Janus kinases: components of multiple signaling pathways

Abstract

Cytoplasmic Janus protein tyrosine kinases (JAKs) are crucial components of diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Evidence to date, indicates that JAK kinase function may integrate components of diverse signaling cascades. While it is likely that activation of STAT proteins may be an important function attributed to the JAK kinases, it is certainly not the only function performed by this key family of cytoplasmic tyrosine kinases. Emerging evidence indicates that phosphorylation of cytokine and growth factor receptors may be the primary functional attribute of JAK kinases. The JAK-triggered receptor phosphorylation can potentially be a rate-limiting event for a successful culmination of downstream signaling events. In support of this hypothesis, it has been found that JAK kinase function is required for optimal activation of the Src-kinase cascade, the Ras-MAP kinase pathway, the PI3K-AKT pathway and STAT signaling following the interaction of cytokine/interferon receptors with their ligands. Aberrations in JAK kinase activity, that may lead to derailment of one or more of the above mentioned pathways could disrupt normal cellular responses and result in disease states. Thus, over-activation of JAK kinases has been implicated in tumorigenesis. In contrast, loss of JAK kinase function has been found to result in disease states such as severe-combined immunodeficiency. In summary, optimal JAK kinase activity is a critical determinant of normal transmission of cytokine and growth factor signals.

Tyrosine kinases and signal transduction

Signal transduction involves multiple sets of proteins interacting in a harmonious fashion to integrate diverse cues emanating from the extracellular milieu resulting in changes in gene expression. Majority of the mammalian signal transduction processes are initiated as a result of ligand receptor interactions (Kishimoto et al., 1994). These interactions result in biochemical changes, which are processed and delivered to the nucleus to bring about changes in gene expression. Molecular cloning of cytokine receptors and subsequent structure-function studies has revealed that unlike growth-factor receptors, cytokine receptors lack a cytoplasmic kinase domain. Nevertheless, interaction of a cytokine with its receptor has been found to rapidly induce tyrosine phosphorylation of the receptors and a variety of cellular proteins suggesting that these receptors transmit their signals through cellular tyrosine kinases (Kishimoto et al., 1994). During the past decade, new evidence has emerged to indicate that most cytokines transmit their signals via a new family of tyrosine kinases termed JAK kinases (Ihle et al., 1995, 1997; Darnell, 1998; Darnell et al., 1994; Schindler, 1999; Schindler and Darnell, 1995; Ward et al., 2000; Pellegrini and Dusanter-Fourt, 1997; Leonard and O'Shea, 1998; Leonard and Lin, 2000; Heim, 1999).

Conventional protein tyrosine kinases (PTKs) possess catalytic domains ranging from 250 to 300 amino acids, corresponding to about 30 kDa (Hanks et al., 1988). The location of the kinase catalytic domain, in most enzymes, lies near the carboxy terminus of the molecule, whereas the amino terminus performs a regulatory role (Figure 1). The catalytic domains comprise of a characteristic signature of conserved amino acid residues. PTKs can be further grouped as members of either the Src subfamily, Tec subfamily or one of the three different growth factor receptor subfamilies, the EGF-receptor subfamily, the insulin receptor subfamily or the PDGF-receptor subfamily. The protein tyrosine kinases encoded by the c-abl and c-fes/fps genes are distantly related to the Src subfamily. At least nine of the protein tyrosine kinases including c-fgr, c-src, c-abl and c-fes have been transduced by retroviruses, where they perform a transforming function (Pierce, 1989). Apart from the kinase catalytic domains, PTKs possess certain other 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 domain (PH), (d) a negative regulatory tyrosine in the carboxy terminus and (e) myristylation or palmitoylation lipid modification sites at the N-terminus (Cooper and Howell, 1993; Pawson, 1995). The PH and the lipid modification sites are considered to be important for attachment of the kinases to membranes. The PH domain may also facilitate association of other signaling proteins to membranes thereby bringing them in close proximity to the kinases. The JAK kinase family, which constitutes a distinct family of tyrosine kinases is a relatively new member of tyrosine gene families and has been the focus of intensive investigation during the past few years. While it was originally thought that this family of kinases is mainly involved in interferon and cytokine mediated signal transduction pathways, recent evidence suggests that these kinases act as mediators of multiple signaling pathways and are essential for the normal function of the mammalian organism.

Figure 1
figure1

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 kinases

Discovery of the Janus kinases (JAKs) occurred at a time when a variety of approaches were being tested in attempts to identify novel protein tyrosine kinases. The first JAK kinase, which was named TYK2 (tyrosine kinase 2), was obtained upon screening a T-cell library using low stringency hybridization techniques (Krolewski et al., 1990; Firmbach-Kraft et al., 1990). The unique structure and function of TYK2 became obvious only after other members of the JAK family were identified and characterized. Polymerase chain reaction (PCR) using degenerate oligonucleotides spanning the heavily conserved kinase domains of members of the Src family of protein tyrosine kinases resulted in identification of partial cDNA clones of JAK1 and JAK2 (Wilks, 1989). Full length cDNA clones of JAK1 and JAK2 were subsequently cloned using the partial cDNA fragments as probes (Wilks, 1991; Harpur et al., 1992). Their discovery, along with several other members of the tyrosine kinase family resulted in the usage of the acronym JAK (Just Another Kinase). Subsequent sequencing studies revealed that the JAK family of protein tyrosine kinases (PTKs) differs markedly from other classes of PTKs by the presence of an additional kinase domain. To denote this unique structural feature, these kinases were renamed as ‘Janus kinases’ in reference to an ancient two-faced Roman God of gates and doorways (Darnell, 1998; Darnell et al., 1994; Ihle et al., 1995, 1997; Pellegrini and Dusanter-Fourt, 1997; Schindler and Darnell, 1995; Heim, 1999; Leonard and O'Shea, 1998; Schindler, 1999; Leonard and Lin, 2000; Ward et al., 2000).

Expression studies indicated that JAK1, JAK2 and TYK2 are ubiquitously expressed and are encoded by transcripts of 5.4 kb (JAK1), two transcripts of 5.3 and 5.0 kb (JAK2), and a 4.4 kb transcript (TYK2), respectively. Several independent groups identified and cloned a fourth member of the JAK family, JAK3 (Johnston et al., 1994; Rane et al., 1994; Kawamura et al., 1994; Takahashi and Shirasawa, 1994; Witthuhn et al., 1994). JAK3 is encoded by a 4.2 kb transcript that codes for a 120 kD protein that is expressed predominantly in cells of hematopoietic origin. Recently, reports describing JAK3 expression in normal and transformed human cell lines of various origins have been published (Lai et al., 1995; Verbsky et al., 1996).

The chromosomal locations of the murine and human JAKs have been determined. In humans, the JAK1, JAK2 and TYK2 genes have been mapped to chromosomes 1p13.3 (Pritchard et al., 1992), 9p24 (Pritchard et al., 1992) and 19p13.2 (Firmback-Kraft et al., 1990) respectively. However, using interspecific hybrids JAK2 has been reportedly localized to murine chromosome 19 at a region which corresponds to the human chromosome 10q23-q24.1 and not to 9p24 as previously reported (Ihle et al., 1995). JAK3 has been mapped to human chromosome 19p13.1 (Kumar et al., 1996; Riedy et al., 1996). The corresponding murine genes have also been mapped with JAK1 on mouse chromosome 4, JAK2 on mouse chromosome 19 and JAK3 on mouse chromosome 8 (Kono et al., 1996).

The unique structure of the JAK kinases clearly distinguishes them from other members of the protein tyrosine kinase family (Figure 1). The most intriguing feature of these proteins is the presence of two domains (JH1 and JH2), with extensive homology to the tyrosine kinase domains. A second interesting feature is the absence of any SH2 or SH3 domains. Instead, these proteins encode a group of well-conserved domains termed as JAK homology (JH1-JH7) domains that follow a non-conserved amino terminus of about 30–50 amino acids. Of the dual kinase domains identified only the JH1 domain appears to be functional. The JH2 domain, which harbors considerable homology to the tyrosine kinase domains lacks certain critical amino acids required for a functional kinase and does not appear to be associated with a kinase activity. Both the tyrosine kinase domain (JH1) and the pseudo-kinase domain (JH2) are housed at the carboxy terminus of the protein (Figure 1). The other conserved blocks of sequences (JH3-JH7) that are characteristic to members of the JAK family, comprise approximately 600 N-terminal amino-acids residues. The precise functions of the JH3-JH7 domains as well as the pseudokinase domain (JH2) are currently under investigation. The sequences of the JH3-JH7 domains bear no resemblance to any characterized protein motif. Considering the variety of interactions and functions performed by the JAK kinase family members it seems plausible that these domains facilitate some key functions like protein-protein interactions, recruitment of substrates, etc.

N-terminal domain of JAKs mediate their interaction with cytokine/interferon/growth hormone receptors

Recent studies indicate that the amino terminal regions may be involved in receptor binding (Chen et al., 1997; Gauzzi et al., 1997). For example, Cacalano et al. (1999) recently mapped the region on JAK3 that is obligatory for its functional interaction with the IL-2 receptor. These authors identified a single amino acid substitution (Y100C) in the N-terminus of the JH7 domain of in a patient with autosomal severe combined immunodeficiency (SCID). As a result of this mutation, IL-2 responsive signaling was compromised in B-cell lines derived from the patient cells. Furthermore, the authors demonstrated that a region encompassing the JH6 and JH7 domains of JAK3 was sufficient for interaction of the kinase with the proline-rich Box1 region of the IL-2 receptor and was sufficient in reconstituting the IL-2 dependent response. Zhao et al. (1995) using a mutational analysis approach demonstrated 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 membrane proximal region of the beta-chain of the GM-CSF receptor. A similar N-terminal truncation of JAK2 also abolished its interaction with the growth hormone receptor (Frank et al., 1995). Several other studies that utilized diverse receptor systems have further validated the role of the N-terminus of JAK kinases as modular domains that dictate interactions of the kinases with receptors. Thus, the amino terminal region of JAK1 and JAK2 was required for association with the IFN gamma receptor chains and downstream signaling (Kohlhuber et al., 1997).

JH1 and JH2 domains

The kinase like JH2 domain, despite harboring most of the conserved amino acid residues that are a characteristic of the kinase domain, lacks any observable tyrosine kinase activity. It is now clear that this kinase-like domain plays a regulatory role (Luo et al., 1997). Interestingly, Saharinen et al. (2000) recently ascribed a regulatory role to the JAK2 pseudo-kinase domain in modulation of JAK2 kinase activity. In this study, the authors studied the functional significance of the various JAK2 protein domains by mutational analysis. Deletion of the JAK2 pseudo-kinase domain, but not of JH3-JH7 domains, negatively regulated JAK2 catalytic activity as well as STAT5 activation by JAK2. Furthermore, this study indicated that JAK2 kinase inhibition was mediated by an interaction between the JAK2 kinase and the psuedo-kinase domains. The above results were further substantiated when the authors detected an inhibition of JAK2 single-kinase domain activity upon trans expression of the pseudo-kinase domain whereas deletion of the pseudo-kinase domain resulted in de-regulation of JAK2 kinase activity in response to interferon gamma resulting in an increase in STAT activity.

Auto (trans) phosphorylation of conserved tyrosine residues in the JAK kinase activation loop determines the levels of catalytic activation. Such a mode of activation is a conserved feature of tyrosine kinases in general and the tyrosine phosphorylation may allow substrates to gain access to the active site (Duhe and Farrar, 1995). Zhou et al. (1997) who engineered point mutations in the activation loop of the JAK3 kinase domain studied the importance of tyrosine phosphorylation on critical tyrosine residues in modulating JAK kinase catalytic activities. The authors detected multiple auto-phosphorylation sites on JAK3 and mutated two tyrosine residues to phenylalanine (Y980F and/or Y981F). The Y980F mutant exhibited decreased kinase activity whereas the Y981F mutant exhibited significantly increased kinase activity and the double mutant Y980F/Y981F displayed intermediate kinase activity. Furthermore, the authors also demonstrated that optimal phosphorylation of JAK3 on other phosphorylation sites as well as substrate phosphorylation was dependent on Y980 phosphorylation. Similarly, Gauzzi et al. (1996) showed that substitution of two adjacent tyrosine residues in the Tyk2 activation loop with phenylalanine resulted in the abrogation of TYK2 activity in response to IFNα/β.

Activation of JAK kinases by cytokines/interferons and growth hormones

Janus kinases are generally believed to be present in unstimulated cells in an inactive form. Ligand-induced receptor oligomerization, such as cytokine interaction with its specific receptor, serves as a trigger to signal the recruitment of JAKs to close proximity of the receptors. Local aggregation of the JAK kinases and their subsequent activation by either auto or trans phosphorylation is an event which either involves other 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 heterodimers have been implicated in signal transduction processes initiated by a variety of growth factors and cytokines (Table 1). A number of studies show that JAK kinases are involved in signaling by interleukins such as IL-2, IL-4, IL-7, IL-9 and IL-15 (Witthuhn et al., 1994; Zeng et al., 1994; Johnston et al., 1994; Kirken et al., 1994; Tanaka et al., 1994; Yin et al., 1994). JAK2 is activated following erythropoietin stimulation of its receptor (Witthuhn et al., 1993) while JAK1 and JAK2 have been implicated in signaling by IL-3, GM-CSF and IL-5 (Silvennoinen et al., 1993a; Quelle et al., 1994; Lutticken et al., 1994). Stimulation by the cytokines IL-6, CNTF, and LIF results in the activation of JAK1, JAK2 and TYK2 kinases (Stahl et al., 1994; Narazaki et al., 1994; Guschin et al., 1995). Also, JAK2 and TYK2 are activated upon IL-12 stimulation and are proposed to play a critical role in IL-12 mediated T-cell differentiation (Bacon et al., 1995). Similarly, JAK kinases have been shown to play a key role in the signal transduction pathways initiated in response to stimulation by growth hormone (Argetsinger et al., 1993), prolactin (Rui et al., 1994; Dusanter-Fourt et al., 1994) and G-CSF (Nicholson et al., 1994; Shimoda et al., 1994). The interferons also transduce their signals via the JAK kinases, JAK1, JAK2 and TYK2 (Muller et al., 1993; Velazquez et al., 1992, 1995; Watling et al., 1993). JAK3 conveys signals emanating from IL-2, IL-4, IL-7, IL-15 and IL-19. In summary it appears that specific JAK kinases, either alone or in combination with other JAK kinases, may be preferentially activated depending on the type of the receptor that is being activated.

Table 1 Tyrosine kinases, CIS/SOCS proteins and STATs activated by cytokine/growth factor signal transduction pathways

Mode of action of JAKs: receptors are primary substrates of JAK activation

The importance of JAKs in mediating signals from a variety of cytokines/growth factors underscores their importance in signal transduction in general. However, the complexity associated with processing signals from such diverse sources suggests a complicated mechanism of action for the JAK kinases. Although the precise mechanism by which ligand binding results in the activation of JAKs is not known, inferences from results of various studies has allowed the proposition of a model to explain the mechanism of activation of JAK kinases (Figure 2). JAK activation is usually determined by an in vitro kinase assay that measures an increase in tyrosine phosphorylation of substrates. JAKs, when expressed in the baculovirus system are enzymatically active and are phosphorylated on tyrosine residues. Their overexpression in mammalian cells also leads to constitutive activation, most probably due to dimerization (Colamonici et al., 1994a,b; Quelle et al., 1995; Duhe and Farrar, 1995; Yamamoto et al., 1994; Wang et al., 1995; Eilers et al., 1996). On the other hand, a JAK kinase in complex with a native un-liganded receptor is in a catalytically inactive latent state. Receptor dimerization/oligomerization due to ligand binding results in the juxtapositioning of the JAKs, which are in the vicinity either through homo- or heterodimeric interactions. This recruitment of the JAK kinases appears to result in their phosphorylation either via autophosphorylation and/or cross phosphorylation by other JAK kinases or other tyrosine kinase family members. This activation is presumed to result in an increased JAK kinase activity. The activated JAKs then phosphorylate receptors on target tyrosine sites. The phosphotyrosine sites on the receptors can then serve as docking sites that allow the binding of other SH2-domain containing signaling molecules such as STATs, Src-kinases, protein phosphatases and other adaptor signaling proteins such as Shc, Grb2 and Cbl.

Figure 2
figure2

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 specific 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 specific 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

This model is supported by several studies where investigators have demonstrated the ability of cytokines such as Erythropoietin (EPO) to rapidly induce receptor oligomerization leading to JAK2 activation (Watowich et al., 1994). Furthermore, studies with chimeric receptors, which combine the extracellular domains of the EGF receptor with the cytoplasmic domain of the Epo receptor, also provide evidence in support of a role for receptor dimerization/oligomerization in JAK kinase activation. EGF stimulation of cells expressing the EGF-Epo chimeric receptors, leads to receptor oligomerization of the chimeras followed by activation of the JAK2 kinase and subsequent induction of mitogenesis (Ihle et al., 1995). Similar chimeric receptor based studies have elucidated the importance of oligomerization of the βc-chain of the receptors for IL-3/IL-5/GM-CSF in signal transduction by their respective cytokines (Eder et al., 1994; Jubinsky et al., 1993). Also, studies with chimeric receptors for IL-2 suggest a similar role for receptor oligomerization as a prerequisite for signal transduction by IL-2 (Watowich et al., 1994). In these cases, as with the EGF-Epo chimeric receptors, the chimeras were functionally capable of induction of mitogenesis supporting the receptor oligomerization concept for JAK kinase activation. Such a model is readily applicable for both single chain receptors such as those for Epo, prolactin, growth hormone and G-CSF as well as multi-chain receptors such as those for IL-3, IL-5 and GM-CSF. Cytokine binding results in the association of the JAKs with one of the subunits. The receptor associated JAK kinase can then either process the signal or subsequent to the receptor oligomerization can recruit other JAKs in the vicinity. Homo or heterodimerization of the JAKs followed by their activation upon phosphorylation finally results in the propagation of the initial signal.

While the importance of receptor oligomerization in the activation of the JAK kinases was being documented, other studies focused on delineating the regions of the receptors that play a critical role in JAK-receptor interactions. These studies clearly demonstrated the importance of membrane proximal domains of the cytokine receptors containing the Box 1 and Box 2 motifs in receptor/JAK interactions. The Box 1 motif comprises of approximately eight proline-rich amino acids, that are required for interaction of several cytokine/growth factor receptors with JAK2. Thus, the Box 1 region of the receptors for prolactin, growth hormone, erythropoietin, IL-6 and related cytokines, that utilize the gp130 signal transducing receptor subunit, is required for association with JAK2 (Witthuhn et al., 1993; Miura et al., 1994; Tanner et al., 1995; Vanderkuur et al., 1994; Goujon et al., 1994; Hackett et al., 1995; DaSilva et al., 1994). Jiang et al. (1996) used mutational analysis to demonstrate that the Box 1 region of cytokine receptors specifies the region of interaction with JAK kinases. IL-2 stimulation leads to the activation of JAK3 but not of JAK2 whereas erythropoietin conveys its signals by recruiting and activating JAK2. These authors created chimeric receptor molecules by switching the Box 1 region of the EpoR with that of the beta chain of the IL-2R. The EpoR-IL-2R fusion receptor was sufficient to induce activation of JAK2 in response to stimulation with IL-2 thereby confirming the role of Box 1 region in determining the specificity of JAK kinase activation. Mutational analysis of several receptors corroborated such a theory. Mutations in the membrane proximal regions of the Epo receptor resulted in the elimination of JAK2 association and activation as well as abrogation of mitogenesis in response to erythropoietin (Witthuhn et al., 1993). Similar mutational analysis demonstrated the importance of the membrane proximal domains of the prolactin receptor in association and activation of JAK2 (DaSilva et al., 1994). Also, the Box1 and Box2 regions of gp130 were shown to be required for association and activation of JAK1 and JAK2 (Narazaki et al., 1994). The requirement of membrane proximal motifs in JAK kinase interaction is absolute even in the case of receptors that lack Box 1 motifs. JAK1 and JAK3 constitute integral components of the IL-2R signal transduction apparatus. The IL-2R gamma-c chain does not contain a typical Box1 motif and yet can activate the JAK1 and JAK3 kinases. It has been determined that a membrane proximal region of the IL-2R encompassing 52 amino acids is sufficient for JAK3 binding, activation of JAK1 and JAK3 and other downstream signaling events such as induction of transcription factors c-fos and c-myc and cell proliferation (Nelson et al., 1996). These studies indicated that aberrations at any step of the aggregation of ligand-receptor/JAK kinase hetero-oligomeric complexes could be sufficient to abolish activation of the kinases and prevent mitogenesis, most probably by eliminating indispensable downstream signaling events.

Interdependence and hierarchy of JAKs in signaling

The mechanisms by which JAK kinases are activated following cytokine/growth factor stimulation are beginning to unravel. Although the activation of STATs constitutes an important step concurrent to JAK kinase activation, several events prior to STAT activation seem to be unique to JAK kinases. The role of JAK kinases in interferon-α/β and γ signaling exemplifies the inter-dependence in JAK kinase signaling. Consistent with subunit-mediated association of multiple JAK family members, type I (IFN α and β) and type II (IFN γ) interferons bind to two unrelated receptor complexes, each of which utilizes both common and distinct JAK kinases to transmit its signals. Elucidation of the involvement of JAK kinases in interferon signaling has for the first time suggested the involvement of multiple family members, in which individual members, following receptor aggregation, cooperate in the formation of JAK kinase heterodimers (Ihle et al., 1995). Unlike traditional experiments which involve the introduction of systematic deletions within a cDNA and its encoded protein, the mutants which were used to dissect the interferon signaling pathways are naturally occurring and are incapable of responding to either IFN α, β or γ (McKendry et al., 1991; John et al., 1991; Pellegrini et al., 1989). One cellular mutant, designated as U1, fails to respond to IFN α or IFN β, but remains responsive to IFN γ. The gene responsible for the restoration of a cellular response to IFN α/β, following expression cloning, was found to be TYK2 (Velazquez et al., 1992). Another set of mutants, the γ mutant cell lines, fail to respond to IFN γ, but retain their IFN α/β responsiveness. Ectopic expression of JAK2, but not TYK2 or JAK1, in these cells results in the restoration of a cellular response to IFN γ; subsequent experiments demonstrated JAK2 tyrosine phosphorylation and activation in cells following treatment with IFN γ (Watling et al., 1993; Shuai et al., 1993; Silvennoinen et al., 1993b).

Thus far the involvement of JAK kinases in interferon signaling seemed relatively simple and complete, in which an individual JAK kinase family member was responsible for the transduction of signals from a given receptor. A new twist in JAK signaling originated from the analysis of the U4 mutant, which, under normal conditions, is incapable of responding to each of the above interferons. Using an approach analogous to that used to identify the genetic lesion within the γ mutants, ectopic expression of JAK1 in these cells results in a cell type which is fully capable of transducing IFN-generated signals (Muller et al., 1993). Thus, while both type I and II IFN receptors appear to associate with JAK1, it appears that TYK2 and JAK2 are associated with one chain of the IFN α/β and IFN γ receptors, respectively (Soh et al., 1994; Novick et al., 1994; Colamonici et al., 1994a,b; Igarashi et al., 1994).

The possibility of existence of a JAK kinase cascade in interferon-α/β and γ signaling, by virtue of one JAK phosphorylating and activating the other JAK kinase seemed intriguing at the time. Such a hypothesis was tested and excluded, since it was observed that both JAK kinases were required and functionally active for tyrosine phosphorylation and activation of each other. However, separate studies have attempted to analyse the temporal order of activation of JAK kinases using the interferon model system. JAK1 was shown to be obligatory to initiate phosphorylation and activation of TYK2 in an interferon-α/β receptor complex (Gauzzi et al., 1996). On the other hand, in the case of the interferon-γ receptor complex, JAK2 serves as the initiator kinase which phosphorylates JAK1 as part of the signaling cascade (Briscoe et al., 1996). Signaling via receptors that contain gp130 indicated that JAK1 is the obligatory kinase and JAK2 and TYK2 served as additional components. Interestingly, in this system it was shown that the absence of one JAK kinase did not affect phosphorylation of the other JAKs leading to the speculation that JAKs could serve as substrates for other tyrosine kinases, such as the Src-family of PTKs (Guschin et al., 1995).

It is presumed that the precise basis for the interdependence is the multi-chain/subunit composition of receptors. Such a theory is validated by examination of the structures of interferon-α/β and γ receptors. Both the interferon-α/β and γ receptors consist of at least two chains and it has been shown that the β-chain of the interferon-α/β receptor binds JAK1 (Novick et al., 1994) whereas TYK2 associates with the α-chain (Colamonici et al., 1994a,b). In the case of interferon-γ, co-immunoprecipitation studies indicated an association between JAK1 and the α-chain whereas JAK2 was shown to be associated with the complex upon ligand binding. Therefore, the most plausible theory is that ligand binding to the receptors followed by receptor oligomerization leads to the recruitment of the JAKs into the hetero-oligomeric complex.

Substrates of JAKs

It is becoming increasingly clear that JAKs phosphorylate multiple substrates, the most important of which are the receptors themselves. A number of potential substrates have been identified using different approaches. Thus, GST fusion proteins encoding intracellular domains of a number of cytokine receptors could be readily phosphorylated by activated JAK kinases in vitro (Zhao et al., 1995; Colamonici et al., 1994a; Gauzzi et al., 1996). Similarly, an analysis of proteins phosphorylated following antibody cross-linking of a CD16/JAK2 chimera in BA/F3 cells revealed that STAT-5, Shc and Shp proteins were amongst several proteins that were phosphorylated suggesting that they could be the substrates for JAKs (Sakai et al., 1995). Similarly a chimeric construct containing the extracellular domain of EGF and the JAK2 kinase domain was found to mediate phosphorylation of STAT-5 following EGF stimulation (Nakamura et al., 1996).

STAT signaling pathways

STATs are among the best-characterized JAK substrates and activation of virtually all cytokine/interferon/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 are activated by a given cytokine. It is also interesting to note that a C-terminal deletion mutant of IL-3 βc chain, termed βc541 was found to be deficient in its ability to activate STAT-5, while still being able to activate JAK2 (Smith et al., 1997). These observations also imply that while JAK2 activation is a critical step in IL3/IL5/GMCSF mediated activation of their cognate receptors, it is by itself not adequate for STAT activation.

The signal transducers and activators of transcription

Over the past few years, researchers have deciphered a signaling pathway, which initiates within the cytoplasm but quickly translocates to the nucleus to activate transcription of target genes. This novel signaling pathway features a group of transcription factors named as STATs or Signal transducers and Activators of Transcription (Schindler and Darnell, 1995; Darnell, 1997). These transcription factors were originally described by Darnell and his co-workers (Darnell et al., 1994; Darnell, 1997) as ligand-induced transcription factors in interferon-treated cells. Subsequent studies by a number of groups showed that STATs play a critical role in signal transduction pathways associated with several cytokines and neurokines including the interleukins, the interferons, erythropoietin, prolactin, growth hormone, oncostatin M (OSM), and ciliary neurotrophic factor (CNTF) (Darnell et al., 1994; Darnell, 1997). To-date, six mammalian genes that code for different STATs have been identified, all of which encode for proteins of 750–850 amino acids long and are characterized by the presence of a DNA-binding domain followed by putative SH3 and SH2 domains (Darnell, 1997). In addition, alternative splicing or post-translational proteolytic cleavage reactions appear to generate additional forms for STAT-1,3 and 5, bringing the total number of STATs currently described in literature to eight. Thus, two isoforms of STAT1 have been described, which have been termed as STAT1_ or p91 and STAT 1_ or p84. These two proteins were originally discovered by Darnell's group in association with STAT-2 and a fourth protein termed p48, which constituted the multicomponent transcription factor ISGF3. STAT-3 also exists in two forms termed STAT 3A and STAT 3B with different transcriptional activation functions (Schafer et al., 1995). STAT 5 exists in two isoforms, termed as STAT 5A and STAT 5B, which are encoded by two separate genes, which are tandemly linked on human chromosome 17 and mouse chromosome 11 (Lin et al., 1996; Copeland et al., 1995). The two proteins exhibit extensive sequence homology and differ from each other mainly in the amino and carboxy terminal domains, with the transactivation domain showing most divergence. Both of these genes seem to play a critical role in IL3/IL-5/GMCSF-induced proliferation of hematopoietic cells as well as prolactin-induced proliferation of mammary epithelial cells (Takaki et al., 1994; Quelle et al., 1994; Cornelis et al., 1995; Matsuguchi et al., 1997; Itoh et al., 1996).

Like most transcription factors, STATs exhibit a modular structure with five well defined domains, which include the N-terminal conserved domain, the DNA-binding domain, a putative SH3-like domain, a SH2 domain and C-terminal transactivation domain (Figure 3). The amino terminal region of STATs is well conserved and appears to be critical for STAT function as small deletions in this region were found to eliminate the ability of STATs to be phosphorylated. This region is followed by the DNA binding domain that is usually located between amino acids 400 and 500 (Horvath et al., 1995). This region is highly conserved amongst the STATs, and all STATs with the exception of STAT-2 differentially bind more than 10 related GAS sequences that are characterized by the consensus sequence motif, TTNCNNNAA (Horvath et al., 1995; Xu et al., 1996). Adjacent to the DNA binding domain lies the putative SH3 domain. This domain appears to be least conserved and it is at present unclear as to whether it truly functions as a SH3 domain, since the critical amino acid residues involved in binding to PXXP motifs do not appear to be well conserved. Adjacent to this putative SH3 domain lies the SH2 domain, which is highly conserved amongst the STATs and appears to play a very important role in STAT signaling. Thus, it is critical for the recruitment of STATs to the activated receptor complexes and is required for the interaction with JAK and Src kinases. In addition, it is required for STAT homo and hetero dimerization, which in turn appears to be critical for nuclear localization and DNA binding activities. It is also possible that this domain participates in other protein-protein interactions that have not yet been fully deciphered. Immediately downstream of the SH2 domain, around position 700, all STATs contain a tyrosine residue, which plays a critical role in STAT activation. Phosphorylation of this tyrosine residue has been found to be essential for the activation and dimerization of STATs. Phosphorylation of this tyrosine appears to be achieved by growth factor receptors as well as JAK and Src kinases, depending on the nature of the cell type and the ligand/receptor interactions. The C-terminal domain of STATs is required for transcriptional transactivation. Phosphorylation of a single serine residue in the TA domains of STAT1, STAT3, STAT4 and STAT5, can enhance the transcriptional activity. Differentially spliced STATs such as STAT 1_, lacking the C-terminal TA domain can still bind DNA but do not activate transcription.

Figure 3
figure3

STAT protein structure. Cytokines signal via members of the STAT family of proteins. The various STAT proteins, many of which are derived as a result of alternative splicing events (e.g.: Stat 1, Stat 3, Stat 4 and Stat 5) comprise of approximately 800 amino acid residues. Sequence analysis indicates that the STAT proteins harbor conserved motifs, such as the DNA binding region (DNAB), SH2 and putative SH3 domains that allow DNA-protein and protein-protein interactions. The transactivation (TA) domain and the highly conserved phosphorylatable tyrosine (pY) and serine (pS) residues that are essential for optimal STAT activation reside at the carboxy terminus of the proteins

STATs, which are normally localized in the cytoplasm are activated when phosphorylated on the tyrosine located around residue 700, which facilitates their dimerization and translocation to the nucleus (Darnell et al., 1994; Schindler and Darnell, 1995). The phosphorylation of STATs is known to occur immediately after the binding of growth factors or interferons to their receptors. Studies with interferon (IFN) receptor signaling have revealed that JAK family kinases are involved in IFN-specific gene expression in cooperation with STATs. Studies with mutant cell lines which were unable to respond to interferons, described earlier, clearly established a critical role for JAKs in the interferon signaling pathways.

Since the cytokine and interferon receptor-ligand interactions were found to result in the activation of JAK kinases, which often exist in association with cytokine receptors, and this activation was obligatory for the activation of STATs, it was proposed that STATs might be substrates for JAK kinases. This notion was further supported by studies, which showed in vitro phosphorylation of purified STAT-5 by JAK-2 (Flores-Morales et al., 1998). In this study, the investigators expressed STAT-5 in Sf9 insect cells using the baculovirus expression system and showed that this recombinant protein can be phosphorylated by JAK-2 that confers DNA binding property to STAT-5. In addition, these investigators showed STAAT-5 in its non-phosphorylated form was able to form a stable complex with activated JAK2, while non-activated JAK2 and phosphorylated STAT-5 were unable to participate in complex formation. A second line of evidence, which suggested that JAKs might be activators of STATs, came from the use of dominant negative mutants of JAK-2. Over-expression of dominant negative JAK1 and TYK2 mutants in human cells suppressed transcriptional activation of a luciferase reporter gene downstream from an ISRE. In addition, these dominant negative mutants were found to inhibit STAT-1 and STAT-2 phosphorylation (Krishnan et al., 1997).

However, the activated JAK kinases do not seem to exhibit specificity for a particular STAT as different receptors activate a common STAT, even though they activate distinctively different JAK kinase (Kohlhuber et al., 1997; Darnell, 1997). In addition chimeric receptor molecules with different JAK binding sites but containing the same STAT-binding site were found to activate the same STAT (Kotenko et al., 1996; Kohlhuber et al., 1997). Thus, the specificity for STAT phosphorylation appears to be determined by the docking sites for STATs that are present in the receptor molecules and not JAK kinases.

The two isoforms of STAT5, STAT5a and STAT5b as well as STAT6 are activated by receptors for IL-3, GM-CSF and IL-5 (Quelle et al., 1995; Azam et al., 1995). IL-3 stimulation leads to activation of JAK2 and phosphorylation and activation of STAT5a (via tyrosine phosphorylation of Y694) and STAT5b (via tyrosine phosphorylation of Y699). Mutational studies with the IL-3, GM-CSF and IL-5 receptors have shown that JAK2 is essential for STAT activation (Kouro et al., 1996; Matsuguchi et al., 1997; Caldenhoven et al., 1995; Mui et al., 1995a,b; Smith et al., 1997). However, a C-terminal mutant of the βc chain has been described that can activate JAK2 but is unable to activate STAT5 indicating that activation of JAK2 is not sufficient for STAT5 activation (Smith et al., 1997).

A role for Src kinases in STAT activation was first suggested by studies aimed at investigating the molecular mechanisms associated with Src-mediated transformation of fibroblasts and hematopoietic cell lines. Thus, Src-transformed NIH/3T3 cells were found to express a tyrosine phosphorylated form of STAT-3 in a constitutive manner (Yu et al., 1995; Cao et al., 1996). In addition, results from Cao et al. (1996) showed that v-Src can bind to STAT-3 and phosphorylate this protein in vitro. Transfection of an expression vector coding for a dominant negative mutant of STAT-3 into v-Src-transformed NIH/3T3 cells resulted in a block to v-Src-mediated transformation of cells, further enforcing the notion that STAT-3 signaling plays a critical role in this transformation process.

In our studies aimed at delineating the molecular mechanisms associated with v-Src-mediated transformation of hematopoietic cells, we used 32Dc13 cells that are derived from normal mouse bone marrow and require IL-3 for proliferation and survival. These cells, when cultured in the presence of G-CSF were found to terminally differentiate into mature granulocytes in a period of 10–12 days (Valtieri et al., 1987). When these cells were grown in a medium lacking IL-3 or G-CSF, they were found to undergo apoptosis in a period of 24–48 h. This cell line, when transformed with expression vectors containing v-abl or bcr-abl or v-src was found to grow indefinitely in the absence of IL-3 and to form tumors in nude mouse assays (Kruger and Anderson, 1991; Laneuville et al., 1991; Rovera et al., 1987). In addition, expression of either v-src, v-abl or bcr-abl was found to block the ability of 32Dc13 cells to undergo GCSF-induced differentiation (Rovera et al., 1987). Interestingly, several other tyrosine kinases closely related to v-src failed to induce such cytokine-independence of these cells suggesting a very specific target for v-src in these cells. Thus, when v-fgr or an activated form of c-fgr which lacks the C-terminal 12 amino acids (c-fgrD) were expressed in this cell line, they failed to render these cells IL-3 independent for growth (Chaturvedi et al., 1997).

To understand the possible mechanisms underlying the IL-3 independent growth of v-src-transformed 32Dc13 cells, we examined the phosphorylation status of JAK-1, JAK-2 and JAK-3 kinases in the v-src and v-fgr-transformed cells. These studies showed that none of the JAK kinases were phosphorylated by v-Src or v-Fgr. However, v-src-transformed cells were found to express constitutively phosphorylated forms of STAT-1, 3 and 5, which exhibited appropriate DNA binding activities. Our results also showed that STAT-3 co-immunoprecipitates with v-Src suggesting that the activation of STAT-3 occurs due to direct association with v-Src (Chaturvedi et al., 1998). Since hematopoietic cells express high levels of c-Src and since Src-kinases associate with IL-3 receptor following its activation by IL-3, this observation suggested that c-Src might play a role in the activation of STAT-3 in normal hematopoietic cells following IL-3 stimulation. This was further confirmed by our studies where we examined the phosphorylation status of JAKs and STAT-3 in 32Dc13 cells following IL-3 stimulation. These studies showed that IL-3 stimulation induces rapid tyrosine phosphorylation of JAK-1 and JAK-2 in 32Dc13 cells. In addition, our results show that interaction of IL-3 with its receptor leads to the phosphorylation and nuclear translocation of STAT-1, STAT-3 and STAT-5. In addition, this ligand/receptor interaction leads to the activation of c-Src kinase activity, which in turn facilitates the binding of c-Src to STAT-3. This association leads to the phosphorylation of STAT-3, allowing STAT3 to translocate to the nucleus. Expression of a dominant negative mutant of src (AMSrc) in these cells results in a block to IL-3 mediated phosphorylation of STAT-3, and its ability to bind to DNA (Chaturvedi et al., 1998). On the other hand, expression of a dominant negative mutant of JAK2 (JAK2KE) had no effect on Il-3-mediated activation of STAT-3. Our results also show that AMSrc does not affect the phosphorylation of JAK2, suggesting that two independent pathways mediate JAK and STAT-3 phosphorylation events. These results suggest that Src family kinases mediate the phosphorylation of STAT-3 mediated by IL3/receptor interactions and play a critical role in signal transduction pathways associated with myeloid cell proliferation. These results suggest that activation of STAT-3 following cytokine stimulation of certain hematopoietic cells might follow a very different pathway from that seen with STAT-5. These results taken together indicate that while one or both isoforms of STAT-5 interact directly with JAK-2, which in turn mediates their phosphorylation, STAT-3 activation might require its interaction with c-Src, which in turn mediates its phosphorylation (Chaturvedi et al., 1998). Based on these results, we proposed the JAK-SRC-STAT model, where JAK kinases may be more crucial to phosphorylation of the cytokine/growth factor receptors. Moreover, JAK-mediated phosphorylation may create docking sites on the receptors for binding of SH2-contacting proteins such as STATs, Src-kinases and other signaling intermediates (Reddy et al., 2000). JAKs or Src-kinases, depending on the nature of STAT that is being activated then induce tyrosine phosphorylation and activation of STAT proteins.

The notion that different STATs might be phosphorylated by different tyrosine kinases under different conditions is suggested by two other observations. Like v-Src, v-Abl and BCR-ABL have been shown to transform hematopoietic cells and render them cytokine-independent for growth (Pierce et al., 1985; Cook et al., 1985; Mathey-Prevot et al., 1986). Thus, v-Abl encoded by the Abelson Murine Leukemia Virus was found to transform both B-cells and myeloid cells resulting in cytokine-independent growth of these cells (Pierce et al., 1985; Cook et al., 1985; Mathey-Prevot et al., 1986). Examination of the molecular mechanisms associated with v-Abl mediated transformation show that B-cells transformed by this oncogene exhibit constitutively activated forms of JAK-1, JAK-3 as well as STAT-1,3 and 5 (Danial et al., 1995, 1998). In addition, activated JAK-1 in these cells was found to be associated with the v-Abl protein. Mapping the JAK-1 interaction domain in v-Abl showed that a region of the C-terminal domain of v-Abl protein binds to JAK-1 and a mutant of v-Abl that lacks this JAK-1 binding domain failed to activate JAK-1 and STAT proteins. In addition, a dominant negative mutant of JAK-1 was found to inhibit STAT activation mediated by v-Abl suggesting that the v-Abl protein activates STATs via activation of JAK-1.

In sharp contrast, the BCR-ABL oncogene, a gene closely related to the v-abl oncogene was found to constitutively activate STAT-1 and STAT-5 with very little or no activation of JAKs (Carlesso et al., 1996; Ilaria et al., 1996; Frank and Varticovski, 1996; Niebrowska-Skorska et al., 1999). Unlike with v-abl transformed B-cells, over-expression of dominant negative JAK kinase mutants in these cell lines had no effect on STAT phosphorylation (Ilaria et al., 1996). Similarly, K562 cells, which are Philadelphia-chromosome positive express constitutively activated STAT-5 and over-expression of a dominant negative STAT-5 could suppress the transformed state of these cells. Activation of STATs by BCR-ABL oncogene was shown to be dependent on the presence of the SH2 and SH3 domains (Niebrowska-Skorska et al., 1999), suggesting a Src-like interaction reported by us (Chaturvedi et al., 1998). These observations would also provide a molecular basis for the inability of v-Abl to directly activate STATs, since this oncoprotein lacks the SH3 domain that is present in the BCR-ABL oncogene (Reddy et al., 1983).

A second line of evidence comes from the study of Kazansky et al. (1999) who studied the DNA-binding and tyrosine phosphorylation of STAT-5A and STAT-5B following prolactin activation or Src activation. Their results show that following prolactin activation, both STAT5A and STAT5B were rapidly phosphorylated and translocated to the nucleus. Similar to prolactin activation, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. Furthermore, overexpression of a dominant negative mutant of JAK-2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B. In sharp contrast, over-expression of dominant negative JAK-2 had no effect on Src-mediated phosphorylation of STATs. In contrast, there was a modest increase in the levels of Src-mediated phosphorylation of STAT5 in the presence of dominant JAK-2. These studies again suggest that there exist two independent pathways that mediate STAT activation, one that is dependent on JAKs and one that is independent of Src kinases. The context of the cytokine and cell type appears to play a critical role in determining as to which pathway is utilized by the cell.

Cross-talk among JAKs and components of other signaling pathways

Signal transduction in response to a variety of ligands involves the activation of the Ras signaling pathway (Egan and Weinberg, 1993). In the Ras pathway, typified by signal transduction in response to Erythropoietin as well as IL-3, cytokine stimulation results in the recruitment and tyrosine phosphorylation of Shc. Following phosphorylation, Grb2 associates with Shc and with Sos. This results in an increase in GTP-bound Ras, activation of Raf-1, followed by activation of the mitogen-activated (MAP) kinases and induction of primary response genes such as c-myc, c-fos, etc. Recently, several studies have explored the interdependence between the JAK kinase signaling pathway and the Ras-linked to MAP kinases pathway as well as other key signaling molecules such as Vav, PI-3 kinase and the protein tyrosine phosphatases such as SH-PTP1. Evidence from these studies suggests that there may be interactions amongst the diverse components of the cells' signal transduction repertoire. Also, data indicates that cells have equipped themselves with ‘safety routes’ in the event of malfunction of a particular ‘primary’ signaling pathway. The availability of an optional signaling route, available via interactions with multiple signaling molecules, enable cells to survive the loss of the ‘primary’ signaling route. Thus, although the Ras and JAK kinase pathways mediate distinct signals it is becoming evident that there may be interdependence among the two pathways. Thus, phosphorylation of the receptors by JAK kinases creates potential docking sites for adaptor molecules such as Shc, the p85 subunit of PI-3K, STAT proteins and other kinases such as Src-kinases which can associate with the phosphorylated receptors via their SH2 domains. Once recruited to the receptor complex, the JAK kinases can phosphorylate multiple downstream signaling molecules to further propagate the Ras and JAK kinase pathways.

The abilities of various receptors to integrate activation of the JAK kinase pathway to elements of the Ras pathway have been extensively documented (Alam et al., 1995; Bates et al., 1996; Kumar et al., 1994). Carboxy terminal truncations in the IL-3 receptor β-chain resulted in the loss of activation of the Ras pathway, although the ability to activate JAK2 and induce mitogenesis was retained providing evidence for the existence of alternate signaling pathways in cytokine signaling (Quelle et al., 1994). In a similar study, it was demonstrated that distinct regions of the G-CSF receptor are required for tyrosine phosphorylation of JAK2, STAT3 as well as p42, p44 MAP kinases. MAP kinase tyrosine phosphorylation correlated with both the proliferative response and JAK2 activation (Nicholson et al., 1995). Recently, Mizuguchi et al. (2000) described the JAK kinase mediated activation of both the Ras and STAT pathways in cell proliferation. Conditional activation of JAK kinases confers IL-3 independence to BA/F3 cells that requires functional Ras and STAT5 proteins. This observation indicates an obligatory role for the Ras and STAT proteins as targets of JAK activity. In agreement with the above studies, overexpression of Ras or STAT5 alone does not confer IL-3 independence whereas concomitant activation of Ras and STAT5 is sufficient to confer IL-3 independence. The same group also, elucidated the role of JAK kinase activity in Ras signaling using a TYK2 protein that was modified by the addition of a membrane localization sequence and a chemical dimerizer (coumermycin)-dependent dimerization sequence (Mizuguchi and Hatakeyama, 1998). The modified TYK2, upon activation by dimerization, conferred IL-3 independence to pro-B lymphoid cells that was abolished by expression of dominant negative Ras indicating the mandatory role for Ras proteins as downstream targets of JAK kinase signals. Interferon stimulation led to activation of the Ras pathways that also depend on JAK kinase activities (Sakatsume et al., 1998; Stancato et al., 1997). Sakatsume et al. (1998) showed that activation of the Raf-1 protein by interferon gamma was Ras-independent but required the kinase activity of JAK1. Similarly, Stancato et al. (1997) reported that activation of Raf-1 and the MAP kinase pathways, upon stimulation of cells with interferon beta and oncostatin M, required JAK1 kinase activity. The above studies, taken together indicate that JAK kinases target both the Ras and STAT pathways to exert their biological effects.

Activation of Ras and PI3Kinase as well as STAT proteins leads to increased induction of transcription factors such as c-jun. Also, c-jun levels are elevated in B-lymphoid cells exposed to ionizing radiation (IR). Goodman et al. recently demonstrated that the induction of c-jun in IR treated cells required the activation of JAK3 but was impervious to the activation status of the Btk, Syk and Lyn tyrosine kinases (Goodman et al., 1998).

Interaction of JAK2 with the SH2 domain of p95 Vav, a protein expressed in hematopoietic cells, was reported in cells stimulated with GM-CSF (Matsuguchi et al., 1995). Also, in IL-7 stimulated T cells, members of the JAK family were shown to regulate the activity of PI-3 kinase. Coupling of the JAK signaling pathway and Shc phosphorylation was demonstrated in signal transduction events from c-Mpl, a member of the cytokine receptor family and the receptor for thrombopoietin (Gurney et al., 1995). Integration of the JAK and Ras pathways was evident in IL-6 induced signals in a B-cell line AF10 in which JAK1, p52Shc, Raf-1 and MEK-1 were activated (Kumar et al., 1994). Studies involving signal transduction by growth hormone (GH) indicated that JAK2, Ras and Raf are required for activation of the MAP kinases (Winston and Hunter, 1995). Based on this study, it was suggested that JAK kinases might represent a common component during activation of the ERK2/MAPK and STAT signaling pathways, which appeared to bifurcate upstream of Ras activation but converged with the phosphorylation of STATs by the ERK/MAPK proteins. The role of JAK kinases in insulin signaling demonstrated that JAK1 and JAK2 constitutively associate with Grb2 via interactions with the SH3 domains of Grb2 (Giorgetti-Peraldi et al., 1995).

Several studies have reported the involvement of JAK kinase activity in the modulation of PI-3 Kinase function. GM-CSF stimulation of neutrophils triggers the activation of Jak2, STAT3, STAT5B, and P13K. Treatment of these cells with a JAK2 inhibitor AG-490 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 indicated that the p85 subunit associated with JAK2 and this interaction was not dependent on the two SH2 domain of PI-3K. Also, Yamauchi et al. (1998) using cells either deficient in JAK2 or harboring dominant negative JAK2, showed that the insulin-receptor substrate (IRS) proteins, IRS-1, IRS-2 and IRS-3, are phosphorylated by JAK2. Furthermore, these authors elegantly demonstrated that upon phosphorylation by JAK2, the phosphorylated IRS proteins serve as scaffolding intermediates to allow docking and subsequent activation of PI3-Kinase. Stimulation of cardiac myocytes by LIF promotes interaction between JAK1 and PI-3K that leads to increased PI-3K activity in JAK1 immunoprecipitates suggesting that JAK1 mediates the activation of the PI3 pathway through the gp130 subunit (Oh et al., 1998). Taken together, these studies indicate that the cellular signal transduction machinery is programmed to respond to changes in stimulus by integrating diverse signaling pathways to generate an orchestrated response.

Negative regulation of JAK activity

The involvement of JAK kinases in multiple signal transduction cascades in response to a diverse group of cytokines and growth factors warrants a requirement of appropriate negative regulation of the JAK kinases. Such a negative regulatory mechanism may be necessary to preclude the possibility of constitutive activation of JAK kinases that may lead to an aberrant activation of downstream signals and inappropriate gene expression. Such a scenario is validated by observations that JAK kinase signaling has been implicated in disease states such as leukemia and other hematological malignancies (Leonard and O'Shea, 1998; Ward et al., 2000).

Involvement of proteasome-mediated degradation pathways

Proteasome mediated degradation pathways can modulate the activity of JAK kinases. This is consistent with the observation that degradation of STATs and cytokine/growth factor receptors is also seen in the regulation of signaling (Kim and Maniatis, 1996; Strous et al., 1996). Thus, it has been recently documented that proteasome inhibitors potentiate the IL-2 and IL-3 induced activation of the JAK kinase pathways (Callus and Mathey-Prevot, 1998; Yu and Burakoff, 1997). Yu and Burakoff (1997) showed that the proteasome inhibitor MG132 was able to stabilize the IL-2 induced tyrsoine phosphorylation of Jak1 and Jak3. Also, treatment of IL-3 stimulated Ba/F3 with proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), resulted in a stabilization of tyrosine phosphorylation of the IL-3 receptor-beta common chain, STAT5, Shc, and mitogen activated protein kinases (MAPKs) (Callus and Mathey-Prevot, 1998). Furthermore, these authors showed that these stable phosphorylation events were the result of a prolonged activation of JAK1 and JAK2 kinases.

Negative regulation by protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) regulate the kinase activities of tyrosine kinases by de-phosphorylating tyrosine residues involved in catalytic function (Fischer et al., 1991; Pallen et al., 1992). Optimal activation of JAK kinases is positively regulated by phosphorylation of a critical tyrosine residue in the kinase-activating domain (Gauzzi et al., 1996; Feng et al., 1997; Zhou et al., 1997; Liu et al., 1997; Weiss and Schlessinger, 1998). Several studies have demonstrated the role of protein tyrosine phosphatases in the regulation of JAK signaling pathways (David et al., 1995; Klingmuller et al., 1995; Yetter, 1995). PTPs such as SHP-1 have been shown to inhibit tyrosine phosphorylation of JAK kinases following their recruitment to receptor complexes which is facilitated by their binding to the receptors via their SH2 domains. These phosphatases have also been shown to bind JAK kinases directly and mediate their dephosphorylation (Weiss and Schlessinger, 1998; Haque et al., 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 demonstrated in signaling by interferons (David et al., 1995). SH-PTP1 was also shown to be involved in inactivation of JAK2 and consequently abrogating the proliferative signals initiated by erythropoietin (Klingmuller et al., 1995). Similarly, PTPeC, has been shown to inhibit JAK kinase phosphorylation resulting in inhibition of differentiation and apoptosis in M1 cells stimulated by IL-6 and LIF (Tanuma et al., 2000). Bittorf et al. (1999) recently showed that SHP1 inhibited erythropoietin-induced erythroid differentiation and inhibition of apoptosis in an erythroleukemic cell line. This effect of SHP1 was a result of inhibition of both the JAK kinase and MAP kinase pathways. Also, You et al. recently documented an important role for SHP-2 in negative regulation of JAK kinases in interferon stimulated cells (You et al., 1999). Furthermore, Yin et al. (1997) performed a detailed molecular characterization of specific interactions between SHP-2 and JAK kinases. These authors demonstrated that SHP-2 is tyrosine phosphorylated by JAK1 and JAK2 but not JAK3, on Y304 and Y327 via direct association. The SHP-2 and JAK kinase association does not require the SH2 domain of SHP-2 or the kinase-like domain in JAKs but does not require the N-terminal region of JAK proteins and regions encompassing amino acids residues 232 and 272 on the SHP-2 protein. Interestingly, SHP-2 phosphatase activity seems to be non-essential for JAK-SHP-2 interactions as mutations that render SHP-2 phosphatase inactive do not abolish the interaction between SHP-2 and JAK kinases.

The CIS/JAB/SOCS/SIS protein family modulates the activity of JAK signaling pathways

Recently, a new family of cytokine-inducible proteins has been characterized, that plays a critical role in negative regulation of cytokine signals processed by JAK kinases. These proteins, referred to as either CIS (cytokine-induced SH2 containing proteins) or SOCS (suppressors of cytokine signaling) or SIS (STAT-induced STAT inhibitor) or JAB (JAK-binding protein), possess SH2 domains that allow protein-protein interactions with members of the cytokine receptor family and other signaling components. For the sake of convenience, and taking into account the current confusing nomenclature, we will refer to these proteins as SOCS (suppressors of cytokine signaling) proteins. While a comprehensive analysis of this novel protein family is beyond the scope of this review, we present a brief narrative regarding the salient features of this important family and its plausible mode of action. For additional details readers are referred to several recent excellent reviews on the topic (Naka et al., 1999; Yoshimura, 1998a,b; Nicholson and Hilton, 1998; Yasukawa et al., 2000).

The SOCS proteins, which were cloned by several independent groups around the same time, are small proteins which posses SH2 domains and a conserved SOCS/CIS box (Naka et al., 1997; Endo et al., 1997; Starr et al., 1997). The SOCS family, at the current time, is represented by at least eight members that play a critical role in the modulation of signals propagated by diverse cytokine receptors (Table 1; Naka et al., 1999; Yoshimura, 1998a,b; Nicholson and Hilton, 1998; Yasukawa et al., 2000). CIS1, the first identified member of the SOCS family, was cloned as an early response gene for IL-2, IL-3 and erythropoietin and was found to be associated with these receptors (Yoshimura et al., 1995; Uchida et al., 1997; Matsumoto et al., 1997; Verdier et al., 1998). Moreover, overexpression of CIS1 led to a suppression of signaling in response to IL-3 and Epo. SOCS1 (JAB or SSI1) was identified as a potent inhibitor of JAK kinases (Endo et al., 1997). SOCS1 can interact with the JAK2 kinase domain and suppress IL-6 signal transduction pathways (Starr et al., 1997). Recently, Sasaki et al. showed that SOCS3 suppresses erythropoietin mediated signaling pathways by binding to the Epo-receptor and JAK2 (Sasaki et al., 2000). Deletion analysis experiments demonstrated that a cytoplasmic 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-terminal to the SH2 domain of SOCS3 were necessary for binding to JAK2 and Epo-R.

Nicholson et al. (1999) also demonstrated that optimal activity of SOCS proteins and the consequential inhibition of cytokine activity requires both the SH2 and the N-terminal regions of the SOCS proteins. Deletion of the N-terminal 51–78 amino acid residues as well as mutations in the SH2 domains resulted in the prevention of inhibition of LIF signaling. Narazaki et al. (1998) mapped three distinct domains of the SOCS proteins that perform obligatory roles in inhibition of cytokine signaling. Mutation of the conserved C-terminal region of the SOCS proteins (SC-motif), the pre-SH2 and SH2 domains indicated that they were critical for the suppression of IL-6 signaling and collaborated in the mediation of inhibitory signals. Furthermore, they demonstrated that the pre-SH2 domain was crucial for SOCS functional activity, the SH2-domain was critical for interaction with JAK kinases and the SC-motif imparted stability to the SOCS proteins preventing them from proteasomal degradation.

Yasukawa et al. (1999) recently conducted further structure-function studies with a goal of understanding the mechanism of action of the SOCS proteins and found that at least one of the SOCS family members, SOCS1 (JAB/SSI1), inhibits JAK kinase activity by directly binding to the JAK kinase activation loop. SOCS1 specifically binds to the Y1007, that is part of the activation domain and is required for optimal JAK kinase activity. These authors also demonstrated that optimal JAK/SOCS binding required the SH2 domain of SOCS as well as the 12 amino acids region adjacent to the SH2 domain that house two residues (Ile68 and Leu75) that are conserved in SOCS proteins. High affinity binding and inhibition of the JAK2 kinase also requires an N-terminal 12 amino-acid region on SOCS1. Zhang et al. (1999) provided an alternate mode of action for the SOCS proteins that implicated the SOCS box domain in proteasomal degradation of target proteins. In this study, the authors showed that the SOCs box mediated interactions of SOCS family proteins with cytoskeletal proteins, elongin B and elongin C. The authors suggested that the SOCS/Elongin interaction may target the SOCS proteins and their associated substrates, which could be critical to propagate the cytokine responses, to proteasomal degradation pathways.

Thus, the CIS/JAB/SOCS/SIS family of proteins could regulate JAK signaling pathways by multiple mechanisms (Ward et al., 2000). Some of these proteins such as JAB and CIS3 are able to bind to the kinase domain of JAK2 leading to the inhibition of its catalytic activity. As has been mentioned earlier, this interaction requires both the SH2 domain along with additional 12 N-terminal amino acid residues. This extended SH2 domain appears to bind the phospho-tyrosine residue Y1007 in the activation loop of JAK2, which is critical for the kinase activity of JAK2. Other members of this protein family seem to directly bind to receptors, where they may block the interaction of receptors with other signaling molecules. Alternatively, they might target the receptors and other interacting proteins for proteasomal degradation.

JAKs as mediators of apoptosis signals

Observations that JAK kinases mediate proliferative responses from a variety of cytokines/growth factors is indicative of their indispensability to the overall growth stimulus provided by the cytokine/growth factor signal transduction pathways. This observation also suggests that JAK kinase activity may play an important role in the prevention of cell death or apoptosis. JAK kinase activity, in conjunction with other pathways that dictate proliferation such as the Ras, PI3K, MAPK pathways, may contribute to the overall proliferative stimulus. On the other hand, JAK kinase activity may also have an underlying influence on the levels of cell survival and apoptosis. Several recent studies that implicate JAK kinase activity in the modulation of rates of cell survival and apoptosis have corroborated this hypothesis.

Proteins represented by the Bcl-2 family genes modulate the levels of apoptosis and cell survival by either positively or negatively influencing the cell death and cell survival machinery (Antonsson and Martinou, 2000; Pellegrini and Strasser, 1999; Reed, 1997; Adams and Corey, 1998). Thus, the family members Bcl-2, Bcl-XL and Mcl-1 inhibit apoptosis whereas Bax, Bad and Bak proteins accelerate apoptosis. Signaling cascades emanating from diverse pathways regulate the level of expression of Bcl-2 family members. Packham et al. (1998) recently implicated the JAK signaling pathway in the modulation of cell survival and apoptosis by regulating members of the Bcl-2 family. This study demonstrated that regulation of the cell death effector Bcl-XL is mediated by the JAK kinase pathway. Moreover, the authors portray the specificity of the role of JAK kinases by providing evidence that regulation of Bcl-XL protein levels was independent of the status of other signaling components such as STAT proteins, PI-3 Kinases and Ras. The same group also highlighted the role of JAK kinase signaling in mediating the rescue of p53-dependent cell cycle arrest and apoptosis in cytokine treated cells (Quelle et al., 1998). This study evaluated the role of signal transduction pathways in gamma-radiation (IR) induced p53 dependent apoptosis and p53-independent cell cycle arrest. The authors showed that the IR-induced cell cycle arrest and apoptosis was inhibited by cytokines such as erythropoietin and that these effects were dependent on the activation of JAK kinases. Using mutants of the erythropoietin receptors (Epo-R) this study delineated the functional domains on the Epo-R that were responsible for mediating the cell cycle arrest and apoptosis signals. While the membrane proximal domain of the Epo-R was sufficient to prevent IR-induced cell death a membrane distal domain was required for subverting the growth arrest associated with IR-induced DNA damage. Furthermore, that the activation of JAK kinase signaling was necessary and sufficient for these signals was underscored by the observation that cell survival by Epo was not dependent on the activation of other signaling pathways such as the PI3-Kinase, PLC-gamma, Ras or STAT pathways. JAK kinase mediated cell survival, however, required the participation of Bcl-2 family members such as Bcl-XL. Sakai and Kraft (1997) also illustrated the role of the JAK2 kinase domain in induction of Bcl-2 protein that mediated cell survival and delay of hematopoietic cell death.

Implication of aberrant JAK activity in disease states

Mutations in JAK3 predispose to certain forms of severe combined immunodeficiency syndrome (SCID)

Mutations in the γc chain of the IL-2 receptor has been known to be responsible for the X-linked severe combined immunodeficiency syndrome, X-SCID (O'Shea et al., 1997). X-SCID is an inherited disorder that is typified by a rampant defect in the body's immune system. γc chain specifically associates with JAK3 kinase and therefore it was postulated that mutations in JAK3 may predispose to some form of SCID (Russell et al., 1994; Noguchi et al., 1993; Leonard, 1996; O'Shea et al., 1997). In confirmation of this hypothesis, several patients with autosomal recessive SCID have been identified, that harbor mutations in their JAK3 locus (Candotti et al., 1997; Russell et al., 1995; Macchi et al., 1995; Cacalano et al., 1999). Furthermore, to elucidate the role of JAK3 in development several groups engineered JAK3 deficient mice (Thomis et al., 1995; Nosaka et al., 1995; Park et al., 1995). JAK3 nullizygous mice are immunodeficient, similar to γc-deficient mice (Cao et al., 1995; DiSanto et al., 1995). These mice have severely depleted B-cell repertoire and a slight increase in peripheral T-cells. This observation is in contrast to that observed in human SCID patients with mutations in JAK3 and the γc, who exhibit severely depleted T-cells with normal or slightly increased numbers of B cells. The observed defect in lymphoid development is, at least in part, due to lack of IL-7 signal transduction. Cacalano et al. (1999) reported a single Y100C amino acid substitution in the N-terminus JH7 domain of JAK3 that was identified in a patient with autosomal severe combined immunodeficiency (SCID). IL-2 responsive signaling was compromised in B-cell lines derived from patient cells. Furthermore, the authors demonstrated that a region encompassing the JH6 and JH7 domains of JAK3 was sufficient for interaction of the kinase with the proline-rich Box1 region of the IL-2 receptor and was sufficient in reconstituting the IL-2 dependent response.

Aberrant activation of JAK kinases in leukemia

Aberrant activation of JAK kinase activity has been implicated in several hematological malignancies (Ward et al., 2000). Thus, a t(9;12)(p24;p13) chromosomal translocation has been detected in a patient with T cell childhood acute lymphoblastic leukemia (Lacronique et al., 1997). This translocation results in the fusion of the C-terminal kinase catalytic region of JAK2 to the N-terminal region of the ETS-transcription family member, TEL, to generate an overactive TEL-JAK2 tyrosine kinase that could impart cytokine independence to an IL-3 dependent cell line, Ba/F3. Peeters et al. (1997) detected similar translocations involving the JAK2 kinase at 9p24 and TEL (the ETV6 gene at 12p13) in both myeloid and lymphoid leukemias. A t(9;12)(p24;p13) translocation was detected in a case of early pre-B acute lymphoid leukemia and a t(9;15;12)(p24;q15;p13) translocation was detected in atypical chronic myelogenous leukemia. In either case the authors detected different fusion mRNA transcripts and one fusion protein product that consisted of the helix-loop-helix (HLH) domain of the TEL (ETV6) gene and the tyrosine kinase domain of JAK2. A Glycine 341 to Glutamic acid amino acid substitution in the Drosophila hopscotch gene was shown to cause leukemia-like hematopoietic defect (Luo et al., 1995; Harrison et al., 1995). The only known Drosophila member of the JAK kinase family was identified during the cloning of hopscotch (Binari and Perrimon, 1994). hopscotch is required maternally for the establishment of the normal array of embryonic segments in Drosophila. It was observed that the hopscotch gene was involved in the control of pair-rule gene transcription in a stripe-specific manner thereby providing the first evidence for stripe-specific regulation of pair-rule genes by a tyrosine kinase. Furthermore, a Glycine 341 to Glutamic acid amino acid substitution in the hopscotch gene was shown to cause leukemia-like hematopoietic defects (Luo et al., 1995; Harrison et al., 1995). This study was the first study, which indicated that a mutant JAK kinase can cause leukemia-like abnormalities. These studies, taken together, indicate that aberrant activation of JAK kinases can result in hematological abnormalities.

Role of JAKs in development

The role of JAK kinases in development and disease have been examined using gene targeting approaches in mice (Rodig et al., 1998; Neubauer et al., 1998; Parganas et al., 1998; Park et al., 1995; Thomis et al., 1995; Nosaka et al., 1995). Mice carrying a disruption of the JAK1 locus are susceptible to perinatal lethality primarily due to defective suckling arising from neurological deficits (Rodig et al., 1998). Furthermore, JAK1 deficient cells derived from these mice are defective in signaling via multiple cytokine receptors families such as the gp130 family, the interferon receptor family and gamma chain containing receptors. Mice deficient in JAK2 succumb to embryonic lethality and die at embryonic day E12.5 (Neubauer et al., 1998; Parganas et al., 1998). The embryonic lethality is mostly due to defects in erythropoiesis and cells from JAK2 nullizygous mice have defective signaling response to cytokines from the IL-3, interferon, and single chain (excluding G-CSFR) receptor families. JAK3 deficient mice exhibit severe defects in lymphoid development and show aberrations in B-cell maturation and T-lymphocyte activation (Park et al., 1995; Thomis et al., 1995; Nosaka et al., 1995). Apart from the defects in B cell and T-cell development, JAK3 deficient mice also show defects in the development of natural killer (NK) cells. Moreover, Grossman et al. (1999) recently demonstrated that JAK3 nullizygous mice display dysregulated myelopoiesis. Their results demonstrate that JAK3 deficient mice show evidence of increased immature neutrophil and monocyte counts in peripheral blood smears along with splenomegaly. Specific cell-surface marker analysis also indicated an expansion of cells of the myeloid lineage. Taken together, these observations indicate that JAK3 has an important role in the development of cells destined for both the myeloid and lymphoid lineages. Further detailed analysis of the role of JAK3 in cells of lymphoid and myeloid origin may yield important clues regarding the role of JAK3 in the development of these cell lineages.

Perspective and future directions

While we have learnt a great deal about the role of JAK kinases in signaling, a number of questions about these kinases remain unanswered. For example, the role of the unique structural domains (JH3-JH7) 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 proteins that interact with these domains is likely to further our understanding of the role played by these kinases in cell signaling. Like other growth factor/receptor interactions, cytokine/receptor interactions lead to the formation of multi-protein complexes which includes JAKs, Src family kinases, STATs, Ras, MAP and P13Kinase family of proteins, protein phosphatases as well as CIS/JAB/SOCS family of proteins. It is intriguing to note that different cytokine and interferon receptors interact with very similar sets of signaling molecules such as JAKs, STATs, serine/threonine kinases, dual kinases, phosphatases and their negative regulators and yet each cytokine elicits very distinctive biological and biochemical responses from a given cell. It is clear that the exact stoichiometry of the complexes that are formed following cytokine/receptor interactions plays a critical role in eliciting these distinctive responses.

The observation that different cytokines and interferons activate similar STAT complexes that bind to very related sequences and yet elicit very different biological responses raises the question as to how specific gene expression is achieved. It is possible that STATs themselves do not dictate the phenotype that results from cytokine/interferon stimulation. It is likely, that it is the combination of different pathways that lead to the activation of different transcription factors which act in concert with STATs that dictate the phenotype produced by a given cytokine/receptor interaction. It is clear that JAK kinases play a critical role in the activation of these multiple pathways and a detailed understanding of the mode of action of JAK kinases is likely to lead to a better understanding of the complex biological processes regulated by interleukins, interferons and other growth hormones.

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Acknowledgements

This work was supported by grants from the NIH (ES09225-02 and CA68239-04).

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Correspondence to E Premkumar Reddy.

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Rane, S., Reddy, E. Janus kinases: components of multiple signaling pathways. Oncogene 19, 5662–5679 (2000). https://doi.org/10.1038/sj.onc.1203925

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Keywords

  • JAK kinases
  • STATs
  • ras
  • PI3K
  • CIS/JAB/SOCS/SIS protein family
  • signal transduction

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