Human SLFN5 is a transcriptional co-repressor of STAT1-mediated interferon responses and promotes the malignant phenotype in glioblastoma.

We provide evidence that the IFN-regulated member of the Schlafen (SLFN) family of proteins, SLFN5, promotes the malignant phenotype in glioblastoma multiforme (GBM). Our studies indicate that SLFN5 expression promotes motility and invasiveness of GBM cells, and that high levels of SLFN5 expression correlate with high-grade gliomas and shorter overall survival in patients suffering from GBM. In efforts to uncover the mechanism by which SLFN5 promotes GBM tumorigenesis, we found that this protein is a transcriptional co-repressor of STAT1. Type-I IFN treatment triggers the interaction of STAT1 with SLFN5, and the resulting complex negatively controls STAT1-mediated gene transcription via interferon stimulated response elements. Thus, SLFN5 is both an IFN-stimulated response gene and a repressor of IFN-gene transcription, suggesting the existence of a negative-feedback regulatory loop that may account for suppression of antitumor immune responses in glioblastoma.

The role of Fas-associated phosphatase 1 in leukemia stem cell persistence during tyrosine kinase inhibitor treatment of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by expression of Bcr-abl, a tyrosine kinase oncogene. Clinical outcomes in CML were revolutionized by development of Bcr-abl-targeted tyrosine kinase inhibitors (TKIs), but CML is not cured by these agents. CML leukemia stem cells (LSCs) are relatively TKI insensitive and persist even in remission. LSC persistence results in relapse upon TKI discontinuation, or drug resistance or blast crisis (BC) during prolonged treatment. We hypothesize that increased expression of Fas-associated phosphatase 1 (Fap1) in CML contributes to LSC persistence and BC. As Fap1 substrates include Fas and glycogen synthase kinase-3ß (Gsk3ß), increased Fap1 activity in CML is anticipated to induce Fas resistance and stabilization of ß-catenin protein. Resistance to Fas-induced apoptosis may contribute to CML LSC persistence, and ß-catenin activity increases during BC. In the current study, we directly tested the role of Fap1 in CML LSC persistence using in an in vivo murine model. In TKI-treated mice, we found that inhibiting Fap1, using a tripeptide or small molecule, prevented TKI resistance, BC and relapse after TKI discontinuation; all events observed with TKI alone. In addition, Fap1 inhibition increased Fas sensitivity and decreased ß-catenin activity in CD34(+) bone marrow cells from human subjects with CML. Therapeutic Fap1 inhibition may permit TKI discontinuation and delay in progression in CML.

Regulation of CDX4 gene transcription by HoxA9, HoxA10, the Mll-Ell oncogene and Shp2 during leukemogenesis.

Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in human myeloid leukemias.

Beta interferon regulation of glucose metabolism is PI3K/Akt dependent and important for antiviral activity against coxsackievirus B3.

UNLABELLED: An effective type I interferon (IFN)-mediated immune response requires the rapid expression of antiviral proteins that are necessary to inhibit viral replication and virus spread. We provide evidence that IFN-ß regulates metabolic events important for the induction of a rapid antiviral response: IFN-ß decreases the phosphorylation of AMP-activated protein kinase (AMPK), coincident with an increase in intracellular ATP. Our studies reveal a biphasic IFN-ß-inducible uptake of glucose by cells, mediated by phosphatidylinositol 3-kinase (PI3K)/Akt, and IFN-ß-inducible regulation of GLUT4 translocation to the cell surface. Additionally, we provide evidence that IFN-ß-regulated glycolytic metabolism is important for the acute induction of an antiviral response during infection with coxsackievirus B3 (CVB3). Last, we demonstrate that the antidiabetic drug metformin enhances the antiviral potency of IFN-ß against CVB3 both in vitro and in vivo. Taken together, these findings highlight an important role for IFN-ß in modulating glucose metabolism during a virus infection and suggest that the use of metformin in combination with IFN-ß during acute virus infection may result in enhanced antiviral responses. IMPORTANCE: Type I interferons (IFN) are critical effectors of an antiviral response. These studies describe for the first time a role for IFN-ß in regulating metabolism--glucose uptake and ATP production--to meet the energy requirements of a robust cellular antiviral response. Our data suggest that IFN-ß regulates glucose metabolism mediated by signaling effectors similarly to activation by insulin. Interference with IFN-ß-inducible glucose metabolism diminishes the antiviral response, whereas treatment with metformin, a drug that increases insulin sensitivity, enhances the antiviral potency of IFN-ß.

The evolution of the TOR pathway and its role in cancer.

The target of rapamycin (TOR) pathway is highly conserved among eukaryotes and has evolved to couple nutrient sensing to cellular growth. TOR is found in two distinct signaling complexes in cells, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). These complexes are differentially regulated and act as effectors for the generation of signals that drive diverse cellular processes such as growth, proliferation, protein synthesis, rearrangement of the cytoskeleton, autophagy, metabolism and survival. Mammalian TOR (mTOR) is very important for development in embryos, while in adult organisms it is linked to aging and lifespan effects. In humans, the mTOR pathway is implicated in the tumorigenesis of multiple cancer types and its deregulation is associated with familial cancer syndromes. Because of its high biological relevance, different therapeutic strategies have been developed to target this signaling cascade, resulting in the emergence of unique pharmacological inhibitors that are either already approved for use in clinical oncology or currently under preclinical or clinical development. Multimodal treatment strategies that simultaneously target multiple nodes of the pathway and/or negative feedback regulatory loops may ultimately provide the best therapeutic advantage in targeting this pathway for the treatment of malignancies.

Apigenin induces apoptosis via downregulation of S-phase kinase-associated protein 2-mediated induction of p27Kip1 in primary effusion lymphoma cells.

OBJECTIVE: The mechanisms that regulate mitogenic and antiapoptotic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we assessed the effect of apigenin (4',5,7-trihydroxyflavone), a flavonoid on a panel of PEL cell lines. MATERIALS AND METHODS: We studied the effect of apigenin on four PEL cell lines. Apoptosis was measured by annexin V/PI dual staining and DNA laddering. Protein expression was measured by immunoblotting. RESULTS: Apigenin induced apoptosis in PEL cell lines in a dose dependent manner. Such effects of apigenin appeared to result from suppression of constitutively active kinase AKT resulting in down-regulation of SKP2, hypo-phosphorylation of Rb and accumulation of p27Kip1. Apigenin treatment of PEL cells caused dephosphorylation of p-Bad protein leading to down regulation of the anti-apoptotic protein, Bcl-2 and an increase in Bax/Bcl2 ratio. Apigenin treatment also triggered Bax conformational change and subsequently translocation from cytosole to mitochondria causing loss of mitochondrial membrane potential with subsequent release of cytochrome c. Released cytochrome c onto the cytosole activated caspase-9 and caspase-3, followed by polyadenosin-5'-diphosphate-ribose polymerase (PARP) cleavage. Finally, treatment of PEL cells with apigenin down-regulated the expression of inhibitor of apoptosis protein (IAPs). CONCLUSIONS: Altogether, these data suggest a novel function for apigenin, acting as a suppressor of AKT/PKB pathway in PEL cells, and raise the possibility that this agent may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.

GRIM-19 associates with the serine protease HtrA2 for promoting cell death.

We have isolated a novel interferon (IFN)-retinoid regulated cell death regulatory protein genes associated with retinoid-IFN-induced mortality (GRIM)-19 earlier. To understand its mechanism of action, we have employed a yeast-two-hybrid screen and identified serine protease HtrA2 as its binding partner. GRIM-19 physically interacts with HtrA2 and augments cell death in an IFN/all-trans retinoic acid (RA)-dependent manner. In the presence of GRIM-19, the HtrA2-driven destruction of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP) is augmented. These interactions were disrupted by an human herpes virus-8 (HHV-8)-coded oncoprotein, vIRF1, and conferred resistance to IFN/RA-induced cell death. These data show a critical role of HtrA2 in a cytokine-induced cell death response for the first time and its inhibition by a viral protein.

Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias.

Curcumin has been shown to possess variety of biological functions including anti-tumor activity. The mechanism by which curcumin inhibit cell proliferation remains poorly understood. In the present report, we investigated the effect of curcumin on the activation of apoptotic pathway in T-cell acute lymphoblastic leukemia (T-ALL) malignant cells. Our data demonstrate that curcumin causes dose dependent suppression of proliferation in several T cell lines. Curcumin treatment causes the de-phosphorylation/inactivation of constitutively active AKT, FOXO transcription factor and GSK3. Curcumin also induces release of cytochrome c accompanied by activation of caspase-3 and PARP cleavage. In addition, zVAD-fmk, a universal inhibitor of caspases, prevents caspase-3 activation and abrogates cell death induced by curcumin treatment. Finally, treatment of T-ALL cells with curcumin down-regulated the expression of inhibitor of apoptosis protein (IAPs). Taken together, our finding suggest that curcumin suppresses constitutively activated targets of PI3'-kinase (AKT, FOXO and GSK3) in T cells leading to the inhibition of proliferation and induction of caspase-dependent apoptosis.

Poxvirus infection rapidly activates tyrosine kinase signal transduction.

Viruses have evolved a number of strategies to gain entry and replicate in host target cells that, for human immunodeficiency virus (HIV) and the poxvirus, myxoma virus, involve appropriating chemokine receptors. In this report we demonstrate that activation of multiple intracellular tyrosine phosphorylation events rapidly ensues following virus adsorption to NIH 3T3.CD4.CCR5 cells and affects the ultimate level of myxoma virus replication. UV-inactivated myxoma virus induces the rapid phosphorylation of CCR5 on tyrosine residues, the association of CCR5 with Jaks and p56(lck), and their phosphorylation-activation within minutes of virus adsorption. Additionally, we provide evidence for myxoma virus-inducible signal transducers and activators of transcription (Stat) and insulin receptor substrate (IRS) activation. In contrast to CCR5 activation effected by HIV Env protein, these myxoma virus-inducible phosphorylation events are not sensitive to pertussis toxin treatment. Moreover, in cells that are non-permissive for myxoma virus infection, we provide evidence that myxoma virus fails to invoke this tyrosine phosphorylation cascade. Consistent with the observation that infection of CCR5-expressing cells is blocked by herbimycin A and the Jak 2 inhibitor, tyrophostin AG490, we infer that viral infectivity may be dependent on non-G-protein-coupled signal transduction pathways triggered by the infecting myxoma virus particle. This provides a novel post-binding mechanism by which viruses can co-opt a cellular receptor to permit productive virus infection.

Protein kinase C-delta regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase.

The procoagulant thrombin promotes the adhesion of polymorphonuclear leukocytes to endothelial cells by a mechanism involving expression of intercellular adhesion molecule 1 (ICAM-1) via an NF-kappaB-dependent pathway. We now provide evidence that protein kinase C-delta (PKC-delta) and the p38 mitogen-activated protein (MAP) kinase pathway play a critical role in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. We observed the phosphorylation of PKC-delta and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-delta inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-delta. Inhibition of PKC-delta or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-kappaB activity and resultant ICAM-1 expression. The effects of PKC-delta inhibition were secondary to inhibition of IKKbeta activation and of subsequent NF-kappaB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IkappaBalpha degradation without affecting the DNA binding function of nuclear NF-kappaB. Thus, PKC-delta signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKbeta, which mediates NF-kappaB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-kappaB p65 (RelA).

The p38 MAPK pathway mediates the growth inhibitory effects of interferon-alpha in BCR-ABL-expressing cells.

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.

Rantes activates Jak2 and Jak3 to regulate engagement of multiple signaling pathways in T cells.

The chemokine RANTES (regulated on activation normal T cell expressed and secreted) and its cognate receptor CC chemokine receptor 5 (CCR5) have been implicated in regulating immune cell function. Previously we reported that in T cells, RANTES activation of CCR5 results in Stat1 and Stat3 phosphorylation-activation, leading to Stat1:1 and Stat1:3 dimers that exhibit DNA binding activity and the transcriptional induction of a Stat-inducible gene, c-fos. Given that RANTES and CCR5 have been implicated in T cell activation, we have studied RANTES-induced signaling events in a CCR5-expressing T cell line, PM1. RANTES treatment of PM1 T cells results in the rapid phosphorylation-activation of CCR5, Jak2, and Jak3. RANTES-inducible Jak phosphorylation is insensitive to pertussis toxin inhibition, indicating that RANTES-CCR5-mediated tyrosine phosphorylation events are not coupled directly to Galpha(i) protein-mediated events. In addition to Jaks, several other proteins are rapidly phosphorylated on tyrosine residues in a RANTES-dependent manner, including the Src kinase p56(lck), which associates with Jak3. Additionally our data confirm that the amino-terminally modified RANTES proteins, aminooxypentane-RANTES and Met-RANTES, are agonists for CCR5 and induce early tyrosine phosphorylation events that are indistinguishable from those inducible by RANTES with similar kinetics. Our data also demonstrate that RANTES activates the p38 mitogen-activated protein (MAP) kinase pathway. This is evidenced by the rapid RANTES-dependent phosphorylation and activation of p38 MAP kinase as well as the activation of the downstream effector of p38, MAP kinase-activated protein (MAPKAP) kinase-2. Pharmacological inhibition of RANTES-dependent p38 MAP kinase activation blocks MAPKAP kinase-2 activity. Thus, activation of Jak kinases and p38 MAP kinase by RANTES regulates the engagement of multiple signaling pathways.

Engagement of the CrkL adaptor in interferon alpha signalling in BCR-ABL-expressing cells.

Interferon alpha (IFNalpha) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR-ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR-ABL-expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNalpha-signalling in the CML-derived KT-1 cell line, which expresses BCR-ABL and is sensitive to the growth inhibitory effects of IFNalpha. CrkL is constitutively associated with BCR-ABL in these cells and treatment with IFNalpha had no effect on the BCR-ABL/CrkL interaction. After IFNalpha stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNalpha-dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N-terminus SH3 domains of CrkL. The resulting CrkL-Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the beta-casein promoter or the promoter of the PML gene, an IFNalpha-inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT-1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR-ABL in CML-derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti-proliferative effects of IFNalpha in CML.

ERK1 and ERK2 activate CCAAAT/enhancer-binding protein-beta-dependent gene transcription in response to interferon-gamma.

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.

Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to all-trans-retinoic acid.

Several signaling pathways are activated by all-trans-retinoic acid (RA) to mediate induction of differentiation and apoptosis of malignant cells. In the present study we provide evidence that the p38 MAP kinase pathway is activated in a RA-dependent manner in the NB-4, acute pro-myelocytic leukemia, and the MCF-7, breast carcinoma, cell lines. RA treatment of cells induces a time- and dose-dependent phosphorylation of p38, and such phosphorylation results in activation of its catalytic domain. p38 activation is not inducible by RA in a variant NB-4 cell line, NB-4.007/6, which is resistant to the effects of RA, suggesting a role for this pathway in the induction of RA responses. Our data also demonstrate that the small G-protein Rac1 is activated by RA and functions as an upstream regulator of p38 activation, whereas the MAPKAPK-2 serine kinase is a downstream effector for the RA-activated p38. To obtain information on the functional role of the Rac1/p38/MAPKAPK-2 pathway in RA signaling, the effects of pharmacological inhibition of p38 on RA-induced gene transcription and cell differentiation were determined. Our results indicate that treatment of cells with the SB203580 inhibitor does not inhibit RA-dependent gene transcription via retinoic acid response elements or induction of Stat1 protein expression. However, treatment with SB203580 or SB202190 strongly enhances RA-dependent induction of cell differentiation and RA-regulated growth inhibitory responses. Altogether, our findings demonstrate that the Rac1/p38 MAP kinase pathway is activated in a RA-dependent manner and exhibits negative regulatory effects on the induction of differentiation.

CD5+ immunophenotype in the bone marrow but not in the peripheral blood in a patient with hairy cell leukemia.

We report the case of a patient with rare CD5+ hairy cell leukemia, which was unusual, as there was also a discrepancy between the bone marrow and peripheral blood immunophenotypes. We propose that some degree of maturation within the malignant clone and the predominance of more mature CD5- hairy cells in the peripheral blood can explain this observation. The patient was treated with a conventional course of cladribine (2'-chlorodeoxyadenosine) and achieved complete remission. We conclude that the unusual CD5+ immunophenotype of this patient did not affect prognosis or change the response to standard therapy.

Engagement of the CrkL adapter in interleukin-5 signaling in eosinophils.

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.

Activation of the Akt/FKHRL1 pathway mediates the antiapoptotic effects of erythropoietin in primary human erythroid progenitors.

Erythropoietin (Epo), stem cell factor (SCF), and insulin-like growth factor-1 (IGF-1) are key regulators of erythroid cell proliferation and differentiation. To understand the mechanisms of generation of signals by each of these growth factors, we determined the activation of the PI3-kinase/Akt pathway during proliferation and differentiation of primary human erythroid progenitors. Our results demonstrate that PKB/Akt is activated by Epo and SCF, but not by IGF-1 in human primary erythroid progenitors. In addition, Epo treatment of erythroid progenitors induces phosphorylation of a member of the Forkhead family (FH) of transcription factors FKHRL1, downstream of activation of the Akt kinase. Such Epo-dependent activation of FKHRL1 apparently regulates the generation of Epo-dependent antiapoptotic signals as evidenced by the induction of apoptosis of erythroid progenitors during treatment of cells with the PI3-kinase (PI3K) inhibitor LY294002. Thus, the PI3K/Akt/FKHRL1 pathway is essential for inhibition of apoptosis in response to Epo and SCF, while the IGF-1 receptor utilizes a different pathway.

The Rac1/p38 mitogen-activated protein kinase pathway is required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins.

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.

All-trans-retinoic acid induces tyrosine phosphorylation of the CrkL adapter in acute promyelocytic leukemia cells.

OBJECTIVE: All-trans-retinoic acid (RA) is a potent inducer of differentiation of acute promyelocytic leukemia (APL) cells in vitro and in vivo. It also exhibits synergistic effects with interferons on the induction of differentiation and growth inhibition in vitro. Recent studies showed that interferons engage a signaling pathway involving the CBL proto-oncogene and the CrkL adapter, which mediates interferon-induced growth inhibitory signals. The objective of this study was to determine whether the CBL-CrkL pathway is activated by treatment of the NB-4 and HL-60 acute leukemia cell lines with RA. MATERIALS AND METHODS: The effects of RA treatment on CBL and CrkL phosphorylation, as well as on protein-protein interactions, were determined in studies involving immunoprecipitations of cell extracts with specific antibodies and Western blots. In addition, glutathione-S-transferase fusion proteins were used in binding studies to determine whether the SH2 domain of CrkL interacts with CBL in a RA-dependent manner and whether Rapl is activated by RA. RESULTS: Treatment of NB-4 or HL-60 cells with RA resulted in strong tyrosine phosphorylation of CBL, which was time and dose dependent. Similarly, RA induced tyrosine phosphorylation of the CrkL adapter and the association of CrkL with CBL. The RA-dependent interaction of CrkL with CBL was mediated by binding of the SH2 domain of CrkL to tyrosine phosphorylated CBL, suggesting that CBL provides a docking site for engagement of CrkL in a RA-activated cellular pathway. The guanine exchange factor C3G was found to be associated with CrkL at similar levels before and after RA treatment, but Rapl activation downstream of C3G was not inducible by RA. CONCLUSIONS: These findings demonstrate that the CBL-CrkL pathway is one of the mediators of the effects of RA on APL cells and suggest that one of the mechanisms of synergy between RA and interferons may involve regulation of components of this signaling cascade.