptor resulted in the elimination JAK-receptor interactions. These studies clearly de- of JAK2 association and activation as well as monstrated the importance of membrane proximal abrogation of mitogenesis in response to erythropoietin domains of the cytokine receptors containing the Box (Witthuhn et al., 1993). Similar mutational analysis 1 and Box 2 motifs in receptor/JAK interactions. The demonstrated the importance of the membrane prox- Box 1 motif comprises of approximately eight proline- imal domains of the prolactin receptor in association rich amino acids, that are required for interaction of and activation of JAK2 (DaSilva et al., 1994). Also, several cytokine/growth factor receptors with JAK2. the Box1 and Box2 regions of gp130 were shown to be Thus, the Box 1 region of the receptors for prolactin, required for association and activation of JAK1 and growth hormone, erythropoietin, IL-6 and related JAK2 (Narazaki et al., 1994). The requirement of cytokines, that utilize the gp130 signal transducing membrane proximal motifs in JAK kinase interaction receptor subunit, is required for association with JAK2 is absolute even in the case of receptors that lack Box 1 (Witthuhn et al., 1993; Miura et al., 1994; Tanner et motifs. JAK1 and JAK3 constitute integral compo- al., 1995; Vanderkuur et al., 1994; Goujon et al., 1994; nents of the IL-2R signal transduction apparatus. The Hackett et al., 1995; DaSilva et al., 1994). Jiang et al. IL-2R gamma-c chain does not contain a typical Box1

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5667 motif and yet can activate the JAK1 and JAK3 transducing IFN-generated signals (Muller et al., 1993). kinases. It has been determined that a membrane Thus, while both type I and II IFN receptors appear to proximal region of the IL-2R encompassing 52 amino associate with JAK1, it appears that TYK2 and JAK2 acids is sucient for JAK3 binding, activation of are associated with one chain of the IFN a/b and IFN JAK1 and JAK3 and other downstream signaling g receptors, respectively (Soh et al., 1994; Novick et al., events such as induction of transcription factors c-fos 1994; Colamonici et al., 1994a,b; Igarashi et al., 1994). and c-myc and cell proliferation (Nelson et al., 1996). The possibility of existence of a JAK kinase cascade These studies indicated that aberrations at any step of in interferon-a/b and g signaling, by virtue of one JAK the aggregation of ligand-receptor/JAK kinase hetero- phosphorylating and activating the other JAK kinase oligomeric complexes could be sucient to abolish seemed intriguing at the time. Such a hypothesis was activation of the kinases and prevent mitogenesis, most tested and excluded, since it was observed that both probably by eliminating indispensable downstream JAK kinases were required and functionally active for signaling events. tyrosine phosphorylation and activation of each other. However, separate studies have attempted to analyse the temporal order of activation of JAK kinases using Interdependence and hierarchy of JAKs in signaling the interferon model system. JAK1 was shown to be obligatory to initiate phosphorylation and activation of The mechanisms by which JAK kinases are activated TYK2 in an interferon-a/b receptor complex (Gauzzi et following cytokine/growth factor stimulation are al., 1996). On the other hand, in the case of the beginning to unravel. Although the activation of interferon-g receptor complex, JAK2 serves as the STATs constitutes an important step concurrent to initiator kinase which phosphorylates JAK1 as part of JAK kinase activation, several events prior to STAT the signaling cascade (Briscoe et al., 1996). Signaling activation seem to be unique to JAK kinases. The role via receptors that contain gp130 indicated that JAK1 is of JAK kinases in interferon-a/b and g signaling the obligatory kinase and JAK2 and TYK2 served as exempli®es the inter-dependence in JAK kinase signal- additional components. Interestingly, in this system it ing. Consistent with subunit-mediated association of was shown that the absence of one JAK kinase did not multiple JAK family members, type I (IFN a and b) a€ect phosphorylation of the other JAKs leading to the and type II (IFN g) interferons bind to two unrelated speculation that JAKs could serve as substrates for receptor complexes, each of which utilizes both other tyrosine kinases, such as the Src-family of PTKs common and distinct JAK kinases to transmit its (Guschin et al., 1995). signals. Elucidation of the involvement of JAK kinases It is presumed that the precise basis for the in interferon signaling has for the ®rst time suggested interdependence is the multi-chain/subunit composition the involvement of multiple family members, in which of receptors. Such a theory is validated by examination individual members, following receptor aggregation, of the structures of interferon-a/b and g receptors. cooperate in the formation of JAK kinase heterodimers Both the interferon-a/b and g receptors consist of at (Ihle et al., 1995). Unlike traditional experiments which least two chains and it has been shown that the b-chain involve the introduction of systematic deletions within of the interferon-a/b receptor binds JAK1 (Novick et a cDNA and its encoded protein, the mutants which al., 1994) whereas TYK2 associates with the a-chain were used to dissect the interferon signaling pathways (Colamonici et al., 1994a,b). In the case of interferon-g, are naturally occurring and are incapable of respond- co-immunoprecipitation studies indicated an associa- ing to either IFN a, b or g (McKendry et al., 1991; tion between JAK1 and the a-chain whereas JAK2 was John et al., 1991; Pellegrini et al., 1989). One cellular shown to be associated with the complex upon ligand mutant, designated as U1, fails to respond to IFN a or binding. Therefore, the most plausible theory is that IFN b, but remains responsive to IFN g. The gene ligand binding to the receptors followed by receptor responsible for the restoration of a cellular response to oligomerization leads to the recruitment of the JAKs IFN a/b, following expression cloning, was found to be into the hetero-oligomeric complex. TYK2 (Velazquez et al., 1992). Another set of mutants, the g mutant cell lines, fail to respond to IFN g, but retain their IFN a/b responsiveness. Ectopic expression Substrates of JAKs of JAK2, but not TYK2 or JAK1, in these cells results in the restoration of a cellular response to IFN g; It is becoming increasingly clear that JAKs phosphor- subsequent experiments demonstrated JAK2 tyrosine ylate multiple substrates, the most important of which phosphorylation and activation in cells following are the receptors themselves. A number of potential treatment with IFN g (Watling et al., 1993; Shuai et substrates have been identi®ed using di€erent ap- al., 1993; Silvennoinen et al., 1993b). proaches. Thus, GST fusion proteins encoding intra- Thus far the involvement of JAK kinases in cellular domains of a number of cytokine receptors interferon signaling seemed relatively simple and could be readily phosphorylated by activated JAK complete, in which an individual JAK kinase family kinases in vitro (Zhao et al., 1995; Colamonici et al., member was responsible for the transduction of signals 1994a; Gauzzi et al., 1996). Similarly, an analysis of from a given receptor. A new twist in JAK signaling proteins phosphorylated following antibody cross- originated from the analysis of the U4 mutant, which, linking of a CD16/JAK2 chimera in BA/F3 cells under normal conditions, is incapable of responding to revealed that STAT-5, Shc and Shp proteins were each of the above interferons. Using an approach amongst several proteins that were phosphorylated analogous to that used to identify the genetic lesion suggesting that they could be the substrates for JAKs within the g mutants, ectopic expression of JAK1 in (Sakai et al., 1995). Similarly a chimeric construct these cells results in a cell type which is fully capable of containing the extracellular domain of EGF and the

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5668 JAK2 kinase domain was found to mediate phosphor- ylation of STAT-5 following EGF stimulation (Naka- mura et al., 1996).

STAT signaling pathways

STATs are among the best-characterized JAK sub- strates and activation of virtually all cytokine/interfer- on/growth hormone receptors lead to the activation of one or more STATs. For example, IL-3 activation of hematopoietic cells appears to lead to the activation of multiple STATs, which include STAT-1, STAT-3, STAT-5 and STAT-6 (Reddy et al., 2000). The nature of STATs that are activated appear to be more dependent of the cell line that is used in the study, rather than the cytokine or the nature of JAK activated by cytokine/receptor interactions. This observation suggests that neither the cytokine receptors nor the JAKs by themselves dictate the nature of STATs that Figure 3 STAT protein structure. Cytokines signal via members are activated by a given cytokine. It is also interesting to of the STAT family of proteins. The various STAT proteins, note that a C-terminal deletion mutant of IL-3 bc chain, many of which are derived as a result of alternative splicing termed bc541 was found to be de®cient in its ability to events (e.g.: Stat 1, Stat 3, Stat 4 and Stat 5) comprise of activate STAT-5, while still being able to activate JAK2 approximately 800 amino acid residues. Sequence analysis indicates that the STAT proteins harbor conserved motifs, such (Smith et al., 1997). These observations also imply that as the DNA binding region (DNAB), SH2 and putative SH3 while JAK2 activation is a critical step in IL3/IL5/ domains that allow DNA-protein and protein-protein interac- GMCSF mediated activation of their cognate receptors, tions. The transactivation (TA) domain and the highly conserved it is by itself not adequate for STAT activation. phosphorylatable tyrosine (pY) and serine (pS) residues that are essential for optimal STAT activation reside at the carboxy terminus of the proteins The signal transducers and activators of transcription

Over the past few years, researchers have deciphered a 3B with di€erent transcriptional activation functions signaling pathway, which initiates within the cytoplasm (Schafer et al., 1995). STAT 5 exists in two isoforms, but quickly translocates to the nucleus to activate termed as STAT 5A and STAT 5B, which are encoded transcription of target genes. This novel signaling by two separate genes, which are tandemly linked on pathway features a group of transcription factors human chromosome 17 and mouse chromosome 11 named as STATs or Signal transducers and Activators (Lin et al., 1996; Copeland et al., 1995). The two of Transcription (Schindler and Darnell, 1995; Darnell, proteins exhibit extensive sequence homology and 1997). These transcription factors were originally di€er from each other mainly in the amino and described by Darnell and his co-workers (Darnell et carboxy terminal domains, with the transactivation al., 1994; Darnell, 1997) as ligand-induced transcription domain showing most divergence. Both of these genes factors in interferon-treated cells. Subsequent studies seem to play a critical role in IL3/IL-5/GMCSF- by a number of groups showed that STATs play a induced proliferation of hematopoietic cells as well as critical role in signal transduction pathways associated prolactin-induced proliferation of mammary epithelial with several cytokines and neurokines including the cells (Takaki et al., 1994; Quelle et al., 1994; Cornelis interleukins, the interferons, erythropoietin, prolactin, et al., 1995; Matsuguchi et al., 1997; Itoh et al., 1996). growth hormone, oncostatin M (OSM), and ciliary Like most transcription factors, STATs exhibit a neurotrophic factor (CNTF) (Darnell et al., 1994; modular structure with ®ve well de®ned domains, Darnell, 1997). To-date, six mammalian genes that which include the N-terminal conserved domain, the code for di€erent STATs have been identi®ed, all of DNA-binding domain, a putative SH3-like domain, a which encode for proteins of 750 ± 850 amino acids SH2 domain and C-terminal transactivation domain long and are characterized by the presence of a DNA- (Figure 3). The amino terminal region of STATs is well binding domain followed by putative SH3 and SH2 conserved and appears to be critical for STAT function domains (Darnell, 1997). In addition, alternative as small deletions in this region were found to splicing or post-translational proteolytic cleavage eliminate the ability of STATs to be phosphorylated. reactions appear to generate additional forms for This region is followed by the DNA binding domain STAT-1,3 and 5, bringing the total number of STATs that is usually located between amino acids 400 and currently described in literature to eight. Thus, two 500 (Horvath et al., 1995). This region is highly isoforms of STAT1 have been described, which have conserved amongst the STATs, and all STATs with been termed as STAT1_ or p91 and STAT 1_ or p84. the exception of STAT-2 di€erentially bind more than These two proteins were originally discovered by 10 related GAS sequences that are characterized by the Darnell's group in association with STAT-2 and a consensus sequence motif, TTNCNNNAA (Horvath et fourth protein termed p48, which constituted the al., 1995; Xu et al., 1996). Adjacent to the DNA multicomponent transcription factor ISGF3. STAT-3 binding domain lies the putative SH3 domain. This also exists in two forms termed STAT 3A and STAT domain appears to be least conserved and it is at

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5669 present unclear as to whether it truly functions as a dominant negative JAK1 and TYK2 mutants in human SH3 domain, since the critical amino acid residues cells suppressed transcriptional activation of a lucifer- involved in binding to PXXP motifs do not appear to ase reporter gene downstream from an ISRE. In be well conserved. Adjacent to this putative SH3 addition, these dominant negative mutants were found domain lies the SH2 domain, which is highly conserved to inhibit STAT-1 and STAT-2 phosphorylation amongst the STATs and appears to play a very (Krishnan et al., 1997). important role in STAT signaling. Thus, it is critical However, the activated JAK kinases do not seem to for the recruitment of STATs to the activated receptor exhibit speci®city for a particular STAT as di€erent complexes and is required for the interaction with JAK receptors activate a common STAT, even though they and Src kinases. In addition, it is required for STAT activate distinctively di€erent JAK kinase (Kohlhuber homo and hetero dimerization, which in turn appears et al., 1997; Darnell, 1997). In addition chimeric to be critical for nuclear localization and DNA binding receptor molecules with di€erent JAK binding sites activities. It is also possible that this domain but containing the same STAT-binding site were found participates in other protein-protein interactions that to activate the same STAT (Kotenko et al., 1996; have not yet been fully deciphered. Immediately Kohlhuber et al., 1997). Thus, the speci®city for STAT downstream of the SH2 domain, around position phosphorylation appears to be determined by the 700, all STATs contain a tyrosine residue, which plays docking sites for STATs that are present in the a critical role in STAT activation. Phosphorylation of receptor molecules and not JAK kinases. this tyrosine residue has been found to be essential for The two isoforms of STAT5, STAT5a and STAT5b the activation and dimerization of STATs. Phosphor- as well as STAT6 are activated by receptors for IL-3, ylation of this tyrosine appears to be achieved by GM-CSF and IL-5 (Quelle et al., 1995; Azam et al., growth factor receptors as well as JAK and Src 1995). IL-3 stimulation leads to activation of JAK2 kinases, depending on the nature of the cell type and and phosphorylation and activation of STAT5a (via the ligand/receptor interactions. The C-terminal do- tyrosine phosphorylation of Y694) and STAT5b (via main of STATs is required for transcriptional tyrosine phosphorylation of Y699). Mutational studies transactivation. Phosphorylation of a single serine with the IL-3, GM-CSF and IL-5 receptors have residue in the TA domains of STAT1, STAT3, STAT4 shown that JAK2 is essential for STAT activation and STAT5, can enhance the transcriptional activity. (Kouro et al., 1996; Matsuguchi et al., 1997; Di€erentially spliced STATs such as STAT 1_, lacking Caldenhoven et al., 1995; Mui et al., 1995a,b; Smith the C-terminal TA domain can still bind DNA but do et al., 1997). However, a C-terminal mutant of the bc not activate transcription. chain has been described that can activate JAK2 but is STATs, which are normally localized in the unable to activate STAT5 indicating that activation of cytoplasm are activated when phosphorylated on the JAK2 is not sucient for STAT5 activation (Smith et tyrosine located around residue 700, which facilitates al., 1997). their dimerization and translocation to the nucleus A role for Src kinases in STAT activation was ®rst (Darnell et al., 1994; Schindler and Darnell, 1995). The suggested by studies aimed at investigating the phosphorylation of STATs is known to occur molecular mechanisms associated with Src-mediated immediately after the binding of growth factors or transformation of ®broblasts and hematopoietic cell interferons to their receptors. Studies with interferon lines. Thus, Src-transformed NIH/3T3 cells were found (IFN) receptor signaling have revealed that JAK family to express a tyrosine phosphorylated form of STAT-3 kinases are involved in IFN-speci®c gene expression in in a constitutive manner (Yu et al., 1995; Cao et al., cooperation with STATs. Studies with mutant cell lines 1996). In addition, results from Cao et al. (1996) which were unable to respond to interferons, described showed that v-Src can bind to STAT-3 and phosphor- earlier, clearly established a critical role for JAKs in ylate this protein in vitro. Transfection of an expression the interferon signaling pathways. vector coding for a dominant negative mutant of Since the cytokine and interferon receptor-ligand STAT-3 into v-Src-transformed NIH/3T3 cells resulted interactions were found to result in the activation of in a block to v-Src-mediated transformation of cells, JAK kinases, which often exist in association with further enforcing the notion that STAT-3 signaling cytokine receptors, and this activation was obligatory plays a critical role in this transformation process. for the activation of STATs, it was proposed that In our studies aimed at delineating the molecular STATs might be substrates for JAK kinases. This mechanisms associated with v-Src-mediated transfor- notion was further supported by studies, which showed mation of hematopoietic cells, we used 32Dc13 cells in vitro phosphorylation of puri®ed STAT-5 by JAK-2 that are derived from normal mouse bone marrow and (Flores-Morales et al., 1998). In this study, the require IL-3 for proliferation and survival. These cells, investigators expressed STAT-5 in Sf9 insect cells using when cultured in the presence of G-CSF were found to the baculovirus expression system and showed that this terminally di€erentiate into mature granulocytes in a recombinant protein can be phosphorylated by JAK-2 period of 10 ± 12 days (Valtieri et al., 1987). When that confers DNA binding property to STAT-5. In these cells were grown in a medium lacking IL-3 or G- addition, these investigators showed STAAT-5 in its CSF, they were found to undergo apoptosis in a period non-phosphorylated form was able to form a stable of 24 ± 48 h. This cell line, when transformed with complex with activated JAK2, while non-activated expression vectors containing v-abl or bcr-abl or v-src JAK2 and phosphorylated STAT-5 were unable to was found to grow inde®nitely in the absence of IL-3 participate in complex formation. A second line of and to form tumors in nude mouse assays (Kruger and evidence, which suggested that JAKs might be Anderson, 1991; Laneuville et al., 1991; Rovera et al., activators of STATs, came from the use of dominant 1987). In addition, expression of either v-src,v-abl or negative mutants of JAK-2. Over-expression of bcr-abl was found to block the ability of 32Dc13 cells

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5670 to undergo GCSF-induced di€erentiation (Rovera et create docking sites on the receptors for binding of SH2- al., 1987). Interestingly, several other tyrosine kinases contacting proteins such as STATs, Src-kinases and closely related to v-src failed to induce such cytokine- other signaling intermediates (Reddy et al., 2000). JAKs independence of these cells suggesting a very speci®c or Src-kinases, depending on the nature of STAT that is target for v-src in these cells. Thus, when v-fgr or an being activated then induce tyrosine phosphorylation activated form of c-fgr which lacks the C-terminal 12 and activation of STAT proteins. amino acids (c-fgrD) were expressed in this cell line, The notion that di€erent STATs might be phos- they failed to render these cells IL-3 independent for phorylated by di€erent tyrosine kinases under di€erent growth (Chaturvedi et al., 1997). conditions is suggested by two other observations. Like To understand the possible mechanisms underlying v-Src, v-Abl and BCR-ABL have been shown to the IL-3 independent growth of v-src-transformed transform hematopoietic cells and render them cyto- 32Dc13 cells, we examined the phosphorylation status kine-independent for growth (Pierce et al., 1985; Cook of JAK-1, JAK-2 and JAK-3 kinases in the v-src and v- et al., 1985; Mathey-Prevot et al., 1986). Thus, v-Abl fgr-transformed cells. These studies showed that none of encoded by the Abelson Murine Leukemia Virus was the JAK kinases were phosphorylated by v-Src or v-Fgr. found to transform both B-cells and myeloid cells However, v-src-transformed cells were found to express resulting in cytokine-independent growth of these cells constitutively phosphorylated forms of STAT-1, 3 and 5, (Pierce et al., 1985; Cook et al., 1985; Mathey-Prevot et which exhibited appropriate DNA binding activities. al., 1986). Examination of the molecular mechanisms Our results also showed that STAT-3 co-immunopreci- associated with v-Abl mediated transformation show pitates with v-Src suggesting that the activation of that B-cells transformed by this oncogene exhibit STAT-3 occurs due to direct association with v-Src constitutively activated forms of JAK-1, JAK-3 as well (Chaturvedi et al., 1998). Since hematopoietic cells as STAT-1,3 and 5 (Danial et al., 1995, 1998). In express high levels of c-Src and since Src-kinases addition, activated JAK-1 in these cells was found to associate with IL-3 receptor following its activation by be associated with the v-Abl protein. Mapping the IL-3, this observation suggested that c-Src might play a JAK-1 interaction domain in v-Abl showed that a role in the activation of STAT-3 in normal hematopoie- region of the C-terminal domain of v-Abl protein binds tic cells following IL-3 stimulation. This was further to JAK-1 and a mutant of v-Abl that lacks this JAK-1 con®rmed by our studies where we examined the binding domain failed to activate JAK-1 and STAT phosphorylation status of JAKs and STAT-3 in proteins. In addition, a dominant negative mutant of 32Dc13 cells following IL-3 stimulation. These studies JAK-1 was found to inhibit STAT activation mediated showed that IL-3 stimulation induces rapid tyrosine by v-Abl suggesting that the v-Abl protein activates phosphorylation of JAK-1 and JAK-2 in 32Dc13 cells. STATs via activation of JAK-1. In addition, our results show that interaction of IL-3 In sharp contrast, the BCR-ABL oncogene, a gene with its receptor leads to the phosphorylation and closely related to the v-abl oncogene was found to nuclear translocation of STAT-1, STAT-3 and STAT- constitutively activate STAT-1 and STAT-5 with very 5. In addition, this ligand/receptor interaction leads to little or no activation of JAKs (Carlesso et al., 1996; the activation of c-Src kinase activity, which in turn Ilaria et al., 1996; Frank and Varticovski, 1996; facilitates the binding of c-Src to STAT-3. This Niebrowska-Skorska et al., 1999). Unlike with v-abl association leads to the phosphorylation of STAT-3, transformed B-cells, over-expression of dominant allowing STAT3 to translocate to the nucleus. Expres- negative JAK kinase mutants in these cell lines had sion of a dominant negative mutant of src (AMSrc) in no e€ect on STAT phosphorylation (Ilaria et al., 1996). these cells results in a block to IL-3 mediated Similarly, K562 cells, which are Philadelphia-chromo- phosphorylation of STAT-3, and its ability to bind to some positive express constitutively activated STAT-5 DNA (Chaturvedi et al., 1998). On the other hand, and over-expression of a dominant negative STAT-5 expression of a dominant negative mutant of JAK2 could suppress the transformed state of these cells. (JAK2KE) had no e€ect on Il-3-mediated activation of Activation of STATs by BCR-ABL oncogene was STAT-3. Our results also show that AMSrc does not shown to be dependent on the presence of the SH2 and a€ect the phosphorylation of JAK2, suggesting that two SH3 domains (Niebrowska-Skorska et al., 1999), independent pathways mediate JAK and STAT-3 suggesting a Src-like interaction reported by us phosphorylation events. These results suggest that Src (Chaturvedi et al., 1998). These observations would family kinases mediate the phosphorylation of STAT-3 also provide a molecular basis for the inability of v-Abl mediated by IL3/receptor interactions and play a critical to directly activate STATs, since this oncoprotein lacks role in signal transduction pathways associated with the SH3 domain that is present in the BCR-ABL myeloid cell proliferation. These results suggest that oncogene (Reddy et al., 1983). activation of STAT-3 following cytokine stimulation of A second line of evidence comes from the study of certain hematopoietic cells might follow a very di€erent Kazansky et al. (1999) who studied the DNA-binding pathway from that seen with STAT-5. These results and tyrosine phosphorylation of STAT-5A and STAT- taken together indicate that while one or both isoforms 5B following prolactin activation or Src activation. of STAT-5 interact directly with JAK-2, which in turn Their results show that following prolactin activation, mediates their phosphorylation, STAT-3 activation both STAT5A and STAT5B were rapidly phosphory- might require its interaction with c-Src, which in turn lated and translocated to the nucleus. Similar to mediates its phosphorylation (Chaturvedi et al., 1998). prolactin activation, src activation resulted in tyrosine Based on these results, we proposed the JAK-SRC- phosphorylation and DNA binding of both STAT5A STAT model, where JAK kinases may be more crucial to and STAT5B. Furthermore, overexpression of a phosphorylation of the cytokine/growth factor recep- dominant negative mutant of JAK-2 prevented pro- tors. Moreover, JAK-mediated phosphorylation may lactin-induced tyrosine phosphorylation and nuclear

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5671 translocation of STAT5A and STAT5B. In sharp tion of JAK2, STAT3 as well as p42, p44 MAP contrast, over-expression of dominant negative JAK-2 kinases. MAP kinase tyrosine phosphorylation corre- had no e€ect on Src-mediated phosphorylation of lated with both the proliferative response and JAK2 STATs. In contrast, there was a modest increase in the activation (Nicholson et al., 1995). Recently, Mizugu- levels of Src-mediated phosphorylation of STAT5 in chi et al. (2000) described the JAK kinase mediated the presence of dominant JAK-2. These studies again activation of both the Ras and STAT pathways in cell suggest that there exist two independent pathways that proliferation. Conditional activation of JAK kinases mediate STAT activation, one that is dependent on confers IL-3 independence to BA/F3 cells that requires JAKs and one that is independent of Src kinases. The functional Ras and STAT5 proteins. This observation context of the cytokine and cell type appears to play a indicates an obligatory role for the Ras and STAT critical role in determining as to which pathway is proteins as targets of JAK activity. In agreement with utilized by the cell. the above studies, overexpression of Ras or STAT5 alone does not confer IL-3 independence whereas concomitant activation of Ras and STAT5 is sucient Cross-talk among JAKs and components of other to confer IL-3 independence. The same group also, signaling pathways elucidated the role of JAK kinase activity in Ras signaling using a TYK2 protein that was modi®ed by Signal transduction in response to a variety of ligands the addition of a membrane localization sequence and involves the activation of the Ras signaling pathway a chemical dimerizer (coumermycin)-dependent dimer- (Egan and Weinberg, 1993). In the Ras pathway, ization sequence (Mizuguchi and Hatakeyama, 1998). typi®ed by signal transduction in response to Erythro- The modi®ed TYK2, upon activation by dimerization, poietin as well as IL-3, cytokine stimulation results in conferred IL-3 independence to pro-B lymphoid cells the recruitment and tyrosine phosphorylation of Shc. that was abolished by expression of dominant negative Following phosphorylation, Grb2 associates with Shc Ras indicating the mandatory role for Ras proteins as and with Sos. This results in an increase in GTP-bound downstream targets of JAK kinase signals. Interferon Ras, activation of Raf-1, followed by activation of the stimulation led to activation of the Ras pathways that mitogen-activated (MAP) kinases and induction of also depend on JAK kinase activities (Sakatsume et al., primary response genes such as c-myc,c-fos, etc. 1998; Stancato et al., 1997). Sakatsume et al. (1998) Recently, several studies have explored the interdepen- showed that activation of the Raf-1 protein by dence between the JAK kinase signaling pathway and interferon gamma was Ras-independent but required the Ras-linked to MAP kinases pathway as well as the kinase activity of JAK1. Similarly, Stancato et al. other key signaling molecules such as Vav, PI-3 kinase (1997) reported that activation of Raf-1 and the MAP and the protein tyrosine phosphatases such as SH- kinase pathways, upon stimulation of cells with PTP1. Evidence from these studies suggests that there interferon beta and oncostatin M, required JAK1 may be interactions amongst the diverse components of kinase activity. The above studies, taken together the cells' signal transduction repertoire. Also, data indicate that JAK kinases target both the Ras and indicates that cells have equipped themselves with STAT pathways to exert their biological e€ects. `safety routes' in the event of malfunction of a Activation of Ras and PI3Kinase as well as STAT particular `primary' signaling pathway. The availability proteins leads to increased induction of transcription of an optional signaling route, available via interac- factors such as c-jun. Also, c-jun levels are elevated in tions with multiple signaling molecules, enable cells to B-lymphoid cells exposed to ionizing radiation (IR). survive the loss of the `primary' signaling route. Thus, Goodman et al. recently demonstrated that the although the Ras and JAK kinase pathways mediate induction of c-jun in IR treated cells required the distinct signals it is becoming evident that there may be activation of JAK3 but was impervious to the interdependence among the two pathways. Thus, activation status of the Btk, Syk and Lyn tyrosine phosphorylation of the receptors by JAK kinases kinases (Goodman et al., 1998). creates potential docking sites for adaptor molecules Interaction of JAK2 with the SH2 domain of p95 such as Shc, the p85 subunit of PI-3K, STAT proteins Vav, a protein expressed in hematopoietic cells, was and other kinases such as Src-kinases which can reported in cells stimulated with GM-CSF (Matsuguchi associate with the phosphorylated receptors via their et al., 1995). Also, in IL-7 stimulated T cells, members SH2 domains. Once recruited to the receptor complex, of the JAK family were shown to regulate the activity the JAK kinases can phosphorylate multiple down- of PI-3 kinase. Coupling of the JAK signaling pathway stream signaling molecules to further propagate the and Shc phosphorylation was demonstrated in signal Ras and JAK kinase pathways. transduction events from c-Mpl, a member of the The abilities of various receptors to integrate cytokine receptor family and the receptor for throm- activation of the JAK kinase pathway to elements of bopoietin (Gurney et al., 1995). Integration of the JAK the Ras pathway have been extensively documented and Ras pathways was evident in IL-6 induced signals (Alam et al., 1995; Bates et al., 1996; Kumar et al., in a B-cell line AF10 in which JAK1, p52Shc, Raf-1 1994). Carboxy terminal truncations in the IL-3 and MEK-1 were activated (Kumar et al., 1994). receptor b-chain resulted in the loss of activation of Studies involving signal transduction by growth the Ras pathway, although the ability to activate JAK2 hormone (GH) indicated that JAK2, Ras and Raf and induce mitogenesis was retained providing evi- are required for activation of the MAP kinases dence for the existence of alternate signaling pathways (Winston and Hunter, 1995). Based on this study, it in cytokine signaling (Quelle et al., 1994). In a similar was suggested that JAK kinases might represent a study, it was demonstrated that distinct regions of the common component during activation of the ERK2/ G-CSF receptor are required for tyrosine phosphoryla- MAPK and STAT signaling pathways, which appeared

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5672 to bifurcate upstream of Ras activation but converged the proteasome inhibitor MG132 was able to stabilize with the phosphorylation of STATs by the ERK/ the IL-2 induced tyrsoine phosphorylation of Jak1 and MAPK proteins. The role of JAK kinases in insulin Jak3. Also, treatment of IL-3 stimulated Ba/F3 with signaling demonstrated that JAK1 and JAK2 consti- proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl- tutively associate with Grb2 via interactions with the norleucinal (LLnL), resulted in a stabilization of SH3 domains of Grb2 (Giorgetti-Peraldi et al., 1995). tyrosine phosphorylation of the IL-3 receptor-beta Several studies have reported the involvement of common chain, STAT5, Shc, and mitogen activated JAK kinase activity in the modulation of PI-3 Kinase protein kinases (MAPKs) (Callus and Mathey-Prevot, function. GM-CSF stimulation of neutrophils triggers 1998). Furthermore, these authors showed that these the activation of Jak2, STAT3, STAT5B, and P13K. stable phosphorylation events were the result of a Treatment of these cells with a JAK2 inhibitor AG-490 prolonged activation of JAK1 and JAK2 kinases. resulted in abrogation of phosphorylation of the p85 subunit of PI-3K in response to GM-CSF stimulation (Al-Shami and Naccache, 1999). Further studies Negative regulation by protein tyrosine phosphatases indicated that the p85 subunit associated with JAK2 and this interaction was not dependent on the two SH2 Protein tyrosine phosphatases (PTPs) regulate the kinase domain of PI-3K. Also, Yamauchi et al. (1998) using activities of tyrosine kinases by de-phosphorylating cells either de®cient in JAK2 or harboring dominant tyrosine residues involved in catalytic function (Fischer negative JAK2, showed that the insulin-receptor et al., 1991; Pallen et al., 1992). Optimal activation of substrate (IRS) proteins, IRS-1, IRS-2 and IRS-3, are JAK kinases is positively regulated by phosphorylation phosphorylated by JAK2. Furthermore, these authors of a critical tyrosine residue in the kinase-activating elegantly demonstrated that upon phosphorylation by domain (Gauzzi et al., 1996; Feng et al., 1997; Zhou et JAK2, the phosphorylated IRS proteins serve as al., 1997; Liu et al., 1997; Weiss and Schlessinger, 1998). sca€olding intermediates to allow docking and sub- Several studies have demonstrated the role of protein sequent activation of PI3-Kinase. Stimulation of tyrosine phosphatases in the regulation of JAK signaling cardiac myocytes by LIF promotes interaction between pathways (David et al., 1995; Klingmuller et al., 1995; JAK1 and PI-3K that leads to increased PI-3K activity Yetter, 1995). PTPs such as SHP-1 have been shown to in JAK1 immunoprecipitates suggesting that JAK1 inhibit tyrosine phosphorylation of JAK kinases follow- mediates the activation of the PI3 pathway through the ing their recruitment to receptor complexes which is gp130 subunit (Oh et al., 1998). Taken together, these facilitated by their binding to the receptors via their SH2 studies indicate that the cellular signal transduction domains. These phosphatases have also been shown to machinery is programmed to respond to changes in bind JAK kinases directly and mediate their depho- stimulus by integrating diverse signaling pathways to sphorylation (Weiss and Schlessinger, 1998; Haque et al., generate an orchestrated response. 1998; Migone et al., 1998; David et al., 1995; Jiao et al., 1996). Regulation of JAK kinases by SH-PTP1 (also referred to as PTP1C, SHP, HCP, PTP1) has been Negative regulation of JAK activity demonstrated in signaling by interferons (David et al., 1995). SH-PTP1 was also shown to be involved in The involvement of JAK kinases in multiple signal inactivation of JAK2 and consequently abrogating the transduction cascades in response to a diverse group of proliferative signals initiated by erythropoietin (Kling- cytokines and growth factors warrants a requirement muller et al., 1995). Similarly, PTPeC, has been shown to of appropriate negative regulation of the JAK kinases. inhibit JAK kinase phosphorylation resulting in inhibi- Such a negative regulatory mechanism may be tion of di€erentiation and apoptosis in M1 cells necessary to preclude the possibility of constitutive stimulated by IL-6 and LIF (Tanuma et al., 2000). activation of JAK kinases that may lead to an aberrant Bittorf et al. (1999) recently showed that SHP1 inhibited activation of downstream signals and inappropriate erythropoietin-induced erythroid di€erentiation and gene expression. Such a scenario is validated by inhibition of apoptosis in an erythroleukemic cell line. observations that JAK kinase signaling has been This e€ect of SHP1 was a result of inhibition of both the implicated in disease states such as leukemia and other JAK kinase and MAP kinase pathways. Also, You et al. hematological malignancies (Leonard and O'Shea, recently documented an important role for SHP-2 in 1998; Ward et al., 2000). negative regulation of JAK kinases in interferon stimulated cells (You et al., 1999). Furthermore, Yin et al. (1997) performed a detailed molecular characteriza- Involvement of proteasome-mediated degradation tion of speci®c interactions between SHP-2 and JAK pathways kinases. These authors demonstrated that SHP-2 is tyrosine phosphorylated by JAK1 and JAK2 but not Proteasome mediated degradation pathways can mod- JAK3, on Y304 and Y327 via direct association. The ulate the activity of JAK kinases. This is consistent SHP-2 and JAK kinase association does not require the with the observation that degradation of STATs and SH2 domain of SHP-2 or the kinase-like domain in JAKs cytokine/growth factor receptors is also seen in the but does not require the N-terminal region of JAK regulation of signaling (Kim and Maniatis, 1996; proteins and regions encompassing amino acids residues Strous et al., 1996). Thus, it has been recently 232 and 272 on the SHP-2 protein. Interestingly, SHP-2 documented that proteasome inhibitors potentiate the phosphatase activity seems to be non-essential for JAK- IL-2 and IL-3 induced activation of the JAK kinase SHP-2 interactions as mutations that render SHP-2 pathways (Callus and Mathey-Prevot, 1998; Yu and phosphatase inactive do not abolish the interaction Burako€, 1997). Yu and Burako€ (1997) showed that between SHP-2 and JAK kinases.

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5673 The CIS/JAB/SOCS/SIS protein family modulates the collaborated in the mediation of inhibitory signals. activity of JAK signaling pathways Furthermore, they demonstrated that the pre-SH2 domain was crucial for SOCS functional activity, the Recently, a new family of cytokine-inducible proteins SH2-domain was critical for interaction with JAK has been characterized, that plays a critical role in kinases and the SC-motif imparted stability to the SOCS negative regulation of cytokine signals processed by proteins preventing them from proteasomal degradation. JAK kinases. These proteins, referred to as either CIS Yasukawa et al. (1999) recently conducted further (cytokine-induced SH2 containing proteins) or SOCS structure-function studies with a goal of understanding (suppressors of cytokine signaling) or SIS (STAT- the mechanism of action of the SOCS proteins and induced STAT inhibitor) or JAB (JAK-binding found that at least one of the SOCS family members, protein), possess SH2 domains that allow protein- SOCS1 (JAB/SSI1), inhibits JAK kinase activity by protein interactions with members of the cytokine directly binding to the JAK kinase activation loop. receptor family and other signaling components. For SOCS1 speci®cally binds to the Y1007, that is part of the sake of convenience, and taking into account the the activation domain and is required for optimal JAK current confusing nomenclature, we will refer to these kinase activity. These authors also demonstrated that proteins as SOCS (suppressors of cytokine signaling) optimal JAK/SOCS binding required the SH2 domain proteins. While a comprehensive analysis of this novel of SOCS as well as the 12 amino acids region adjacent protein family is beyond the scope of this review, we to the SH2 domain that house two residues (Ile68 and present a brief narrative regarding the salient features Leu75) that are conserved in SOCS proteins. High of this important family and its plausible mode of anity binding and inhibition of the JAK2 kinase also action. For additional details readers are referred to requires an N-terminal 12 amino-acid region on several recent excellent reviews on the topic (Naka et SOCS1. Zhang et al. (1999) provided an alternate al., 1999; Yoshimura, 1998a,b; Nicholson and Hilton, mode of action for the SOCS proteins that implicated 1998; Yasukawa et al., 2000). the SOCS box domain in proteasomal degradation of The SOCS proteins, which were cloned by several target proteins. In this study, the authors showed that independent groups around the same time, are small the SOCs box mediated interactions of SOCS family proteins which posses SH2 domains and a conserved proteins with cytoskeletal proteins, elongin B and SOCS/CIS box (Naka et al., 1997; Endo et al., 1997; elongin C. The authors suggested that the SOCS/ Starr et al., 1997). The SOCS family, at the current time, Elongin interaction may target the SOCS proteins is represented by at least eight members that play a and their associated substrates, which could be critical critical role in the modulation of signals propagated by to propagate the cytokine responses, to proteasomal diverse cytokine receptors (Table 1; Naka et al., 1999; degradation pathways. Yoshimura, 1998a,b; Nicholson and Hilton, 1998; Thus, the CIS/JAB/SOCS/SIS family of proteins Yasukawa et al., 2000). CIS1, the ®rst identi®ed member could regulate JAK signaling pathways by multiple of the SOCS family, was cloned as an early response gene mechanisms (Ward et al., 2000). Some of these proteins for IL-2, IL-3 and erythropoietin and was found to be such as JAB and CIS3 are able to bind to the kinase associated with these receptors (Yoshimura et al., 1995; domain of JAK2 leading to the inhibition of its Uchida et al., 1997; Matsumoto et al., 1997; Verdier et catalytic activity. As has been mentioned earlier, this al., 1998). Moreover, overexpression of CIS1 led to a interaction requires both the SH2 domain along with suppression of signaling in response to IL-3 and Epo. additional 12 N-terminal amino acid residues. This SOCS1 (JAB or SSI1) was identi®ed as a potent inhibitor extended SH2 domain appears to bind the phospho- of JAK kinases (Endo et al., 1997). SOCS1 can interact tyrosine residue Y1007 in the activation loop of JAK2, with the JAK2 kinase domain and suppress IL-6 signal which is critical for the kinase activity of JAK2. Other transduction pathways (Starr et al., 1997). Recently, members of this protein family seem to directly bind to Sasaki et al. showed that SOCS3 suppresses erythro- receptors, where they may block the interaction of poietin mediated signaling pathways by binding to the receptors with other signaling molecules. Alternatively, Epo-receptor and JAK2 (Sasaki et al., 2000). Deletion they might target the receptors and other interacting analysis experiments demonstrated that a cytoplasmic proteins for proteasomal degradation. region of the Epo-R that contains the Y401 is responsible for SOCS3 binding and is required for optimal SOCS3 inhibitory activity. Furthermore, regions both N- and C- JAKs as mediators of apoptosis signals terminal to the SH2 domain of SOCS3 were necessary for binding to JAK2 and Epo-R. Observations that JAK kinases mediate proliferative Nicholson et al. (1999) also demonstrated that optimal responses from a variety of cytokines/growth factors is activity of SOCS proteins and the consequential indicative of their indispensability to the overall growth inhibition of cytokine activity requires both the SH2 stimulus provided by the cytokine/growth factor signal and the N-terminal regions of the SOCS proteins. transduction pathways. This observation also suggests Deletion of the N-terminal 51 ± 78 amino acid residues that JAK kinase activity may play an important role in as well as mutations in the SH2 domains resulted in the the prevention of cell death or apoptosis. JAK kinase prevention of inhibition of LIF signaling. Narazaki et al. activity, in conjunction with other pathways that (1998) mapped three distinct domains of the SOCS dictate proliferation such as the Ras, PI3K, MAPK proteins that perform obligatory roles in inhibition of pathways, may contribute to the overall proliferative cytokine signaling. Mutation of the conserved C- stimulus. On the other hand, JAK kinase activity may terminal region of the SOCS proteins (SC-motif), the also have an underlying in¯uence on the levels of cell pre-SH2 and SH2 domains indicated that they were survival and apoptosis. Several recent studies that critical for the suppression of IL-6 signaling and implicate JAK kinase activity in the modulation of

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5674 rates of cell survival and apoptosis have corroborated SCID (Russell et al., 1994; Noguchi et al., 1993; this hypothesis. Leonard, 1996; O'Shea et al., 1997). In con®rmation of Proteins represented by the Bcl-2 family genes this hypothesis, several patients with autosomal modulate the levels of apoptosis and cell survival by recessive SCID have been identi®ed, that harbor either positively or negatively in¯uencing the cell death mutations in their JAK3 locus (Candotti et al., 1997; and cell survival machinery (Antonsson and Martinou, Russell et al., 1995; Macchi et al., 1995; Cacalano et 2000; Pellegrini and Strasser, 1999; Reed, 1997; Adams al., 1999). Furthermore, to elucidate the role of JAK3 and Corey, 1998). Thus, the family members Bcl-2, Bcl- in development several groups engineered JAK3 XL and Mcl-1 inhibit apoptosis whereas Bax, Bad and de®cient mice (Thomis et al., 1995; Nosaka et al., Bak proteins accelerate apoptosis. Signaling cascades 1995; Park et al., 1995). JAK3 nullizygous mice are emanating from diverse pathways regulate the level of immunode®cient, similar to gc-de®cient mice (Cao et expression of Bcl-2 family members. Packham et al. al., 1995; DiSanto et al., 1995). These mice have (1998) recently implicated the JAK signaling pathway severely depleted B-cell repertoire and a slight increase in the modulation of cell survival and apoptosis by in peripheral T-cells. This observation is in contrast to regulating members of the Bcl-2 family. This study that observed in human SCID patients with mutations demonstrated that regulation of the cell death e€ector in JAK3 and the gc, who exhibit severely depleted T- Bcl-XL is mediated by the JAK kinase pathway. cells with normal or slightly increased numbers of B Moreover, the authors portray the speci®city of the cells. The observed defect in lymphoid development is, role of JAK kinases by providing evidence that at least in part, due to lack of IL-7 signal transduction. regulation of Bcl-XL protein levels was independent Cacalano et al. (1999) reported a single Y100C amino of the status of other signaling components such as acid substitution in the N-terminus JH7 domain of STAT proteins, PI-3 Kinases and Ras. The same group JAK3 that was identi®ed in a patient with autosomal also highlighted the role of JAK kinase signaling in severe combined immunode®ciency (SCID). IL-2 re- mediating the rescue of p53-dependent cell cycle arrest sponsive signaling was compromised in B-cell lines and apoptosis in cytokine treated cells (Quelle et al., derived from patient cells. Furthermore, the authors 1998). This study evaluated the role of signal demonstrated that a region encompassing the JH6 and transduction pathways in gamma-radiation (IR) in- JH7 domains of JAK3 was sucient for interaction of duced p53 dependent apoptosis and p53-independent the kinase with the proline-rich Box1 region of the IL-2 cell cycle arrest. The authors showed that the IR- receptor and was sucient in reconstituting the IL-2 induced cell cycle arrest and apoptosis was inhibited by dependent response. cytokines such as erythropoietin and that these e€ects were dependent on the activation of JAK kinases. Aberrant activation of JAK kinases in leukemia Using mutants of the erythropoietin receptors (Epo-R) this study delineated the functional domains on the Aberrant activation of JAK kinase activity has been Epo-R that were responsible for mediating the cell implicated in several hematological malignancies (Ward cycle arrest and apoptosis signals. While the membrane et al., 2000). Thus, a t(9;12)(p24;p13) chromosomal proximal domain of the Epo-R was sucient to translocation has been detected in a patient with T cell prevent IR-induced cell death a membrane distal childhood acute lymphoblastic leukemia (Lacronique et domain was required for subverting the growth arrest al., 1997). This translocation results in the fusion of the associated with IR-induced DNA damage. Further- C-terminal kinase catalytic region of JAK2 to the N- more, that the activation of JAK kinase signaling was terminal region of the ETS-transcription family necessary and sucient for these signals was under- member, TEL, to generate an overactive TEL-JAK2 scored by the observation that cell survival by Epo was tyrosine kinase that could impart cytokine indepen- not dependent on the activation of other signaling dence to an IL-3 dependent cell line, Ba/F3. Peeters et pathways such as the PI3-Kinase, PLC-gamma, Ras or al. (1997) detected similar translocations involving the STAT pathways. JAK kinase mediated cell survival, JAK2 kinase at 9p24 and TEL (the ETV6 gene at however, required the participation of Bcl-2 family 12p13) in both myeloid and lymphoid leukemias. A members such as Bcl-XL. Sakai and Kraft (1997) also t(9;12)(p24;p13) translocation was detected in a case of illustrated the role of the JAK2 kinase domain in early pre-B acute lymphoid leukemia and a induction of Bcl-2 protein that mediated cell survival t(9;15;12)(p24;q15;p13) translocation was detected in and delay of hematopoietic cell death. atypical chronic myelogenous leukemia. In either case the authors detected di€erent fusion mRNA transcripts and one fusion protein product that consisted of the helix-loop-helix (HLH) domain of the TEL (ETV6) Implication of aberrant JAK activity in disease states gene and the tyrosine kinase domain of JAK2. A Glycine 341 to Glutamic acid amino acid substitution Mutations in JAK3 predispose to certain forms of severe in the Drosophila hopscotch gene was shown to cause combined immunodeficiency syndrome (SCID) leukemia-like hematopoietic defect (Luo et al., 1995; Mutations in the gc chain of the IL-2 receptor has been Harrison et al., 1995). The only known Drosophila known to be responsible for the X-linked severe member of the JAK kinase family was identi®ed during combined immunode®ciency syndrome, X-SCID the cloning of hopscotch (Binari and Perrimon, 1994). (O'Shea et al., 1997). X-SCID is an inherited disorder hopscotch is required maternally for the establishment that is typi®ed by a rampant defect in the body's of the normal array of embryonic segments in immune system. gc chain speci®cally associates with Drosophila. It was observed that the hopscotch gene JAK3 kinase and therefore it was postulated that was involved in the control of pair-rule gene transcrip- mutations in JAK3 may predispose to some form of tion in a stripe-speci®c manner thereby providing the

Oncogene Signal transduction by JAK kinases SG Rane and EP Reddy 5675 ®rst evidence for stripe-speci®c regulation of pair-rule important clues regarding the role of JAK3 in the genes by a tyrosine kinase. Furthermore, a Glycine 341 development of these cell lineages. to Glutamic acid amino acid substitution in the hopscotch gene was shown to cause leukemia-like Perspective and future directions hematopoietic defects (Luo et al., 1995; Harrison et al., 1995). This study was the ®rst study, which While we have learnt a great deal about the role of indicated that a mutant JAK kinase can cause JAK kinases in signaling, a number of questions about leukemia-like abnormalities. These studies, taken these kinases remain unanswered. For example, the together, indicate that aberrant activation of JAK role of the unique structural domains (JH3-JH7) kinases can result in hematological abnormalities. present in these kinases is yet to be deciphered. Clearly, these domains play a critical role in protein- protein interactions and understanding the nature of Role of JAKs in development proteins that interact with these domains is likely to further our understanding of the role played by these The role of JAK kinases in development and disease kinases in cell signaling. Like other growth factor/ have been examined using gene targeting approaches in receptor interactions, cytokine/receptor interactions mice (Rodig et al., 1998; Neubauer et al., 1998; lead to the formation of multi-protein complexes which Parganas et al., 1998; Park et al., 1995; Thomis et includes JAKs, Src family kinases, STATs, Ras, MAP al., 1995; Nosaka et al., 1995). Mice carrying a and P13Kinase family of proteins, protein phospha- disruption of the JAK1 locus are susceptible to tases as well as CIS/JAB/SOCS family of proteins. It is perinatal lethality primarily due to defective suckling intriguing to note that di€erent cytokine and interferon arising from neurological de®cits (Rodig et al., 1998). receptors interact with very similar sets of signaling Furthermore, JAK1 de®cient cells derived from these molecules such as JAKs, STATs, serine/threonine mice are defective in signaling via multiple cytokine kinases, dual kinases, phosphatases and their negative receptors families such as the gp130 family, the regulators and yet each cytokine elicits very distinctive interferon receptor family and gamma chain containing biological and biochemical responses from a given cell. receptors. Mice de®cient in JAK2 succumb to It is clear that the exact stoichiometry of the complexes embryonic lethality and die at embryonic day E12.5 that are formed following cytokine/receptor interac- (Neubauer et al., 1998; Parganas et al., 1998). The tions plays a critical role in eliciting these distinctive embryonic lethality is mostly due to defects in responses. erythropoiesis and cells from JAK2 nullizygous mice The observation that di€erent cytokines and inter- have defective signaling response to cytokines from the ferons activate similar STAT complexes that bind to IL-3, interferon, and single chain (excluding G-CSFR) very related sequences and yet elicit very di€erent receptor families. JAK3 de®cient mice exhibit severe biological responses raises the question as to how defects in lymphoid development and show aberrations speci®c gene expression is achieved. It is possible that in B-cell maturation and T-lymphocyte activation STATs themselves do not dictate the phenotype that (Park et al., 1995; Thomis et al., 1995; Nosaka et al., results from cytokine/interferon stimulation. It is likely, 1995). Apart from the defects in B cell and T-cell that it is the combination of di€erent pathways that development, JAK3 de®cient mice also show defects in lead to the activation of di€erent transcription factors the development of natural killer (NK) cells. Moreover, which act in concert with STATs that dictate the Grossman et al. (1999) recently demonstrated that phenotype produced by a given cytokine/receptor JAK3 nullizygous mice display dysregulated myelopoi- interaction. It is clear that JAK kinases play a critical esis. Their results demonstrate that JAK3 de®cient role in the activation of these multiple pathways and a mice show evidence of increased immature neutrophil detailed understanding of the mode of action of JAK and monocyte counts in peripheral blood smears along kinases is likely to lead to a better understanding of the with splenomegaly. Speci®c cell-surface marker analysis complex biological processes regulated by interleukins, also indicated an expansion of cells of the myeloid interferons and other growth hormones. lineage. Taken together, these observations indicate that JAK3 has an important role in the development of cells destined for both the myeloid and lymphoid Acknowledgments lineages. Further detailed analysis of the role of JAK3 This work was supported by grants from the NIH in cells of lymphoid and myeloid origin may yield (ES09225-02 and CA68239-04).

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