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Aberrantly activated signal transducer and activator of transcription 3 (Stat3) is implicated in the development of various human cancers. Y705 phosphorylation is conventionally thought to be required for Stat3 signal-dependent activation and seems to play an essential role in some malignancies. Recently, it was shown that Stat3 is activated through novel and noncanonical mechanisms, including phosphorylation at S727. Here, we investigate S727 phosphorylation of Stat3 and its subsequent effects in prostate cancer development, independent of Y705 phosphorylation, using mutated Stat3 in the human prostate cancer cell line LNCaP. We show mutation of S727 to the phosphomimetic residue Glu, and inactivation of Y705 (Y705F/S727E) resulted in a remarkable growth advantage in low-serum, enhanced anchorage-independent growth in soft agar, and increased tumorigenicity in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, possibly by direct activation of downstream proto-oncogenes c-myc, mcl-1, and survivin. Y705F/S727E mutant cells were more invasive than Y705F/S727A (inactivation of Y705 and S727) mutant cells, and more Y705F/S727E mutant Stat3 was localized in the nuclei relative to Y705F/S727A mutant Stat3 at the steady state. Furthermore, the Y705F/S727E but not the Y705F/S727A mutant induced anchorage-independent growth of noncancerous prostate epithelial cells (RWPE-1). We further show that Stat3 is phosphorylated at S727 in 65% of malignant prostate tissues (n = 20) relative to 25% of normal prostate tissues (n = 4). Moreover, there is a positive correlation between phosphoS727-Stat3 expression and Gleason score in these prostate cancer tissues (P = 0.05). Our data suggest for the first time that S727 phosphorylation is sufficient to activate Stat3, thereby driving prostate tumorigenesis independent of Y705 phosphorylation.

The defense of the host from foreign pathogens is the commonly accepted function of the vertebrate immune system. A complex system consisting of many differing cells and structures communicating by both soluble and cell bound ligands, serves to protect the host from infection, and plays a role in preventing the development of certain types of tumours. Numerous signalling pathways are involved in the coordination of the immune system, serving both to activate and attenuate its responses to attack. The ability of the immune system, specifically those cells involved in acute inflammatory responses, to mediate the directed (and sometimes indirect) killing of cells and pathogens, make it a potential threat to host survival. Furthermore, the production and release of various survival factors such as the pleiotropic cytokine IL-6, a major mediator of inflammation and activator of signal transducer and activator of transcription 3, serves to block apoptosis in cells during the inflammatory process, keeping them alive in very toxic environments. Unfortunately, these same pathways serve also to maintain cells progressing towards neoplastic growth, protecting them from cellular apoptotic deletion and chemotherapeutic drugs. Here, we discuss the relationships between cancer and inflammation, and some of the molecular mechanisms involved in mediating the unintended consequences of host defense and tumour survival.

The phosphorylation of an "activation loop" within protein kinases is commonly associated with establishing catalytic competence, and phosphorylation of the Tyr(1007) residue in the activation loop of Janus kinase 2 (JAK2) has been shown to be essential for intracellular propagation of cytokine-initiated signaling. We provide evidence for the presence of a basal activity state of JAK2, which was observed in the absence of activation loop phosphorylation. Phosphorylation of the JAK2 activation loop was essential for conversion to the high-activity state, characterized by high-efficiency ATP utilization during autophosphorylation. Mutagenesis of activation loop tyrosine residues Tyr(1007/1008) to phenylalanine residues impaired, but did not abolish, the enzyme's ability to autophosphorylate. The activation loop mutant JAK2 could also transphosphorylate an inactive JAK2 fragment coexpressed in Sf21 cells, providing evidence of exogenous substrate phosphorylation. The mutant enzyme remained in a basal activity state characterized by low-efficiency ATP utilization during autophosphorylation. Mutagenesis of a critical Lys(882) residue to a glutamate residue abolished all evidence of kinase activity, confirming that the observed activity of Tyr-to-Phe mutants was not due to another kinase. Our data are consistent with the proposal that JAK2 is an inefficient but active enzyme in the absence of activation loop phosphorylation and is capable of conversion to a high-activity state by autophosphorylation under physiological ATP concentrations. This theoretically precludes the need for an upstream activating kinase. The activation process of JAK2 may be envisioned as a multistate process involving at least two kinetically distinct states of activity.

The biochemical and biophysical characteristics of Janus protein-tyrosine kinases (JAKs), which are essential early mediators of cytokine-initiated signal propagation, are virtually undefined. To facilitate the in vitro analysis of JAK-mediated catalysis, we substantially purified a soluble recombinant JAK2 and developed a novel means of quantifying JAK-catalyzed product formation. Glutathione-S-transferase fusion proteins containing active and inactive forms of rat Janus kinase 2 (GST:rJAK2 and GST:rJAK2(CA795)) were highly purified via affinity chromatography. A microtiterplate-based ELISA was used to measure tyrosine phosphorylation of a streptavidin-immobilized biotinylated STAT1-derived peptide. The ELISA data indicated that only about 1% of the enzyme was involved in exogenous substrate phosphorylation. Other immobilized peptides served as apparent substrates with varying efficacy. Traditional radioisotopic autokinase assays demonstrated that the activity of the purified fusion protein was inhibited by a variety of tyrphostin inhibitors. Non-radiolabeled adenine nucleotides, but not guanine nucleotides, inhibited the radioisotopic autokinase assay. These observations verify that the catalytic activity of JAK2 is highly regulated, and are consistent with the suggestion that JAK2 may require additional accessory proteins, such as a potential upstream regulatory kinase, for full catalytic activity.

The precise regulation of both the magnitude and the duration of Janus kinase (JAK) catalytic activity is essential for the cytokine orchestration of many biological processes, and the dysregulation of JAK activity has pathological implications. Immunosuppressive disease states, such as X-linked severe combined immunodeficiency, arise from inappropriate JAK inhibition. In contrast, a limited number of cancers, primarily leukemias, result from constitutive or enhanced activation of JAK activity. JAKs are no longer implicated only in classic cytokine receptor-mediated signaling pathways, but are now also known to integrate indirectly into other receptor-mediated signal transduction processes. Therefore, an increasing number of therapeutic applications exist for biological-response modifiers that can restore aberrant JAK activity to normal levels. Exciting breakthroughs in both physiological and pharmacological methods of selective inhibition of cytokine-JAK-signal transducers and activators of transcription pathways have recently emerged in the form of suppressors of cytokine signaling (also known as cytokine-inducible SH2 protein, JAK-binding protein, or STAT-induced STAT inhibitor) proteins and novel dimethoxyquinazoline derivatives, respectively. The basis of these and other mechanisms of negative regulation of JAK activity, including the suppression of jak expression levels caused by tumor- or pathogen-derived agents, the complex interactions of JAKs with phosphatases, and the redox regulation of JAK catalytic activity, is the focus of this review.

Human T cell leukemia virus type 1 (HTLV-1) transforms cytokine-dependent T lymphocytes and causes adult T cell leukemia. Janus tyrosine kinase (Jak)3 and transcription factors Stat5a and Stat5b are essential for the proliferation of normal T cells and are constitutively hyperactivated in both HTLV-1-transformed human T cell lines and lymphocytes isolated from HTLV-1-infected patients; therefore, a critical role for the Jak3-Stat5 pathway in the progression of this disease has been postulated. We recently reported that tyrphostin AG-490 selectively blocked IL-2 activation of Jak3/Stat5 and growth of murine T cell lines. Here we demonstrate that disruption of Jak3/Stat5a/b signaling with AG-490 (50 microM) blocked the proliferation of primary human T lymphocytes, but paradoxically failed to inhibit the proliferation of HTLV-1-transformed human T cell lines, HuT-102 and MT-2. Structural homologues of AG-490 also inhibited the proliferation of primary human T cells, but not HTLV-1-infected cells. Disruption of constitutive Jak3/Stat5 activation by AG-490 was demonstrated by inhibition of 1) tyrosine phosphorylation of Jak3, Stat5a (Tyr(694)), and Stat5b (Tyr(699)); 2) serine phosphorylation of Stat5a (Ser(726)) as determined by a novel phosphospecific Ab; and 3) Stat5a/b DNA binding to the Stat5-responsive beta-casein promoter. In contrast, AG-490 had no effect on DNA binding by p50/p65 components of NF-kappaB, a transcription factor activated by the HTLV-1-encoded phosphoprotein, Tax. Collectively, these data suggest that the Jak3-Stat5 pathway in HTLV-1-transformed T cells has become functionally redundant for proliferation. Reversal of this functional uncoupling may be required before Jak3/Stat5 inhibitors will be useful in the treatment of this malignancy.

Interleukin (IL) 4 is a potent immunomodulatory cytokine secreted by T-helper 2 (Th2) cells and Th2 mast cells that promotes the commitment of cells. However, unregulated production and release of IL-4 can exacerbate allergic reactions and increase susceptibility to infectious organisms and viruses. Here, we present evidence that AG-490, a Janus tyrosine kinase (JAK) 2-JAK3 inhibitor, effectively blocked IL-4 gene expression and secretion in the Th2 cell line D10 that was not occurring after anti-CD3 antibody stimulation, whereas AG-490 had no inhibitory effect on production of other Th2 cytokines or cytokines synthesized by the corresponding Th1 cell line clone 29. AG-490 potently inhibited IL-4-mediated proliferation of both D10 and the IL-4-dependent cell line CT.4S. Moreover, AG-490 markedly inhibited IL-4 activation of JAK3 and blocked the downstream activation of signal transducer and activator of transcription 6, as judged by tyrosine phosphorylation, DNA binding, and transcription assays. In contrast, AG-490 did not affect tumor necrosis factor alpha activation of NF-kappaB at similar concentrations of drug. These data suggest that tyrosine kinase inhibitors that inhibit JAK3 may have previously unrecognized and selective clinical potential as immunotherapeutic drugs to treat Th2-mediated diseases driven by IL-4. (Blood. 2000;95:3816-3822)

Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase required for T cell development and activated by cytokines that utilize the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)). Genetic inactivation of JAK3 is manifested as severe combined immunodeficiency disease (SCID) in humans and mice. These findings have suggested that JAK3 represents a pharmacological target to control certain lymphoid-derived diseases. Here we provide novel evidence that AG-490 potently inhibits the autokinase activity of JAK3 and tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription 5a and 5b (STAT5a/b). Similar inhibitory effects were observed with other cytokines that use gamma(c). AG-490 also inhibited IL-2-mediated proliferative growth in human T cells with an IC50) = 25 microM that was partially recoverable. Moreover, we demonstrate that this inhibitor prevented tetanus toxoid antigen-specific T cell proliferation and expansion but failed to block activation of Zap70 or p56Lck after anti-CD3 stimulation of human T cells. Taken together, these findings suggest that AG-490 inhibits the JAK3-mediated Type II signaling pathway but not the T cell receptor-derived Type I pathway and possesses therapeutic potential for T cell-derived pathologies such as graft-versus-host disease, allergy, and autoimmune disorders.

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor, these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study, evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation, a non-JAK2-dependent signal, in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates, STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1, -3, -5a, and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT, JAK3/AP-1, and JAK3/MAPK pathways and their cellular consequences. Taken together, these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways.

The activation of Janus kinases (JAKs) is crucial for propagation of the proliferative response initiated by many cytokines. The proliferation of various cell lines, particularly those of hematopoietic origin, is also modulated by mediators of oxidative stress such as nitric oxide and thiol redox reagents. Herein we demonstrate that nitric oxide and other thiol oxidants can inhibit the autokinase activity of rat JAK2 in vitro, presumably through oxidation of crucial dithiols to disulfides within JAK2. The reduced form of JAK2 is the most active form, and the oxidized JAK2 form is inactive. Nitric oxide pretreatment of quiescent Ba/F3 cells also inhibits the interleukin 3-triggered in vivo activation of JAK2, a phenomenon that correlates with inhibited proliferation. Furthermore, we observed that the autokinase activity of JAK3 responds in a similar fashion to thiol redox reagents in vitro and to nitric oxide donors in vivo. We suggest that the thiol redox regulation of JAKs may partially explain the generally immunosuppressive effects of nitric oxide and of other thiol oxidants.

The Janus family of protein-tyrosine kinases has long been known to function in signal transduction pathways initiated by a host of cytokines. A brief overview of the role of Janus kinases (Jaks) in both cytokine and noncytokine signaling pathways highlights the broad physiologic importance of this kinase family. New insights into the structural and mechanistic regulatory aspects of Janus kinases are rapidly emerging. Recent mutational analyses allow the dissection of Jaks into three distinct structural domains governing receptor affiliation, autoregulation, and catalysis. A fourth domain determining substrate specificity is as yet poorly defined and is, therefore, discussed in the context of known substrates and inhibitors, a collection of molecules that have been expanded recently to include Stam and Jab. The proposed mechanism of the interconversion of Janus kinases from inactive to fully active enzymes involves three states of enzymatic activity. Additional layers of regulation can be independently superimposed on this multistate model, providing a simplified description of the behavior of Janus kinases under normal and pathologic circumstances.

Microinjection of expression plasmids encoding either JAK2 or hyperactive (Ndelta661)rJAK2 into serum-starved NIH 3T3 cells resulted in 20-30-fold induction of DNA synthesis. Control microinjections of buffer or parental pcDNA3 vector resulted in only 3-5-fold induction of DNA synthesis. Induction of DNA synthesis was blocked when plasmid encoding JAK2 was microinjected in the presence of the JAK2-selective inhibitor AG-490, whereas AG-490 did not block DNA synthesis induced by microinjected plasmid encoding (Ndelta661)rJAK2. The ability of JAK2 to initiate the G(o)/S cell cycle transition is comparable to that of other proto-oncogenes, and supports a mechanistic role for overexpressed Janus kinases in carcinogenesis.

The lymphocyte growth factors interleukin-2 (IL2), IL4, IL7, IL9 and IL15 use the common IL2 receptor-gamma (IL2R gamma) and activate the IL2R gamma-associated tyrosine kinase JAK3 (Janus kinase 3). IL13 is structurally related to IL4, competes with IL4 for binding to cell surface receptors and exhibits many similar biological effects. The molecular basis for this functional overlap between IL4 and IL13 has been attributed mainly to a shared use of the 140 kDa IL4R alpha, since these cytokines appear to be uniquely different in that, according to several recent reports, IL13 does not recruit the IL2R gamma or JAK3. This notion has been supported by the identification of a novel 70 kDa IL13 receptor in certain IL13-responsive cell lines that lack IL2R gamma. The present study sheds new light on the issue of functional overlap between IL13 and IL4, by demonstrating for the first time that, in cells that express both IL2R gamma and IL4R alpha, IL13 can mimic IL4-induced heterodimerization of IL2R gamma and IL4R alpha, with consequent marked activation of JAK3 and the transcription factor STAT6 (IL4-STAT). Reconstitution experiments in BA/F3 cells showed that both cytokines require the simultaneous presence of IL4R alpha and IL2R gamma to mediate JAK3 and proliferative responses, and analysis of 12 IL4R alpha variants showed that IL4 and IL13 signals were equally affected by mutations of the cytoplasmic domain. We conclude that IL13 activates the IL2R gamma-associated JAK3 tyrosine kinase in appropriate cell types, and propose that IL13 is capable of interacting with multiple receptor subunits in a cell-dependent and combinatorial manner. Consequently, we predict that partial disruption of IL13 signal transduction also contributes to the severe combined immuno-deficiency syndromes associated with inactivation of the IL2R gamma or JAK3 genes.

A number of cytokines and growth factors use the JAK-STAT pathway to signal from the cell membrane to the nucleus. While homodimerizing cytokine receptors may transmit signal via a single form of JAK (i.e. growth hormone receptors), several multicomponent cytokine receptors have been shown to require simultaneous activation of pairs of different JAK kinases (i.e. interferon receptors). Recent evidence for a preferential coupling of JAK3 to interleukin-2 receptor-gamma (IL-2R gamma) and JAK1 to IL-2R beta supports the concept of heterotrans-activation of JAK1 and JAK3 caused by IL-2-induced heterodimerization of their receptor partners. The present study verified the ability of IL-2 to cause tyrosine phosphorylation and activation of JAK1 and JAK3, but demonstrated that IL-2 stimulated JAK3 to a significantly larger extent than JAK1 in human T lymphocytes and the YT cell line. This conclusion was based upon several independent criteria, including more vigorous tyrosine phosphorylation of JAK3, more marked enzymatic activation of JAK3 as well as higher abundance of JAK3 in activated IL-2 receptor complexes. Furthermore, when human IL-2R beta was stably expressed in murine BA/F3 cells, robust IL-2-induced proliferation and JAK3 activation occurred without detectable involvement of either JAK1, JAK2 or TYK2. We therefore propose that IL-2 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL-2-induced heterodimerization of IL-2R beta and IL-2R gamma. Nonetheless, a membrane-proximal region of human IL-2R beta (Asn240-Leu335) was critical for JAK3 activation, and the amount of JAK3 present in activated IL-2 receptor complexes increased with time, suggesting that stabilization of JAK3 binding to the receptor complex relies on both IL-2R beta and IL-2R gamma. Moreover, STAT5 was found to be the predominant STAT transcription factor used by IL-2 in human T cells, and specifically required a COOH-terminal region of IL-2R beta (Ser386-Val525), while STAT5 recruitment was not correlated to activation of IL-2R gamma or JAK3.

Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were produced via the baculovirus expression vector system. Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell line (rJAK2), a carboxyl-terminal deletion mutant lacking the putative catalytic domain (rJAK2(C delta 795)), or an amino-terminal deletion mutant containing the putative catalytic domain ((N delta 661)rJAK2). The proteins produced in infected Sf21 cells were assayed for phosphotyrosine content and autophosphorylating activity. Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection when rJAK2 was initially produced but was apparent 2 or more days postinfection when the rJAK2 level had significantly increased. Tyrosine phosphorylation of rJAK2(C delta 795) was not observed; further, coproduction of rJAK2(C delta 795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consistent with previously published results (Zhuang, H., Patel, S. V., He, T-C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 21411-21414). Mutant (N delta 661)rJAK2 exhibited a robust tyrosine phosphorylation signal. A second 62-kDa tyrosine phosphoprotein co-immunoprecipitated with (N delta 661)rJAK2 but not with rJAK2 or rJAK2(C delta 795). Both rJAK2 and (N delta 661)rJAK2 incorporated phosphate under in vitro kinase assay conditions, but rJAK2(C delta 795) did not. A JAK2 oligomer with interacting catalytic sites and/or inhibitory sites would provide a simple model to describe these results.

A complete cDNA clone encoding the rat JAK2 protein tyrosine kinase was isolated from an Nb2-SP (rat pre-T lymphoma cell line) cDNA library. The nucleotide (nt) and deduced amino acid (aa) sequences for this clone were determined and an open reading frame of 3399 bp, encoding a protein of a deduced mass of 130 kDa, was found. The coding regions of the rat and murine Jak2 clones share 93.4% nt identity and 97.1% aa identity. Northern analysis demonstrated that the 5-kb mRNA is highly abundant in brain and spleen, less abundant in skeletal muscle and testis, and detectable in kidney, heart, lung and liver. Translation of the rat Jak2 mRNA in rabbit reticulocytes results in a protein which is specifically immunoprecipitated by antibodies (Ab) recognizing JAK2, but not by Ab recognizing JAK1.

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

JAK1A is a recently isolated class 3 (nonreceptor) tyrosine kinase that has catalytic domain sequence homology with other kinases and is known to be ubiquitously expressed in all human tissues thus far examined. The gene for this enzyme had previously been localized to chromosome 1 using somatic cell hybrids and linkage analyses. In the present study, fluorescence in situ hybridization was utilized to confirm its localization and regionally assign the gene to chromosome region 1p32.3-->p31.3.

Cytokines that interact with receptors of the hematopoietin super-family have recently been reported to stimulate receptor-associated JAK tyrosine kinases, including PRL activation of JAK2. Unlike other tyrosine kinases, none of the JAK kinases has thus far been implicated in oncogenesis, and their involvement in growth signaling has not been established. Using the PRL-dependent pre-T-cell line Nb2, the present study provided a link between bivalent dimerization of a hematopoietin receptor and activation of its associated JAK kinase, and demonstrated a strong positive correlation between the mitogenic potency of a series of bivalent anti-PRL receptor antibodies and the degree of induced tyrosine phosphorylation of JAK2. Antibody bivalency was required for JAK2 phosphorylation. Monovalent anti-PRL receptor Fab fragments alone were inactive, but their activity could be partially restored by cross-linking with bivalent anti-Fab antibodies. Additional evidence for antibody-induced receptor dimerization was provided by a bell-shaped dose-response curve for the most potent receptor agonist, monoclonal antibody T6. This phenomenon is typically seen at pharmacological concentrations of bivalent ligands, when bound ligand molecules fail to adjoin a second receptor due to occupancy. The present study provided functional support for a model of PRL receptor triggering by ligand-induced receptor homodimerization and subsequent activation of the associated tyrosine kinase JAK2.

Pervanadate has been shown to rapidly increase the level of tyrosine phosphorylation in intact cells. Because one of the most rapidly detectable events following treatment of human T cells with interleukin-2 (IL-2) is tyrosine kinase activation, we were interested to determine whether pervanadate could act to induce IL-2-associated events. We show here that pervanadate does act to induce IL-2 signal transduction pathways as determined by induction of mitogenesis and interferon gamma production in normal human T cells and the factor independent T cell line YT. Analysis of signal transduction events shows that pervanadate induces the activity of the src family of tyrosine kinases lck and fyn and the tyrosine phosphorylation of a major IL-2 responsive protein of 97 kDa. Pervanadate does not, however, induce the activity of tyrosine kinases associated with the IL-2 receptor or the phosphorylation of a major IL-2 responsive protein of 116 kDa (Jak-3). Together these data suggest that src family kinase activation is a down stream event following IL-2 stimulation and is not directly associated with the activation of the IL-2 receptor-associated tyrosine kinase. The data also imply that tyrosine phosphorylation of p116/Jak-3 is strictly associated with activation of tyrosine kinases associated with the IL-2 receptor. With the use of pervanadate as a tool, we have established a dissociation of src family kinases with IL-2 receptor activation and imply the involvement of two distinct tyrosine kinase pathways, a receptor-associated pathway closely coupled with Jak-3 phosphorylation and a downstream pathway involving src family kinase activation.

Janus tyrosine kinase (JAK) has recently been linked to signal transduction by cytokine receptors of the hematopoietin family. We have recently described a 116-kDa tyrosine kinase (p116) present in interleukin-2 (IL-2) receptor complexes in human YT cells that showed functional characteristics of a JAK kinase. These included receptor association, rapid and transient tyrosine phosphorylation kinetics in response to ligand, and in vitro autophosphorylating tyrosine kinase activity (Kirken, R. A., Rui, H., Evans, G. A., and Farrar, W. L. (1993) J. Biol. Chem. 268, 22765-22770). Here we extend these observations by demonstrating structural homologies between IL-2-modulated p116 and prolactin-modulated JAK2 in the rat T cell line Nb2. These include similar net charge as determined by nonequilibrium pH gradient electrofocusing and related primary structure based upon phosphopeptide mapping of V8 protease-digested hyperphosphorylated proteins. This putative JAK kinase underwent marked tyrosine phosphorylation in response to IL-2, IL-4, and IL-7, lymphoid growth factors that use the common IL-2 receptor gamma-chain, but not in response to prolactin. Furthermore, polyclonal antisera to JAK1, JAK2, or tyrosine kinase 2 did not recognize either rat or human p116. However, we identified the IL-2-modulated p116 as the recently cloned novel leukocyte Janus kinase, L-JAK, using an antiserum to a peptide corresponding to the COOH terminus of human L-JAK.

Prolactin (PRL) has recently been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2 in PRL-dependent Nb2 lymphoma cells. The present study represents an initial effort to identify cytoplasmic regions of the PRL receptor (PRLR) that are critical to growth signal generation and JAK2 activation. Variably truncated rat PRLRs were stably expressed in the murine 32D cell line. PRL-induced proliferation was mediated by the full-length receptor and the mutant G328, which contains the membrane-proximal Homology Boxes 1 and 2 characteristic of hematopoietin receptors. In contrast, mutant receptors lacking either Box 2 or both Boxes 1 and 2 were not capable of transmitting a growth signal. The mitogenic capacity of the PRLR variants correlated with JAK2 association and activation, as well as with induction of mRNA levels for the growth-related gene ornithine decarboxylase. We conclude that mitogenic competence of rat PRLRs resides within the first 94 amino acids of the cytoplasmic domain. The data suggest that Homology Box 1/Box 2 region is critical to JAK2 phosphorylation and association with the PRLR. These findings will serve as a basis for more detailed molecular characterization of PRLR domains essential for JAK2 interaction and growth signal generation.

JAK family tyrosine kinases have recently been implicated in intracellular signal transduction by transmembrane cytokine receptors of the interferon (IFN) and hematopoietin receptor families. Using the prolactin (PRL)-dependent rat pre-T cell line Nb2, a PRL receptor-associated, candidate tyrosine kinase of 120-130 kDa was recently characterized (1). In the present work this protein is identified as JAK2, based upon reciprocal anti-JAK2 and anti-phosphotyrosine immunoprecipitation and immunoblotting. JAK2 underwent rapid and transient tyrosine phosphorylation in response to receptor activation, reaching peak levels within 5 min of exposure to 100 nM PRL at 37 degrees C. In vitro tyrosine kinase assays using either [gamma-32P]ATP and autoradiography or unlabeled ATP combined with anti-phosphotyrosine immunoblotting, demonstrated that the activity of JAK2 was stimulated by PRL. Phosphoamino acid analysis of JAK2 after in vitro tyrosine kinase assay revealed that the majority of phosphate was incorporated into tyrosine residues. Furthermore, JAK2 was associated with PRL receptors to a comparable extent before and after PRL binding, as demonstrated by anti-receptor immunoprecipitation and subsequent anti-JAK2 immunoblotting. We propose that binding of ligand to the PRL receptor activates preassociated JAK2, and that this enzyme generates the initial signal in the intracellular communication cascade.

The haematopoietic protein, p95vav, has been shown to be a tyrosine kinase substrate and to have tyrosine kinase-modulated guanine-nucleotide-releasing-factor activity. This implies a function in the control of ras or ras-like proteins. Because ras activation has been shown to be a downstream event following stimulation of the interleukin-2 (IL-2) receptor, we investigated the possibility that vav was involved in IL-2 signal transduction pathways, using human T cells as a model. We found rapid tyrosine phosphorylation of vav in response to IL-2 within 1 min, with maximum increase of phosphorylation of 5-fold occurring by 5 min after treatment in normal human T cells. IL-2 stimulation of the human T-cell line YT and a subclone of the YT cell line (YTlck-) that does not express message for the src-family kinase p56lck also results in a rapid rate of tyrosine phosphorylation of vav of more than 5-fold by 5 min. These results suggest that vav may play an important role in IL-2-stimulated signal transduction and that there is not a strict requirement for the tyrosine kinase p56lck.

The mechanism of action of prolactin (PRL) has remained obscure despite the unveiling of the primary structure of PRL receptors. The present study demonstrates rapid PRL receptor-mediated tyrosine phosphorylation of at least three cellular proteins, designated p120, p97, and p40, in a rat T-lymphoma (Nb2-11C) as revealed by antiphosphotyrosine immunoblotting. One of the phosphotyrosyl proteins, p120, co-purified with activated PRL receptor complexes obtained using either anti-ligand or anti-receptor antibodies. Furthermore, in vitro incubation of affinity-purified PRL receptor complexes from PRL-stimulated cells with ATP in the presence of a tyrosine phosphatase inhibitor, resulted in a 10-15-fold increase in the phosphotyrosine content of p120, as revealed by antiphosphotyrosine immunoblotting. Parallel experiments utilizing [gamma-32P]ATP confirmed a rapid and time-dependent incorporation of phosphate into p120 in the same affinity-purified PRL receptor complexes. These data provide strong evidence for the involvement of a tyrosine kinase in PRL signal transduction and suggest the presence of a tyrosine kinase within the activated PRL receptor complex.

We have examined the signal transduction pathways of a number of cytokines that interact with receptors that are members of the hematopoietin receptor superfamily. A 97-kDa protein was phosphorylated on tyrosine in response to stimulation of appropriate target cells with interleukin (IL)-2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, or erythropoietin. These data suggest that a 97-kDa phosphotyrosylprotein represents a point of convergence for signal transduction by a number of growth factor receptors that do not have homology with any known protein tyrosine kinase. To address the possibility that p97 may represent a tyrosine kinase involved in multiple signal transduction pathways, we tested the capacity of this protein to bind a tyrosine kinase substrate or ATP. Indeed, a 97-kDa phosphotyrosylprotein purified from IL-2-stimulated lymphoid cells as well as granulocyte-macrophage-CSF-stimulated myeloid cells bound to a polymer of glutamic acid and tyrosine which is a tyrosine kinase substrate. Further, a 97-kDa phosphotyrosylprotein present in both lineages also bound 8-azido-ATP. These data indicate that a 97-kDa phosphotyrosylprotein with properties consistent with those of a protein tyrosine kinase is involved in the signal transduction pathways of certain members of the newly identified hematopoietin receptor superfamily and may represent an early point of convergence in the stimulus-response coupling of multiple cytokine receptors.

The IL-2 receptor complex is minimally composed of two genetically unrelated subunits of relative molecular masses 55 and 75 kDa respectively. Structural information deduced from the cDNA sequences of either subunit have not revealed significant information as to the basis of the mechanisms of IL-2 receptor signal transduction. Nevertheless, IL-2 stimulates the activation of one or more tyrosine kinases requiring the functional participation of the p75 member of the receptor complex. Here we have developed the methods to isolate the receptor complex with an associated tyrosine protein kinase. Extracts of membrane glycoproteins from activated normal human T lymphocytes and cell lines demonstrated catalytic activation of tyrosine kinase activity when stimulated with IL-2. Purification of the receptor complex with biotinylated IL-2 revealed the presence of two dominant phosphotyrosyl-proteins of approximate molecular masses 58 and 97 kDa. Denaturation gel electrophoresis followed by renaturation of proteins associated with the IL-2 receptor complex demonstrated that the 97 kDa protein had catalytic autophosphorylation activity. The results indicate that the 58 and 97 kDa phosphotyrosyl-proteins can be found to co-precipitate with the IL-2 receptor complex and that the 97 kDa protein was demonstrated to have protein kinase activity. The association of such kinases with receptors devoid of catalytic structure may represent a unique paradigm of growth-factor receptor mechanisms.

We have examined the signal transduction mechanism of the hematopoietic growth factor erythropoietin (Epo). Epo stimulation of Ba/F3 cells transfected with the Epo receptor resulted in increases in tyrosine phosphorylation of proteins of 97, 75, and 55 kDa. Epo-induced increases in tyrosine phosphorylation of a 97-kDa protein were also detected within the Epo receptor complex, suggesting that a protein tyrosine kinase is associated with the Epo receptor. Protein tyrosine kinase activity was found within the Epo receptor complex and modulation of this activity was observed after treatment of cells with Epo. Furthermore, constitutively high amounts of protein kinase activity were observed in Epo receptor complexes isolated from autonomously growing cells coexpressing the Epo receptor and the leukemogenic glycoprotein gp55. The dominant phosphotyrosylprotein found associated with the Epo receptor was 97 kDa. An Epo receptor-associated protein of identical molecular mass was also found to bind ATP, a characteristic critical for protein kinases. Collectively, these data demonstrate that the Epo receptor is associated with protein tyrosine kinase activity and further suggest that a 97-kDa phosphotyrosylprotein associated with the Epo receptor is a protein tyrosine kinase involved in Epo-mediated signal transduction.

In an effort to identify unique tyrosine kinases found in human leukemia cell lines, we utilized polymerase chain reaction (PCR) technology and degenerate oligonucleotide primers to produce a cDNA library of kinase catalytic domains found in the human monocytic cell line AML-193. This search yielded a member of the class 3 tyrosine kinases closely related to the murine kinase FD-22. Previous work has identified this kinase as JAK1. This class of tyrosine kinases is characterized by being ubiquitously expressed, lacking both a ligand-binding domain and a SH2 domain, while containing a second domain similar to a degenerate kinase domain. Our studies focused on the further characterization of this class 3 tyrosine kinase using Northern blot analysis to demonstrate an increase in steady-state mRNA by interferon-gamma in human monocytes. A human-hamster somatic cell hybrid panel and linkage mapping was used to assign JAK1 (aml-116) to human chromosome 1.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.

We have investigated whether PK-C-regulated events are independent of those biochemical events related to IL-3-induced tyrosine kinase activation by 32Dcl cells. The depletion of functional PK-C isoform activity by prolonged PMA treatment reduced the proliferative response to IL-3 by half that of untreated control cells. PK-C-deficient 32Dcl cells were unable to respond to PMA for the induction of c-myc and ODC mRNA accumulation. PK-C down-regulation did not affect IL-3-induced tyrosine phosphorylation and inhibited IL-3-regulated c-myc and ODC mRNA expression by only 30%. However, PK-C down-regulation had a pronounced inhibitory effect on IL-3 regulation of ODC enzymatic activity. While a PK-C-dependent and -independent pathway for the regulation of c-myc and ODC mRNA expression could be demonstrated, the regulation of ODC enzymatic activity appeared to require an intact PK-C system. The data suggest that the optimum biological and biochemical responses to IL-3 requires both pathways intact, however, tyrosine kinase activation and significant increases in gene products associated with proliferation can be achieved in the absence of a functional PK-C system.

The proliferation of activated T lymphocytes is dependent on the interaction of the polypeptide growth factor interleukin 2 (IL 2) with its heterodimeric receptor, which consists of a p55 alpha subunit and a p70-75 beta subunit. Previously, it was shown that IL 2 stimulates rapid serine phosphorylation of several membrane and cytysolic proteins. Here, using anti-phosphotyrosine antibodies to purify phosphotyrosyl proteins and two-dimensional gel analysis, we show that IL 2 stimulates rapid tyrosine phosphorylation of a variety of cellular proteins, including pp180, pp92, and pp42 in activated human T lymphocytes. In addition, we have examined IL 2-induced tyrosine phosphorylation in the human cell line YT2C2 which expresses mostly the beta subunit of the IL 2 receptor and the gibbon cell line MLA-144 which expresses only the beta subunit. In both of these cell lines, IL 2 induced tyrosine phosphorylation of the same proteins phosphorylated in normal human T lymphocytes in response to IL 2. We conclude that the beta subunit is sufficient to induce tyrosine phosphorylation of the normal cellular target substrates involved in signal transduction.

Interleukin 3 stimulates the proliferation of FDC-P1, a murine myeloid cell line, however the biochemical events subsequent to binding of IL3 have only recently begun to be investigated. We have previously described the activation of protein kinase C (PK-C) and serine/threonine phosphorylation of a 68 kd protein following IL3 treatment of FDC-P1 cells. Here we have used an anti-phosphotyrosine antibody to purify proteins containing phosphotyrosine following IL3 administration to FDC-P1 cells. We find that tyrosine phosphorylation of two proteins of 50 (pp50) and 70 (pp70) kilodaltons occurs rapidly following IL3 treatment. In addition to phosphotyrosine both proteins also contained phosphoserine. Together with previous evidence these results suggest that coactivation of serine/threonine and tyrosine kinase activities which target unique proteins may be an important element in IL3 signal transduction.

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.