Patients suffering from acute myeloid leukemias (AML) bearing FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) have poor outcomes following cytarabine- and anthracyclin-based induction therapy. To a major part this is attributed to drug resistance of FLT3-ITD-positive leukemic cells. Against this background, we have devised an antibody array approach to identify proteins, which are differentially expressed by hematopoietic cells in relation to activated FLT3 signaling. Selective upregulation of antiapoptotic myeloid cell leukemia-1 (MCL-1) was found in FLT3-ITD-positive cell lines and primary mononuclear cells from AML patients as compared with FLT3-wild-type controls. Upregulation of MCL-1 was dependent on FLT3 signaling as confirmed by its reversion upon pharmacological inhibition of FLT3 activity by the kinase inhibitor PKC412 as well as siRNA-mediated suppression of FLT3. Heterologously expressed MCL-1 substituted for FLT3 signaling by conferring resistance of hematopoietic cells to antileukemia drugs such as cytarabine and daunorubicin, and to the proapoptotic BH3 mimetic ABT-737. Conversely, suppression of endogenous MCL-1 by siRNA or by flavopiridol treatment sensitized FLT3-ITD-expressing hematopoietic cells to cytotoxic and targeted therapeutics. In conclusion, MCL-1 is an essential effector of FLT3-ITD-mediated drug resistance. Therapeutic targeting of MCL-1 is a promising strategy to overcome drug resistance in FLT3-ITD-positive AML.

T cell receptor signalling and therefore production of IL-2 upon antigen stimulation, has been shown to be impaired in regulatory T (Treg) cells. Whether the expression and activation of the major transcription factors NFATc2, AP-1 and Nf-kB are affected too, has not yet been determined. We found a strikingly lower expression of all three factors in human Treg cells compared to memory Th cells, but their activation was unharmed. Interestingly, after stimulation with PMA/Ionomycin, thus bypassing upstream signalling events, we found a small Treg cell subset, that was able to overcome its anergic phenotype and produced IL-2. This subpopulation is characterized by higher NFATc2, AP-1 and Nf-kB and lower FOXP3 levels compared to IL-2 nonproducing Treg cells. Our Data suggests that IL-2 production in Treg cells is not switched off by genetic imprinting, but rather the amounts and ratios of the essential transcription factors NFATc2, AP-1, Nf-kB and FOXP3 are essential to prevent IL-2 production in Treg cells and thereby suppport their anergic phenotype despite a very strong stimulation.

1 3 Medical Department, Johannes Gutenberg-University, Mainz, Germany 2 Institute of Molecular Cell Biology, University of Jena, Jena, Germany 3 Experimental Clinical Chemistry, Department of Laboratory Medicine, Lund University, Malmö University Hospital, Malmö, Sweden 4 current address: Department of Medicine (Cancer Research), West German Cancer Center, University Hospital Essen, Essen, Germany 5 both authors contributed equally 6 current address: Department of Hematology/Oncology, Medical Center, Otto-vonGuericke University Magdeburg, Magdeburg, Germany

Naturally occurring CD4+CD25 Tregs (nTregs) are essential for T cell homeostasis and the maintenance of peripheral tolerance. They prevent the activation of autoaggressive T cells in the context of autoimmune diseases and suppress inadequate allergen specific T cells. On the opposite, nTregs inhibit also effective immune responses against tumors such as melanoma. A detailed understanding of molecular mechanisms that control the functional properties of human nTregs is mandatory for the development of novel immunotherapies against allergy, autoimmunity and cancer. Therefore, we initiated a genomic, proteomic and kinome profiling of human nTregs to identify key molecules in human nTregs associated with their functional activation which are responsible for their state of anergy and/or their suppressive activity. We started with large-scale isolation of nTregs using whole leukapheresis products followed by polyclonal stimulation and analysis at different time points. As a result, we identified a distinct molecular activation pattern specific for the activation state of human nTregs. The impact of identified key molecules was tested in functional assays using specific inhibitors and siRNA mediated knockdown of these targets. A general transcriptional network analysis is currently under investigation and will be presented on the meeting. The main objective of our analysis is the identification of novel targets for the immunotherapeutic intervention of dysregulated immune responses in the near future. from 12th Joint Meeting of the Signal Transduction Society (STS). Signal Transduction: Receptors, Mediators and Genes Weimar, Germany. 29–31 October 2008

In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-γ1 (PLC-γ1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-γ1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-γ1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-γ1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-γ1 and Bcr-Abl inhibition. The pivotal role of PLC-γ1 was further confirmed in a mouse leukemogenesis model.

ABSTRACT Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPα promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.

AIMS:Coxsackievirus B3 (CVB3) is a frequent cause of human chronic myocarditis and subsequent fibrosis, leading to dilated cardiomyopathy. The molecular processes underlying the development of fibrosis are poorly understood. Enhanced levels of platelet-derived growth factors (PDGFs), especially PDGF-C, have recently been linked with the development of different forms of fibrosis. Therefore, the expression of PDGF was analysed in hearts of CVB3-infected major histocompatability complex class II knockout mice. The latter were recently established as mouse model mimicking the chronic inflammation and fibrosis characteristic for this disease.METHODS AND RESULTS:Expression of PDGF was analysed by reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry. Hearts of C57BL/6 mice served as controls because infection of these animals leads to acute cardiac inflammation, but the hearts heal without signs of chronic inflammation. In uninfected hearts, basal expression of PDGF, notably PDGF-C, was detectable throughout the heart. The chronic inflammatory process was associated with elevated and sustained expression of all tested PDGF isoforms. Immunostaining and in situ hybridization analysis localized enhanced PDGF levels to areas with highest virus load and inflammatory infiltrations, adjacent to fibrotic areas.CONCLUSION:PDGF may participate in fibrosis development in CVB3-induced myocarditis. Therefore, PDGF signalling may be considered a target for therapeutic interference.

Renaturation permits the detection of protein-tyrosine phosphatase (PTP) activities subsequent to separation by SDS-PAGE in the presence of the (32)P-labeled poly(Glu(4)Tyr) as a macromolecular substrate. An efficient corresponding method has been developed by Burridge and Nelson [Anal. Biochem. 232 (1995) 56]. We describe here the modification of the basic method, its extension to two-dimensional gel electrophoresis, and applications to identify PTPs in signaling complexes and reversibly oxidized PTPs. Particular attention is given to the preparation of samples, to interpretation of the results as well as to advantages and limitations of the technique. Immunodepletion and the use of cells from knockout animals have been successful in the identification of individual PTPs. Readily detectable in cell lysates are PTP-PEST, SHP-2, SHP-1, PTP1B, and T-cell PTP. A much greater complexity of activity bands is, to a large extent, due to the generation of active fragments of PTPs. In-gel detection of PTPs can be employed to monitor cell fractionations and potential PTP activity changes.

The,platelet-derived growth factor (PDGF) beta receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF beta receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF beta receptor, we compared PDGF beta receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF beta receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cgamma1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cgamma1 activity and migratory hyperresponsiveness to PDGF. PDGF beta receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPepsilon ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF beta receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.

UV irradiation causes inflammatory and proliferative cellular responses. We have proposed previously that these effects are, to a large extent, caused by the ligand-independent activation of several receptor tyrosine kinases due to the inactivation of their negative control elements, the protein tyrosine phosphatases (PTPs). We examined the mechanism of this inactivation and found that, in addition to reversible oxidation of PTPs, UV triggers a novel mechanism: induced degradation of PTPs by calpain, which requires both calpain activation and substrate PTP oxidative modification. This as yet unrecognized effect of UV is irreversible, occurs predominantly with UVA and UVB, the range of wavelengths in sunlight that reach the skin surface, and at physiologically relevant doses.

Protein tyrosine phosphatase (PTP) in-gel assays were used to explore association of PTPs with the platelet-derived growth factor beta-receptor (PDGFbetaR). Five PTP activity bands of approximately 120, approximately 70, approximately 60, approximately 53, and approximately 45 kDa could be detected in PDGFbetaR immunoprecipitates and were identified by immunodepletion experiments as PTP-PEST, SHP-2, an active fragment of SHP-2, PTP-1B, and T-cell PTP, respectively. The PTP pattern that was obtained was similar in PDGFbetaR immunoprecipitates from HEK 293 cells overexpressing the human PDGFbetaR and from murine fibroblasts. Association of PTP-1B with the PDGFbetaR was stabilized by pretreatment of the cells with hydrogen peroxide. The epidermal growth factor receptor (EGFR) immunoprecipitated from fibroblasts, and c-Kit isolated from CHRF myeloid cells, were associated with partially overlapping but quantitatively different patterns of PTPs. PTP-PEST was the predominant PTP in EGFR immunoprecipitates, and SHP-1 appeared in c-Kit immunoprecipitates. We propose that the differential association of PTPs with different RTKs is related to their respective contributions to regulation of RTK signaling.

Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro and ex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B(-/-) mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTPlB deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B-fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice.

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