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We provide evidence for a unique pathway engaged by the type II IFN receptor, involving mTORC2/AKT-mediated downstream regulation of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of type II interferon-stimulated genes. Our studies establish that Rictor is essential for the generation of type II IFN-dependent antiviral and antiproliferative responses and that it controls the generation of type II IFN-suppressive effects on normal and malignant hematopoiesis. Together, our findings establish a central role for mTORC2 in IFNγ signaling and type II IFN responses.

The subset of acute myeloid leukemias (AML) with chromosomal translocations involving the MLL gene have a poor prognosis (referred to as 11q23-AML). The MLL fusion proteins that are expressed in 11q23-AML facilitate transcription of a set of HOX genes, including HOXA9 and HOXA10. Because Hox proteins are transcription factors, this suggests the possibility that Hox target genes mediate the adverse effects of MLL fusion proteins in leukemia. Identifying such Hox target genes might provide insights to the pathogenesis and treatment of 11q23-AML. In the current study we found that Mll-Ell (an MLL fusion protein) induced transcriptional activation of the FGF2 gene in a HoxA9- and HoxA10-dependent manner. FGF2 encodes fibroblast growth factor 2 (also referred to as basic fibroblast growth factor). Fgf2 influences proliferation and survival of hematopoietic stem cells and myeloid progenitor cells, and increased Fgf2-expression has been described in AMLs. We determined that expression of Mll-Ell in myeloid progenitor cells resulted in autocrine production of Fgf2 and Fgf2-dependent cytokine hypersensitivity. Therefore, our results implicated increased Fgf2 expression in progenitor proliferation and expansion in 11q23-AML. Because small molecule inhibitors of Fgf-receptors are in human clinical trials, this suggested a potential therapeutic approach to this treatment refractory leukemia.

The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.

Three major laminin and collagen-binding integrins in skin (α6β4, α3β1, and α2β1) are involved in keratinocyte adhesion to the dermis and dissemination of skin cells during wound healing and/or tumorigenesis. Knockdown of α6 integrin in keratinocytes not only results in motility defects but also leads to decreased surface expression of the α2, α3, and β4 integrin subunits. Whereas α2 integrin mRNA levels are decreased in α6 integrin knockdown cells, α3 and β4 integrin mRNAs levels are unaffected. Expression of either α6 or α3 integrin in α6 integrin knockdown cells restores α2 integrin mRNA levels. Moreover, re-expression of α6 integrin increases β4 integrin protein at the cell surface, which results in an increase in α3 integrin expression via activation of initiation factor 4E-binding protein 1. Our data indicate that the α6β4 integrin is a master regulator of transcription and translation of other integrin subunits and underscore its pivotal role in wound healing and cancer.

IFNα exerts potent inhibitory activities against malignant melanoma cells in vitro and in vivo, but the mechanisms by which it generates its antitumor effects remain unknown. We examined the effects of interferon α (IFNα) on the expression of human members of the Schlafen (SLFN) family of genes, a group of cell cycle regulators that mediate growth-inhibitory responses. Using quantitative RT-real time PCR, we found detectable basal expression of all the different human SLFN genes examined (SLFN5, SLFN11, SLFN12, SLFN13, and SLFN14), in malignant melanoma cells and primary normal human melanocytes, but SLFN5 basal expression was suppressed in all analyzed melanoma cell lines. Treatment of melanoma cells with IFNα resulted in induction of expression of SLFN5 in malignant cells, suggesting a potential involvement of this gene in the antitumor effects of IFNα. Importantly, stable knockdown of SLFN5 in malignant melanoma cells resulted in increased anchorage-independent growth, as evidenced by enhanced colony formation in soft agar assays. Moreover, SLFN5 knockdown also resulted in increased invasion in three-dimensional collagen, suggesting a dual role for SLFN5 in the regulation of invasion and anchorage-independent growth of melanoma cells. Altogether, our findings suggest an important role for the SLFN family of proteins in the generation of the anti-melanoma effects of IFNα and for the first time directly implicate a member of the human SLFN family in the regulation of cell invasion.

Type I interferons (IFNs) are cytokines with diverse biological properties, including antiviral, growth inhibitory, and immunomodulatory effects. Although several signaling pathways are activated during engagement of the type I IFN receptor and participate in the induction of IFN responses, the mechanisms of generation of specific signals for distinct biological effects remain to be elucidated. We provide evidence that a novel member of the protein kinase C (PKC) family of proteins is rapidly phosphorylated and activated during engagement of the type I IFN receptor. In contrast to other members of the PKC family that are also regulated by IFN receptors, PKCη does not regulate IFN-inducible transcription of interferon-stimulated genes or generation of antiviral responses. However, its function promotes cell cycle arrest and is essential for the generation of the suppressive effects of IFNα on normal and leukemic human myeloid (colony-forming unit-granulocyte macrophage) bone marrow progenitors. Altogether, our studies establish PKCη as a unique element in IFN signaling that plays a key and essential role in the generation of the regulatory effects of type I IFNs on normal and leukemic hematopoiesis.

We have shown that the mammalian target of rapamycin (mTOR) down-regulates thrombin-induced ICAM-1 expression in endothelial cells by suppressing the activation of NF-kappaB. However, the mechanisms by which mTOR is activated to modulate these responses remain to be addressed. Here, we show that thrombin engages protein kinase C (PKC)-delta and phosphattidylinositol 3-kinase (PI3K)/Akt pathways to activate mTOR and thereby dampens NF-kappaB activation and intercellular adhesion molecule 1 (ICAM-1) expression. Stimulation of human vascular endothelial cells with thrombin induced the phosphorylation of mTOR and its downstream target p70 S6 kinase in a PKC-delta- and PI3K/Akt-dependent manner. Consistent with this, thrombin-induced phosphorylation of p70 S6 kinase was defective in embryonic fibroblasts from mice with targeted disruption of PKC-delta (Pkc-delta(-)(/)(-)), p85alpha and p85beta subunits of the PI3K (p85alpha(-)(/)(-)beta(-)(/)(-)), or Akt1 and Akt2 (Akt1(-)(/)(-)2(-)(/)(-)). Furthermore, we observed that expression of the constitutively active form of PKC-delta or Akt was sufficient to induce NF-kappaB activation and ICAM-1 expression, and that co-expression of mTOR suppressed these responses. In reciprocal experiments, inhibition/depletion of mTOR augmented NF-kappaB activation and ICAM-1 expression induced by PKC-delta or Akt. In control experiments, increasing or impairing mTOR signaling by the above approaches produced similar effects on NF-kappaB activation and ICAM-1 expression induced by thrombin. Thus, these data reveal an important role of PKC-delta and PI3K/Akt pathways in activating mTOR as an endogenous modulator to ensure a tight regulation of NF-kappaB signaling of ICAM-1 expression in endothelial cells.

Arsenic trioxide (As2O3) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As2O3 induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38α MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1–/– or Mnk2–/–, or double Mnk1–/–Mnk2–/– knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.

The interferons (IFNs) are cytokines that play key roles in host defense against viral infections and immune surveillance against cancer. We report that BCR-ABL transformation of hematopoietic cells results in suppression of IFN-dependent responses, including transcription of IFN-inducible genes and generation of IFN-mediated antiviral effects. BCR-ABL transformation suppresses expression of several IFN-regulated genes containing IFN-sensitive response element (ISRE) or GAS elements in their promoters, including Isg15, Irf1, Irf9, and Ifit2 (interferon-induced protein with tetratricopeptide repeats 2). Suppression of transcription of ISRE-containing genes is also seen in cells expressing various BCR-ABL kinase domain mutants, including T315I, H396P, Y253F, and E255K, but not kinase-defective BCR-ABL. Such effects are associated with impaired IFN-dependent phosphorylation of Stat1 on Tyr701 and Stat3 on Tyr705 and defective binding of Stat complexes to ISRE or GAS elements. Beyond suppression of Stat activities, BCR-ABL inhibits IFN-inducible phosphorylation/activation of the p38 MAPK, suggesting a dual mechanism by which this abnormal fusion protein blocks IFN transcriptional responses. The inhibitory activities of BCR-ABL ultimately result in impaired IFNα-mediated protection against encephalomyocarditis virus infection and reversal of IFN-dependent growth suppression. Altogether, our data provide evidence for a novel mechanism by which BCR-ABL impairs host defenses and promotes malignant transformation, involving dual suppression of IFN-activated signaling pathways.

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the mechanisms that mediate such effects are not well understood. We provide evidence that the Akt kinase is phosphorylated/activated during treatment of leukemia cells with As(2)O(3), to regulate downstream engagement of mammalian target of rapamycin (mTOR) and its effectors. Using cells with targeted disruption of both the Akt1 and Akt2 genes, we found that induction of arsenic trioxide-dependent apoptosis is strongly enhanced in the absence of these kinases, suggesting that Akt1/Akt2 are activated in a negative feedback regulatory manner, to control generation of As(2)O(3) responses. Consistent with this, As(2)O(3)-dependent pro-apoptotic effects are enhanced in double knock-out cells for both isoforms of the p70 S6 kinase (S6k1/S6k2), a downstream effector of Akt and mTOR. On the other hand, As(2)O(3)-dependent induction of apoptosis is diminished in cells with targeted disruption of TSC2, a negative upstream effector of mTOR. In studies using primary hematopoietic progenitors from patients with acute myeloid leukemia, we found that pharmacological inhibition of mTOR enhances the suppressive effects of arsenic trioxide on leukemic progenitor colony formation. Moreover, short interfering RNA-mediated inhibition of expression of the negative downstream effector, translational repressor 4E-BP1, partially reverses the effects of As(2)O(3). Altogether, these data provide evidence for a key regulatory role of the Akt/mTOR pathway in the generation of the effects of As(2)O(3), and suggest that targeting this signaling cascade may provide a novel therapeutic approach to enhance the anti-leukemic properties of As(2)O(3).

Transcription factor CCAAT/enhancer-binding protein-beta (C/EBP-beta) regulates a variety of cellular functions in response to exogenous stimuli. We have reported earlier that C/EBP-beta induces gene transcription through a novel interferon (IFN)-response element called gamma-IFN-activated transcriptional element. We show here that IFN-gamma-induced, C/EBP-beta/gamma-IFN-activated transcriptional element-dependent gene expression is regulated by mixed lineage kinases (MLKs), members of the mitogen-activated protein kinase kinase kinase family. MLK3 appears to activate C/EBP-beta in response to IFN-gamma by a mechanism involving decreased phosphorylation of a specific phosphoacceptor residue, Ser(64), within the transactivation domain. Decreased phosphorylation of Ser(64) was independent of IFN-gamma-stimulated ERK1/2 activation and did not require the ERK phosphorylation site Thr(189) located in regulatory domain 2 of C/EBP-beta. Together these studies provide the first evidence that MLK3 is involved in IFN-gamma signaling and identify a novel mechanism of transcriptional activation by IFN-gamma.

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.

Matrix metalloproteinases (MMPs) play an important role in the invasive behavior of a number of cancers including oral squamous cell cancer (OSCC), and increased expression of MMP-9 is correlated with invasive and metastatic OSCC. Because calcium is an important regulator of keratinocyte function, the effect of modulating extracellular calcium on MMP-9 expression in OSCC cell lines was evaluated. Increasing extracellular calcium induced a dose-dependent increase in MMP-9 expression in immortalized normal and premalignant oral keratinocytes, but not in two highly invasive OSCC cell lines. Differential activation of MAPK signaling was also induced by calcium. p38 MAPK activity was down-regulated, whereas ERK1/2 activity was enhanced. Pharmacologic inhibition of p38 MAPK activity or expression of a catalytically inactive mutant of the upstream kinase MAPK kinase 3 (MKK3) increased the calcium induced MMP-9 gene expression, demonstrating that p38 MAPK activity negatively regulated this process. Interestingly blocking p38 MAPK activity enhanced ERK1/2 phosphorylation, suggesting reciprocal regulation between the ERK1/2 and p38 MAPK pathways. Together these data support a model wherein calcium-induced MMP-9 expression is differentially regulated by the ERK1/2 and p38 MAPK pathways in oral keratinocytes, and the data suggest that a loss of this regulatory mechanism accompanies malignant transformation of the oral epithelium.

Protein kinase C-theta (PKC-theta) plays important roles in the activation and survival of lymphocytes and is the predominant PKC isoform expressed in T-cells. Interferons regulate T-cell function and activation, but the precise signaling mechanisms by which they mediate such effects have not been elucidated. We determined whether PKC-theta is engaged in interferon (INF) signaling in T-cells. Both Type I (alpha, beta) and Type II (gamma) IFNs induced phosphorylation of PKC-theta in human T-cell lines and primary human T-lymphocytes. Such phosphorylation of PKC-theta resulted in activation of its kinase domain, suggesting that this kinase plays a functional role in interferon signaling. Consistent with this, inhibition of PKC-theta protein expression using small interfering RNAs (siRNA) abrogated IFN-alpha- and IFN-gamma-dependent gene transcription via GAS elements. Similarly, blocking of PKC-theta kinase activity by overexpression of a dominant-negative PKC-theta mutant also blocked GAS-driven transcription, further demonstrating a requirement for PKC-theta in IFN-dependent transcriptional activation. The effects of PKC-theta on IFN-dependent gene transcription were not mediated by regulation of the IFN-activated STAT pathway, as siRNA-mediated PKC-theta knockdown had no effects on STAT1 phosphorylation and binding of STAT1-containing complexes to SIE/GAS elements. On the other hand, siRNA-mediated PKC-theta inhibition blocked phosphorylation/activation of MKK4, suggesting that interferon-dependent PKC-theta activation regulates downstream engagement of MAP kinase pathways. Altogether, these findings demonstrate that PKC-theta is an interferon-inducible kinase and strongly suggest that it plays an important role in the generation of interferon-responses in T-cells.

Multiple signaling pathways are activated during engagement of the Type I interferon (IFN) receptor to mediate biological responses, including the Jak-Stat and Rac1/p38 Map kinase signaling cascades. In the present study we sought to determine the functional relevance of the p38alpha isoform in IFN signaling, using cells from mouse embryos with targeted disruption of the p38alpha gene. Our data demonstrate that p38alpha activation is essential for Type I IFN-dependent transcriptional regulation via ISRE or GAS elements. On the other hand, the function of p38alpha is not required for IFN-dependent Ser727 or Tyr701 phosphorylation of Stat1 and does not impact on the formation of ISGF3 or SIF nuclear binding complexes. In efforts to identify downstream effectors of p38 that may mediate IFN-dependent transcriptional responses, we found that IFNalpha activates the kinase Msk1, a known regulator of histone phosphorylation and chromatin remodeling. In other studies, we demonstrate that Type I IFN-dependent activation of the kinases MapKapK-2 and MapKapK-3 is defective in the absence of p38alpha, while Type I IFN-dependent antiviral properties are decreased in cells with targeted disruption of the MapKapK-2 gene. Altogether, our data establish that the p38alpha Map kinase pathway regulates activation of downstream effectors that participate in the induction of IFN-dependent gene transcription, to mediate IFN-responses.

The Type I IFN receptor-generated signals required for initiation of mRNA translation and, ultimately, induction of protein products that mediate IFN responses, remain unknown. We have previously shown that IFNalpha and IFNbeta induce phosphorylation of insulin receptor substrate proteins and downstream engagement of the phosphatidylinositol (PI) 3'-kinase pathway. In the present study we provide evidence for the existence of a Type I IFN-dependent signaling cascade activated downstream of PI 3'-kinase, involving p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated on threonine 421 and serine 424 and is activated during treatment of cells with IFNalpha or IFNbeta. Such activation of p70 S6K is blocked by pharmacological inhibitors of the PI 3'-kinase or the FKBP 12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR). Consistent with this, the Type I IFN-dependent phosphorylation/activation of p70 S6K is defective in embryonic fibroblasts from mice with targeted disruption of the p85alpha and p85beta subunits of the PI 3'-kinase (p85alpha-/-beta-/-). Treatment of sensitive cell lines with IFNalpha or IFNbeta also results in phosphorylation/inactivation of the 4E-BP-1 repressor of mRNA translation. Such 4E-BP1 phosphorylation is also PI3'-kinase-dependent and rapamycin-sensitive, indicating that the Type I IFN-inducible activation of PI3'-kinase and FRAP/mTOR results in dissociation of 4E-BP1 from the eukaryotic initiation factor-4E (eIF4E) complex. Altogether, our data establish that the Type I IFN receptor-activated PI 3'-kinase pathway mediates activation of the p70 S6 kinase and inactivation of 4E-BP1, to regulate mRNA translation and induction of Type I IFN responses.

It is well established that engagement of the Type I interferon (IFN) receptor results in activation of JAKs (Janus kinases), which in turn regulate tyrosine phosphorylation of STAT proteins. Subsequently, the IFN-dependent tyrosine-phosphorylated/activated STATs translocate to the nucleus to regulate gene transcription. In addition to tyrosine phosphorylation, phosphorylation of Stat1 on serine 727 is essential for induction of its transcriptional activity, but the IFNalpha-dependent serine kinase that regulates such phosphorylation remains unknown. In the present study we provide evidence that PKC-delta, a member of the protein kinase C family of proteins, is activated during engagement of the Type I IFN receptor and associates with Stat1. Such an activation of PKC-delta appears to be critical for phosphorylation of Stat1 on serine 727, as inhibition of PKC-delta activation diminishes the IFNalpha- or IFNbeta-dependent serine phosphorylation of Stat1. In addition, treatment of cells with the PKC-delta inhibitor rottlerin or the expression of a dominant-negative PKC-delta mutant results in inhibition of IFNalpha- and IFNbeta-dependent gene transcription via ISRE or GAS elements. Interestingly, PKC-delta inhibition also blocks activation of the p38 MAP kinase, the function of which is required for IFNalpha-dependent transcriptional regulation, suggesting a dual mechanism by which this kinase participates in the generation of IFNalpha responses. Altogether, these findings indicate that PKC-delta functions as a serine kinase for Stat1 and an upstream regulator of the p38 MAP kinase and plays an important role in the induction of Type I IFN-biological responses.

Type I interferons (IFNs) are potent regulators of normal hematopoiesis in vitro and in vivo, but the mechanisms by which they suppress hematopoietic progenitor cell growth and differentiation are not known. In the present study we provide evidence that IFN alpha and IFN beta induce phosphorylation of the p38 mitogen-activated protein (Map) kinase in CD34+-derived primitive human hematopoietic progenitors. Such type I IFN-inducible phosphorylation of p38 results in activation of the catalytic domain of the kinase and sequential activation of the MAPK-activated protein kinase-2 (MapKapK-2 kinase), indicating the existence of a signaling cascade, activated downstream of p38 in hematopoietic progenitors. Our data indicate that activation of this signaling cascade by the type I IFN receptor is essential for the generation of the suppressive effects of type I IFNs on normal hematopoiesis. This is shown by studies demonstrating that pharmacological inhibitors of p38 reverse the growth inhibitory effects of IFN alpha and IFN beta on myeloid (colony-forming granulocytic-macrophage) and erythroid (burst-forming unit-erythroid) progenitor colony formation. In a similar manner, transforming growth factor beta, which also exhibits inhibitory effects on normal hematopoiesis, activates p38 and MapKapK-2 in human hematopoietic progenitors, whereas pharmacological inhibitors of p38 reverse its suppressive activities on both myeloid and erythroid colony formation. In further studies, we demonstrate that the primary mechanism by which the p38 Map kinase pathway mediates hematopoietic suppression is regulation of cell cycle progression and is unrelated to induction of apoptosis. Altogether, these findings establish that the p38 Map kinase pathway is a common effector for type I IFN and transforming growth factor beta signaling in human hematopoietic progenitors and plays a critical role in the induction of the suppressive effects of these cytokines on normal hematopoiesis.

The mechanisms by which interferon-alpha (IFN-alpha) mediates its anti-leukemic effects in chronic myelogenous leukemia (CML) cells are not known. We determined whether p38 MAPK is activated by IFN-alpha in BCR-ABL-expressing cells and whether its function is required for the generation of growth inhibitory responses. IFN-alpha treatment induced phosphorylation/activation of p38 in the IFN-alpha-sensitive KT-1 cell line, but not in IFN-alpha-resistant K562 cells. Consistent with this, IFN-alpha treatment of KT-1 (but not K562) cells induced activation of the small GTPase Rac1, which functions as an upstream regulator of p38. In addition, IFN-alpha-dependent phosphorylation/activation of p38 was induced by treatment of primary granulocytes isolated from the peripheral blood of patients with CML. To define the functional role of the Rac1/p38 MAPK pathway in IFN-alpha signaling, the effects of pharmacological inhibition of p38 on the induction of IFN-alpha responses were determined. Treatment of KT-1 cells with the p38-specific inhibitors SB203580 and SB202190 reversed the growth inhibitory effects of IFN-alpha. On the other hand, the MEK kinase inhibitor PD098059 had no effects, further demonstrating the specificity of these findings. To directly determine the significance of IFN-alpha-dependent activation of p38 in the induction of the anti-leukemic effects of IFN-alpha, we evaluated the effects of p38 inhibition on leukemic colony formation in bone marrow samples of patients with CML. IFN-alpha inhibited leukemic granulocyte/macrophage colony formation in a dose-dependent manner, whereas concomitant treatment with p38 inhibitors reversed such an inhibition. Thus, the Rac1/p38 MAPK pathway is activated by IFN-alpha in BCR-ABL-expressing cells and appears to play a key role in the generation of the growth inhibitory effects of IFN-alpha in CML cells.

Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-beta (C/EBP-beta) induces gene transcription through a novel IFN response element called the gamma-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626-12632. Here, we describe a new IFN-gamma-stimulated pathway that operates C/EBP-beta-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-gamma to stimulate C/EBP-beta-dependent expression. Sustained ERK activation directly correlated with C/EBP-beta-dependent gene expression in response to IFN-gamma. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-gamma-stimulated gene induction through the gamma-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-beta-induced gene expression required STAT1, but not JAK1. A C/EBP-beta mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-beta to IFN-gamma signaling through ERKs.

Interleukin-5 (IL-5) drives the terminal differentiation of myeloid progenitors to the eosinophil lineage; blocks eosinophil apoptosis; and primes eosinophils for enhanced functional activities in allergic, parasitic, and other eosinophil-associated diseases. Here we describe a novel signaling pathway activated by the IL-5 receptor in eosinophils involving the CrkL adapter protein. We determined whether IL-5 induces activation of CrkL and STAT5 in eosinophils using both the human eosinophil-differentiated AML14.3D10 cell line and purified peripheral blood eosinophils from normal donors. Stimulation of AML14.3D10 cells or blood eosinophils with IL-5 induced rapid tyrosine phosphorylation of the CrkL adapter and STAT5 and the association of CrkL and STAT5 in vivo as evidenced by the detection of STAT5 in anti-CrkL immunoprecipitates. The resulting CrkL.STAT5 complexes translocated to the nucleus and bound STAT5 consensus DNA-binding sites present in the promoters of IL-5-regulated genes, as shown in gel mobility and antibody supershift assays. IL-5 also induced marked activity of an 8X-GAS (interferon gamma-activated site)-luciferase reporter construct in transient transfections of AML14.3D10 eosinophils, demonstrating that these complexes play a functional role in IL-5 signaling. CrkL was also found to interact, via its N-terminal SH3 domain, with C3G, a guanine exchange factor for the small G-protein Rap1, which was also rapidly activated in an IL-5-dependent manner in these cells, establishing that CrkL mediates downstream activation of at least two signaling cascades in IL-5-stimulated eosinophils. Thus, the CrkL adapter plays an important role in IL-5 signaling in the eosinophil, acting as a nuclear adapter for STAT5 and as an upstream regulator of the C3G-Rap1 signaling pathway.

The p38 mitogen-activated protein (MAP) kinase is activated during engagement of the type I interferon (IFN) receptor and mediates signals essential for IFNalpha-dependent transcriptional activation via interferon-stimulated response elements without affecting formation of the ISGF3 complex. In the present study, we provide evidence that the small GTPase Rac1 is activated in a type I IFN-dependent manner and that its function is required for downstream engagement of the p38 MAP kinase pathway. We also demonstrate that p38 is required for IFNalpha-dependent gene transcription via GAS elements and regulates activation of the promoter of the PML gene that mediates growth inhibitory responses. In studies to determine whether the regulatory effects of p38 are mediated by serine phosphorylation of Stat1 or Stat3, we found that the p38 kinase inhibitors SB203580 or SB202190 or overexpression of a dominant negative p38 mutant do not inhibit phosphorylation of Stat1 or Stat3 on Ser-727 in several IFNalpha-sensitive cell lines. Altogether these data demonstrate that the Rac1/p38 MAP kinase signaling cascade plays a critical role in type I IFN signaling, functioning in cooperation with the Stat-pathway, to regulate transcriptional regulation of IFNalpha-sensitive genes and generation of growth inhibitory responses.

Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRS-related proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3′-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3′-kinase pathways and possibly participate in the generation of the erythropoietin-induced mitogenic responses.

The precise role of the different subunits (alpha/IFNAR1 and betaL/IFNAR2) of the type I interferon receptor (IFN-R) in the activation of signal transducer and activator of transcription (Stat) 1, Stat2, and Stat3 has not yet been established. In this report we demonstrate that there are functionally redundant phosphotyrosine-dependent and -independent binding sites for Stat2 in the alpha and beta subunits of the type I IFN-R. Expression of a type I IFN-R containing only the constitutive Stat2 site or the proximal tyrosines of betaL, but not the docking site on the alpha chain (Tyr466 and Tyr481), supported low levels of Stat2 activation. However, the presence of only one intact Stat2 site did not lead to induction of interferon-stimulated gene factor 3 (ISGF3) or an antiviral state. Normal levels of Stat2 tyrosine phosphorylation, induction of ISGF3, and an antiviral effect always required the proximal tyrosines of betaL and at least one of the other Stat2 sites (Tyralpha466, 481 or betaL404-462). These data suggest that a threshold of Stat2 tyrosine phosphorylation is required for complete activation of ISGF3. Interestingly, a receptor in which all tyrosines were mutated to phenylalanine shows normal Stat3 phosphorylation and low levels of activation of Stat1.

The signaling specificity for cytokines that have common receptor subunits is achieved by the presence of additional cytokine-specific receptor components. In the type I interferon (IFN) family, all 14 subtypes of IFNalpha, IFNbeta, and IFNomega bind to the same alpha and betaL subunits of the type I IFN-R, yet differences in signaling and biological effects exist among them. Our data demonstrate that IFNalpha2 and IFNbeta utilize different regions of the betaL subunit for signaling. Thus, in contrast to other cytokine systems, signal diversity in the type I IFN system can be accomplished within the same receptor complex by utilizing different regions of the same receptor subunits.

All Type I interferons (IFNα, IFNβ, IFNω) bind to the Type I IFN receptor (IFNR) and elicit a common set of signaling events, including activation of the Jak/Stat and IRS pathways. However, IFNβ selectively induces the association of the α subunit of the Type I IFNR with p100, a tyrosyl phosphoprotein, to transduce IFNβ-specific signals. Using antibodies raised against the different components of the Type I IFNR, we identified p100 as the long form of the β subunit (βL subunit) of the Type I IFNR. This was also confirmed in experiments with mouse L-929 cells transfected with truncated forms of βL. Thus, IFNβ stimulation of human cells or mouse L-929 transfectants expressing the human α and βL subunits, selectively induces the formation of a signaling complex containing the α and βL subunits of the receptor. The IFNβ-regulated interaction of the α and βL chains is rapid and transient and follows a similar time course with the tyrosine phosphorylation of these receptor components. These data demonstrate that the signaling specificity for different Type I IFNs is established early in the signaling cascade, at the receptor level, and results from distinct interactions between components of the Type I IFNR.

Binding of interferon alpha (IFN alpha) to its receptor induces activation of the Tyk-2 and Jak-1 tyrosine kinases and tyrosine phosphorylation of multiple downstream signaling elements, including the Stat components of the interferon-stimulated gene factor 3 (ISGF-3). IFN alpha also induces tyrosine phosphorylation of IRS-1, the principle substrate of the insulin receptor. In this study we demonstrate that various Type I IFNs rapidly stimulate tyrosine phosphorylation of IRS-2. This is significant since IRS-2 is the major IRS protein found in hematopoietic cells. The IFN alpha-induced phosphorylated form of IRS-2 associates with the p85 regulatory subunit of the phosphatidylinositol 3'-kinase, suggesting that this kinase participates in an IFN alpha-signaling cascade downstream of IRS-2. We also provide evidence for an interaction of IRS-2 with Tyk-2, suggesting that Tyk-2 is the kinase that phosphorylates this protein during IFN alpha stimulation. A conserved region in the pleckstrin homology domain of IRS-2 may be required for the interaction of IRS-2 with Tyk-2, as shown by the selective binding of glutathione S-transferase (GST) fusion proteins containing the IRS-2-IH1PH or IRS-1-IH1PH domains to Tyk-2 but not other Janus kinases in vitro.

The tyrosine kinase Tyk-2 is physically associated with the Type I interferon (IFN) receptor complex and is rapidly activated during IFN alpha stimulation. We report that Tyk-2 forms stable complexes with the SH2-containing hematopoietic cell phosphatase (HCP) in several hematopoietic cell lines in vivo, and that the IFN alpha-induced tyrosine-phosphorylated form of Tyk-2 is a substrate for the phosphatase activity of HCP in in vitro assays. Furthermore, treatment of cells with the phosphatase inhibitor sodium orthovanadate induces tyrosine phosphorylation of Tyk-2 and an associated 115-kDa protein. Altogether, these data suggest that HCP regulates tyrosine phosphorylation of the Tyk-2 kinase, and thus its function may be important in the transmission of signals generated at the Type I IFN receptor level.

Insulin activates the ras signaling pathway and promotes hematopoietic cell proliferation. One possible mediator in such signaling is the vav proto-oncogene product (p95vav), which is specifically expressed in cells of hematopoietic origin and contains domains typical of guanine nucleotide exchange factors as well as Src homology 2 and Src homology 3 domains. We studied the tyrosine phosphorylation of p95vav in hematopoietic cells expressing insulin receptors. Immunoblotting experiments with an antiphosphotyrosine monoclonal antibody disclosed that insulin induces rapid and transient tyrosine phosphorylation of p95vav in the human U-266 myeloma cell line. These findings were confirmed by immunoprecipitation experiments performed with 32P-labeled cells and phosphoamino acid analysis of the bands corresponding to p95vav. Similarly, insulin-dependent tyrosine phosphorylation of p95vav was observed in the human IM-9 and mouse J558L hematopoietic cell lines. Furthermore, insulin treatment of cells led to the association of the Src homology 2 domain of p95vav with the activated beta-subunit of the insulin receptor in vitro. Altogether, these data suggest that p95vav is a substrate for the insulin receptor tyrosine kinase and may be involved in an insulin signaling pathway linking receptor-generated signals to Ras or other GTP-binding proteins in cells of hematopoietic origin.

We studied the phosphorylation of the alpha and beta subunits of the Type I interferon (IFN) receptor induced by Type I IFNs in the human U-266 and MOLT-4 cell lines. Both IFN-alpha and IFN-beta induced tyrosine phosphorylation of the beta subunit of the receptor. The Type I IFN-induced tyrosine phosphorylation of the beta subunit was rapid and transient, being detectable within 1 min of Type I IFN treatment and gradually diminishing to almost base-line levels by 60 min. All Type I IFNs studied were found to induce tyrosine phosphorylation of the alpha subunit of the Type I IFN receptor, the p135tyk2 and JAK-1 tyrosine kinases, and the ISGF3 alpha components. Interestingly, IFN-beta, but not IFN-alpha or IFN-omega, induced tyrosine phosphorylation of an alpha subunit-associated protein with an apparent molecular mass of approximately 100 kDa (p100). These data suggest the existence of a common signaling pathway(s) for Type I IFNs involving the alpha and beta subunits of the receptor, the tyrosine kinases p135tyk2 and JAK-1, and the ISGF3 alpha components. However, differences between the signaling pathways of different Type I IFNs exist, as suggested by tyrosine phosphorylation of an alpha subunit-associated protein only in response to IFN-beta.

Two different Type I interferon receptors (IFN-R) have been described: the normal and the variant receptors. The alpha subunit of the Type I IFN-R has a molecular mass of 110 kDa in cells expressing normal and variant receptors. The beta subunit has a molecular mass of approximately 100 kDa in cells that express normal receptors and 55 kDa in cells expressing the variant form of the receptor. The IFN alpha-resistant U-937 cell line expresses variant receptors and fails to down-regulate and phosphorylate the alpha subunit on tyrosine residues. We report that two other myelomonocytic cell lines, YK-M2 and ML-2, also expressing the variant form of the receptor, fail to down-regulate and phosphorylate the alpha subunit on tyrosine residues. However, YK-M2 and ML-2 cells are sensitive to the antiproliferative and antiviral effects of IFN alpha 2, indicating that phosphorylation of the alpha subunit is not necessary to elicit an IFN alpha response and that expression of variant receptors is not a source of IFN alpha resistance. We also determined if other proteins involved in the IFN alpha signal transduction pathway had a different phosphorylation pattern. Treatment of cells expressing variant receptors induced tyrosine phosphorylation of the p135tyk2 tyrosine kinase, and the three interferon-stimulated gene factor 3 alpha (ISGF3 alpha) polypeptides (p113, p91, and p84), albeit at lower levels. These results indicate that cells expressing either form of the Type I IFN-R phosphorylate a similar set of proteins, with the exception of the alpha subunit.

The mechanisms of generation of second messengers after binding of interferon alpha (IFN alpha) to its receptor remain unknown. We have studied the phosphorylation of the alpha subunit of the IFN alpha receptor, which is recognized by the monoclonal antibody IFNa receptor 3. Immunoblotting experiments showed that IFN alpha induced rapid tyrosine phosphorylation of the alpha subunit in the IFN alpha-sensitive H-929, U-266, and Daudi cell lines. Immunoprecipitation experiments performed with 32P-labeled cells showed that the alpha subunit is phosphorylated before IFN alpha treatment and that the level of phosphorylation increases after IFN alpha stimulation. Phosphoamino acid analysis confirmed the IFN alpha-induced tyrosine phosphorylation and demonstrated that the base-line phosphorylation corresponded to serine phosphorylation that increased 50% upon IFN alpha treatment. Tyrosine phosphorylation of the alpha subunit was time- and dose-dependent, further demonstrating the specificity of the process. Phosphorylation of the alpha subunit of the receptor occurred rapidly after IFN alpha binding, both at 37 and 4 degrees C. Exposure of the cells to the tyrosine kinase inhibitor genistein blocked the IFN alpha-induced tyrosine phosphorylation of this subunit of the IFN alpha receptor. In contrast H7, a specific protein kinase C inhibitor, and acute and chronic exposure to phorbol esters had no effect on tyrosine phosphorylation, suggesting that protein kinase C does not regulate the tyrosine phosphorylation of the alpha subunit of the IFN alpha receptor. No IFN alpha-induced tyrosine phosphorylation was observed in the IFN alpha-resistant U-937 cell line that expresses a variant IFN alpha receptor. Altogether these data suggest that tyrosine phosphorylation of the alpha subunit may play a role in the signal transduction pathway of IFN alpha.

Type I interferons (IFNalpha and IFNbeta) transduce signals by inducing tyrosine phosphorylation of Jaks and Stats, as well as the CrkL adapter, an SH2/SH3-containing protein which provides a link to downstream pathways that mediate growth inhibition. We report that Stat5 interacts constitutively with the IFN receptor-associated Tyk-2 kinase, and during IFNalpha stimulation its tyrosine-phosphorylated form acts as a docking site for the SH2 domain of CrkL. CrkL and Stat5 then form a complex that translocates to the nucleus. This IFN-inducible CrkL-Stat5 complex binds in vitro to the TTCTAGGAA palindromic element found in the promoters of a subset of IFN-stimulated genes. Thus, during activation of the Type I IFN receptor, CrkL functions as a nuclear adapter protein and, in association with Stat5, regulates gene transcription through DNA binding.