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1.
Oncogene ; 32(35): 4181-8, 2013 Aug 29.
Article in English | MEDLINE | ID: mdl-23045271

ABSTRACT

Cerebellar granule neurons are the most abundant neurons in the brain, and a critical element of the circuitry that controls motor coordination and learning. In addition, granule neuron precursors (GNPs) are thought to represent cells of origin for medulloblastoma, the most common malignant brain tumor in children. Thus, understanding the signals that control the growth and differentiation of these cells has important implications for neurobiology and neurooncology. Our previous studies have shown that proliferation of GNPs is regulated by Sonic hedgehog (Shh), and that aberrant activation of the Shh pathway can lead to medulloblastoma. Moreover, we have demonstrated that Shh-dependent proliferation of GNPs and medulloblastoma cells can be blocked by basic fibroblast growth factor (bFGF). But while the mitogenic effects of Shh signaling have been confirmed in vivo, the inhibitory effects of bFGF have primarily been studied in culture. Here, we demonstrate that mice lacking FGF signaling in GNPs exhibit no discernable changes in GNP proliferation or differentiation. In contrast, activation of FGF signaling has a potent effect on tumor growth: treatment of medulloblastoma cells with bFGF prevents them from forming tumors following transplantation, and inoculation of tumor-bearing mice with bFGF markedly inhibits tumor growth in vivo. These results suggest that activators of FGF signaling may be useful for targeting medulloblastoma and other Shh-dependent tumors.


Subject(s)
Cerebellar Neoplasms/pathology , Cerebellum/growth & development , Fibroblast Growth Factor 2/physiology , Medulloblastoma/pathology , Signal Transduction/physiology , Animals , Cell Cycle , Cell Differentiation , Cerebellar Neoplasms/etiology , Hedgehog Proteins/physiology , Medulloblastoma/etiology , Mice , Mice, Inbred C57BL , Neurons/cytology , Receptor, Fibroblast Growth Factor, Type 1/physiology , Stem Cells/cytology
2.
Eur J Cancer ; 48(18): 3319-27, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22921184

ABSTRACT

BACKGROUND: SF1126 is a peptidic pro-drug inhibitor of pan-PI3K/mTORC. A first-in-human study evaluated safety, dose limiting toxicities (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of SF1126, in patients with advanced solid and B-cell malignancies. PATIENTS AND METHODS: SF1126 was administered IV days 1 and 4, weekly in 28day-cycles. Dose escalation utilised modified Fibonacci 3+3. Samples to monitor PK and PD were obtained. RESULTS: Forty four patients were treated at 9 dose levels (90-1110 mg/m(2)/day). Most toxicity was grade 1 and 2 with a single DLT at180 mg/m(2) (diarrhoea). Exposure measured by peak concentration (C(max)) and area under the time-concentration curve (AUC(0-)(t)) was dose proportional. Stable disease (SD) was the best response in 19 of 33 (58%) evaluable patients. MTD was not reached but the maximum administered dose (MAD) was 1110 mg/m(2). The protocol was amended to enrol patients with CD20+ B-cell malignancies at 1110 mg/m(2). A CLL patient who progressed on rituximab [R] achieved SD after 2 months on SF1126 alone but in combination with R achieved a 55% decrease in absolute lymphocyte count and a lymph node response. PD studies of CLL cells demonstrated SF1126 reduced p-AKT and increased apoptosis indicating inhibition of activated PI3K signalling. CONCLUSION: SF1126 is well tolerated with SD as the best response in patients with advanced malignancies.


Subject(s)
Antineoplastic Agents/therapeutic use , Chromones/therapeutic use , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Oligopeptides/therapeutic use , Phosphoinositide-3 Kinase Inhibitors , Prodrugs/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Proteins/antagonists & inhibitors , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Chromones/administration & dosage , Chromones/adverse effects , Chromones/pharmacokinetics , Chromones/pharmacology , Dose-Response Relationship, Drug , Female , Gastrointestinal Diseases/chemically induced , Hematologic Diseases/chemically induced , Humans , Hypokalemia/chemically induced , Infusions, Intravenous , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/enzymology , Male , Maximum Tolerated Dose , Mechanistic Target of Rapamycin Complex 1 , Middle Aged , Molecular Targeted Therapy , Multiprotein Complexes , Neoplasms/enzymology , Oligopeptides/administration & dosage , Oligopeptides/adverse effects , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Prodrugs/administration & dosage , Prodrugs/adverse effects , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Salvage Therapy , TOR Serine-Threonine Kinases , Young Adult
3.
Curr Pharm Des ; 15(4): 380-8, 2009.
Article in English | MEDLINE | ID: mdl-19199965

ABSTRACT

Angiogenesis is tightly regulated by opposing mechanisms in mammalian cells and is controlled by the angiogenic switch. Other review articles have described a central role for the PTEN/PI-3 kinase/AKT signaling node in the coordinate control of cell division, tumor growth, apoptosis, invasion and cellular metabolism [1, 2]. In this review, we focus on literature that supports the PTEN/PI-3 kinase/AKT signaling node as a major control point for the angiogenic switch in both the on and off positions. We also discuss the rationale for designing small molecule drugs that target the PTEN/PI-3 kinase/AKT signaling node for therapeutic intervention. Our hypothesis is that, instead of inhibiting one cell surface receptor, such as VEGFR2 with bevacizumab (Avastin), thereby leaving a significant number of receptors free to pulse angiogenic signals, a more effective strategy may be to regulate signaling through an intercept node where redundant cell surface receptor signals converge to transmit important signaling events within the cell. This therapeutic configuration brings coordinate control over multiple cell surface receptors in concert with a physiologic response which may combine arrest of cell cycle progression with growth inhibition and the induction of genes involved in specialized functions such as movement, which are all required for the complex process of angiogenesis to occur in a temporal-spatial paradigm.


Subject(s)
Neovascularization, Pathologic , Neovascularization, Physiologic , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Animals , Humans , PTEN Phosphohydrolase/physiology , Phosphatidylinositol 3-Kinases/physiology
4.
Oncogene ; 26(27): 3930-40, 2007 Jun 07.
Article in English | MEDLINE | ID: mdl-17213812

ABSTRACT

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of cell growth and a tumor suppressor. Its growth-attenuating activity is based on the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), an essential second messenger for the phosphoinositide 3-kinase/Akt signaling pathway. This activity may require localization of PTEN to cytoplasmic membranes. Yet PTEN can also localize to the cell nucleus where its functions remain unclear. Here we present data that define a short sequence in the N-terminal region of PTEN required for cytoplasmic localization. We will refer to this sequence as cytoplasmic localization signal (CLS). It could function as a non-canonical signal for nuclear export or as a cytoplasmic retention signal of PTEN. Mutations within the CLS induce nuclear localization and impair growth suppressive activities of PTEN while preserving lipid phosphatase activity. We propose that nuclear localization of PTEN is not compatible with plasma membrane-targeted growth suppressive functions of PTEN.


Subject(s)
Cell Proliferation , Cytoplasm/enzymology , PTEN Phosphohydrolase/metabolism , Amino Acid Sequence , Binding Sites/genetics , Cell Line , Cell Line, Tumor , Cell Nucleus/enzymology , Germ-Line Mutation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Sorting Signals/genetics , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Transfection
5.
Cancer Res ; 61(14): 5587-94, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11454712

ABSTRACT

Increased expression of plasminogen activator inhibitor-1 (PAI-1) in cancer patients is associated with unfavorable outcome, and the reason for this paradox has been poorly understood. We have previously reported elevated levels of PAI-1 in primary tumors of advanced neuroblastomas (Y. Sugiura et al., Cancer Res., 59: 1327-1336, 1999). Here we demonstrate that PAI-1 is coexpressed with the angiogenesis marker alpha(v)beta3 integrin in blood vessels of primary neuroblastoma tumors, suggesting that PAI-1 plays a role in angiogenesis. Using human brain microvascular endothelial cells (HBMECs), we found that PAI-1 inhibits alpha(v)beta3 integrin-mediated cell adhesion to vitronectin but promotes alpha5beta1-mediated migration from vitronectin toward fibronectin. Inhibition of vitronectin adhesion by PAI-1 did not induce HBMEC apoptosis. PAI-1 also inhibited endothelial tube formation on Matrigel in the presence of vitronectin but had a stimulatory effect in the presence of fibronectin. This effect of PAI-1 on microvascular endothelial cells is primarily related to the ability of PAI-1 to bind to vitronectin via its NH2-terminal domain and to interfere with cell adhesion to vitronectin. We propose that PAI-1 acts as a positive switch for angiogenesis by promoting endothelial cell migration away from their vitronectin-containing perivascular space toward fibronectin-rich tumor tissue. These observations provide a novel explanation for the enhancing effect of PAI-1 in cancer progression.


Subject(s)
Cell Movement/drug effects , Endothelium, Vascular/drug effects , Fibronectins/pharmacology , Neovascularization, Pathologic/pathology , Plasminogen Activator Inhibitor 1/pharmacology , Apoptosis/drug effects , Cell Adhesion/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/chemistry , Endothelium, Vascular/cytology , Humans , Immunohistochemistry , Integrins/physiology , Neovascularization, Pathologic/metabolism , Neovascularization, Physiologic/drug effects , Plasminogen Activator Inhibitor 1/biosynthesis , Plasminogen Activator Inhibitor 1/physiology , Recombinant Proteins/pharmacology , Vitronectin/pharmacology
6.
Proc Natl Acad Sci U S A ; 98(8): 4622-7, 2001 Apr 10.
Article in English | MEDLINE | ID: mdl-11274365

ABSTRACT

Mutations of the tumor suppressor PTEN, a phosphatase with specificity for 3-phosphorylated inositol phospholipids, accompany progression of brain tumors from benign to the most malignant forms. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of angiogenesis, a process termed the angiogenic switch. Therefore, we tested whether PTEN regulates tumor progression by modulating angiogenesis. U87MG glioma cells stably reconstituted with PTEN cDNA were tested for growth in a nude mouse orthotopic brain tumor model. We observed that the reconstitution of wild-type PTEN had no effect on in vitro proliferation but dramatically decreased tumor growth in vivo and prolonged survival in mice implanted intracranially with these tumor cells. PTEN reconstitution diminished phosphorylation of AKT within the PTEN-reconstituted tumor, induced thrombospondin 1 expression, and suppressed angiogenic activity. These effects were not observed in tumors reconstituted with a lipid phosphatase inactive G129E mutant of PTEN, a result that provides evidence that the lipid phosphatase activity of PTEN regulates the angiogenic response in vivo. These data provide evidence that PTEN regulates tumor-induced angiogenesis and the progression of gliomas to a malignant phenotype via the regulation of phosphoinositide-dependent signals.


Subject(s)
Neoplasms, Experimental/blood supply , Neovascularization, Pathologic , Phosphoric Monoester Hydrolases/physiology , Tumor Suppressor Proteins , Brain Neoplasms/blood supply , Brain Neoplasms/pathology , Cell Division , Immunohistochemistry , Mutation , Neoplasm Transplantation , Neoplasms, Experimental/pathology , PTEN Phosphohydrolase , Phosphoric Monoester Hydrolases/genetics , Survival Analysis , Thrombospondins/biosynthesis
7.
Clin Exp Rheumatol ; 19(6): 639-46, 2001.
Article in English | MEDLINE | ID: mdl-11791634

ABSTRACT

OBJECTIVE: To evaluate the safety and efficacy of L-asparaginase as an immunosuppressive agent in a mouse model of rheumatoid arthritis. METHODS: Male DBA/1 mice with collagen-induced arthritis (CIA) were treated at different intervals with various doses of native and pegylated L-asparaginase from E. coli. The mice were observed for 4 weeks during which time arthritis was scored. Outcome parameters included effect on severity and progression of established arthritis as well as prevention of disease. In addition, X-rays from the affected joints were obtained for comparison. RESULTS: Both native L-asparaginase at a dose of 50 IU/injection intraperitoneally three days a week and pegylated asparaginase (PEG-L-asparaginase) at a dose of 25 IU/injection twice a week, significantly reduced the mean arthritic score (MAS) in mice with established arthritis (p < 0.001 for PEG-L-asparaginase). When native L-asparaginase was administered before the onset of arthritis (days 14-post immunization) the number of mice developing arthritis as well as the number of arthritic paws and the severity of arthritis in the treatment group were significantly decreased (p < 0.0001). Significant differences were found in the X-ray evaluation between treated and control mice. None of the animals died due to drug related events or showed signs of asparaginase induced toxicity. CONCLUSION: Our data provide the first direct evidence that L-asparaginase is a potent antiarthritic agent and may represent an effective second line agent for future treatment studies in juvenile and adult rheumatoid arthritis.


Subject(s)
Antineoplastic Agents/therapeutic use , Arthritis, Experimental/drug therapy , Asparaginase/therapeutic use , Animals , Arthritis, Experimental/physiopathology , Arthrography , Dose-Response Relationship, Drug , Drug Combinations , Escherichia coli/immunology , Joints/drug effects , Joints/physiopathology , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred DBA , Polyethylene Glycols/therapeutic use , Treatment Outcome
8.
Cancer Res ; 60(3): 712-21, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10676658

ABSTRACT

Inhibition of the RGD-binding integrins, alpha(v)beta3 and alpha(v)beta5, prevents endothelial cell anchorage and induces endothelial apoptosis, which results in disruption of tumor angiogenesis and inhibition of tumor growth in animal models. In this study, we demonstrate by immunohistochemical analysis that integrin alpha(v)beta3 was expressed by 61% (mean) of microvessels in high-risk neuroblastomas (stage IV and MYCN-amplified stage III; n = 28) but only by 18% (mean) of microvessels in low-risk tumors (stages I and II and non-MYCN-amplified stage III; n = 12). Integrin alpha(v)beta5 was found on 60% (mean) of microvessels in 21 Stage IV tumors. These data suggest that neuroblastomas may be targeted for antiangiogenic treatment directed against endothelial integrins alpha(v)beta3 and alpha(v)beta5. In cell culture, inhibition of integrin-dependent endothelial cell anchorage to vitronectin by RGDfV, an RGD function-blocking cyclic peptide, induced apoptosis in bovine brain endothelial cells compared with the control peptide, RADfV (37.5% versus 8.7%, respectively), as detected by chromatin condensation and nuclear fragmentation. Treatment with RGDfV but not with RADfV, which prevented attachment of endothelial cells to vitronectin or fibronectin, was associated with up to a 50% increase in endogenous ceramide, a lipid second messenger that can mediate cell death. Furthermore, exogenous C2-ceramide was cytotoxic to bovine brain endothelial cells and induced activation of C-jun N-terminal kinase (JNK), a MAP kinase that can be activated in stress-induced apoptosis pathways. This suggests that ceramide may function in detachment-induced endothelial cell apoptosis, originating from inhibition of vitronectin binding to integrins such as alpha(v)beta3 and alpha(v)beta5. This is the first report to demonstrate expression of integrins alpha(v)beta3 and alpha(v)beta5 by microvascular endothelium of a childhood tumor and association of their expression with neuroblastoma aggressiveness. Furthermore, our data provide the first suggestion that inhibition of endothelial cell anchorage, resulting from specific blockade of RGD-binding integrins, increases endogenous ceramide, which may contribute to endothelial cell death.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Ceramides/biosynthesis , Endothelium, Vascular/metabolism , Integrins/physiology , JNK Mitogen-Activated Protein Kinases , Neuroblastoma/metabolism , Receptors, Vitronectin/physiology , Apoptosis , Endothelium, Vascular/cytology , Enzyme Activation , Humans , Immunohistochemistry , Integrins/analysis , Integrins/antagonists & inhibitors , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases/metabolism , Neuroblastoma/drug therapy , Receptors, Vitronectin/analysis , Receptors, Vitronectin/antagonists & inhibitors
9.
J Immunol ; 163(11): 6023-34, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10570290

ABSTRACT

We used the U937 cell line to examine the modulation of adaptor protein interactions (Shc, Grb2, and Cbl) after high affinity IgG receptor (FcgammaRI) cross-linking, leading to the formation of the Grb2-Sos complex, the activation of Ras, and the regulation of the respiratory burst. Cross-linking of FcgammaRI induced the conversion of GDP-Ras to GTP-Ras reaching a maximum 5 min after stimulation. Concomitant with Ras activation, Sos underwent an electrophoretic mobility shift and the Sos-Grb2 association was increased (6-fold). The Grb2-Sos complex was present only in the membrane fraction and was augmented after FcgammaRI stimulation. Tyrosine-phosphorylated Shc, mainly the p52 isoform, was observed to transiently onload to the membrane Grb2-Sos complex on FcgammaRI stimulation. Cross-linking of FcgammaRI induces the tyrosine phosphorylation of Cbl, which forms a complex with Grb2 and Shc via the Cbl C terminus. Kinetic experiments confirm that Cbl-Grb2 is relatively stable, whereas Grb2-Sos, Grb2-Shc, and Cbl-Shc interactions are highly inducible. The Src family tyrosine kinase inhibitor, PP1, was shown to completely inhibit Shc tyrosine phosphorylation, the Shc-Grb2 interaction, and the FcgammaR-induced respiratory burst. Our results provide the first evidence that the upstream activation of Src kinases is required for the modulation of the Shc-Grb2 interaction and the myeloid NADPH oxidase response.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , NADPH Oxidases/metabolism , Proteins/metabolism , Son of Sevenless Proteins/metabolism , Ubiquitin-Protein Ligases , src Homology Domains , src-Family Kinases/metabolism , Cell Compartmentation , Cell Membrane/metabolism , GRB2 Adaptor Protein , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-cbl , Receptors, IgG , Shc Signaling Adaptor Proteins , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1 , Tyrosine/metabolism , U937 Cells , ras Proteins/metabolism
10.
Blood ; 94(6): 2112-20, 1999 Sep 15.
Article in English | MEDLINE | ID: mdl-10477741

ABSTRACT

Cross-linking of Fc receptors for IgA, FcalphaR (CD89), on monocytes/macrophages is known to enhance phagocytic activity and generation of oxygen free radicals. We provide evidence here that the FcalphaR signals through the gamma subunit of FcepsilonRI in U937 cells differentiated with interferon gamma (IFNgamma). Our results provide the first evidence that FcalphaR-mediated signals modulate a multimolecular adaptor protein complex containing Grb2, Shc, SHIP, CrkL, Cbl, and SLP-76. Cross-linking of FcalphaRI using anti-FcalphaRI induces the phosphorylation of the gamma subunit as detected by mobility retardation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Stimulation of FcalphaRI induced the tyrosine phosphorylation of Shc and increased the association of Grb2 with Shc and CrkL. Grb2 associates constitutively with Sos, and the latter undergoes mobility shift upon FcalphaRI stimulation. The complex adapter proteins, Cbl and SLP-76, are physically associated in myeloid cells and both proteins undergo tyrosine phosphorylation upon FcalphaR stimulation. These data indicate that the stimulation of FcalphaR results in the modulation of adaptor complexes containing tyrosine-phosphorylated Cbl, Shc, SHIP, Grb2, and Crkl. Experiments performed with the Src kinase inhibitor, PP1, provide the first evidence that Src kinase activation is required for FcalphaRI-induced production of superoxide anions and provide insight into the mechanism for FcalphaR-mediated activation of downstream oxidant signaling in myeloid cells.


Subject(s)
Adaptor Proteins, Signal Transducing , Antigens, CD/physiology , Interferon-gamma/pharmacology , Receptors, Fc/physiology , Antibodies/pharmacology , Cell Differentiation , Cross-Linking Reagents/pharmacology , ErbB Receptors/physiology , GRB2 Adaptor Protein , Humans , Macromolecular Substances , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation , Proteins/physiology , Recombinant Proteins , Signal Transduction , U937 Cells , src Homology Domains
11.
J Leukoc Biol ; 65(4): 523-34, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10204582

ABSTRACT

Cbl is a cytosolic protein that is rapidly tyrosine phosphorylated in response to Fc receptor activation and binds to the adaptor proteins Grb2, CrkL, and Nck. A few reports describe Cbl interactions in primary human hematopoietic cells. We show evidence that Cbl participates in signaling initiated by Fc gammaRI receptor cross-linking in human primary macrophages, and functions downstream of Src family kinases in this pathway. Fc gammaRI stimulation in human macrophages was associated with rapid and transient tyrosine phosphorylation of the Cbl adaptor protein. Immunoprecipitated Cbl was complexed with several tyrosine phosphorylated proteins, the most prominent of which was a 38-kDa band identified as the CrkL adaptor protein. CrkL associated with tyrosine-phosphorylated Cbl and itself became tyrosine phosphorylated after Fc gammaRI cross-linking. SLP-76, a recently cloned Grb2-associated protein, was strongly tyrosine phosphorylated after Fc gammaRI stimulation and was associated with both Cbl and Grb2. Grb2 and Cbl binding to SLP-76 were inducible after Fc gammaRI stimulation of the macrophages. Nck was inducibly bound to Cbl after Fc gammaRI stimulation, whereas Grb2 was constitutively associated with it. Shc was also inducibly tyrosine phosphorylated and bound to Grb2 after Fc gammaRI stimulation of the macrophages. PP1, a specific inhibitor of Src kinases, inhibited the Fc gammaRI-induced respiratory burst, as well as the tyrosine phosphorylation of Cbl and its inducible association with CrkL. These results suggest a fundamental role for the tyrosine phosphorylation of Cbl, CrkL, SLP-76, and Shc and the association of Cbl with CrkL, SLP-76, and Nck in Fc gammaRI signaling in human macrophages. Experiments performed with PP1, the specific Src kinase inhibitor, demonstrate the first evidence that Cbl and the Cbl-Crkl interaction are downstream targets for myeloid Src kinases required for the activation of myeloid NADPH oxidase activity.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Macrophages/metabolism , Proto-Oncogene Proteins/physiology , Receptors, IgG/physiology , Signal Transduction/physiology , Ubiquitin-Protein Ligases , src-Family Kinases/physiology , Bone Marrow Cells , Cells, Cultured , ErbB Receptors/metabolism , ErbB Receptors/physiology , GRB2 Adaptor Protein , Humans , Macrophages/enzymology , Oncogene Proteins/metabolism , Oncogene Proteins/physiology , Phosphoproteins/metabolism , Phosphoproteins/physiology , Phosphorylation , Proteins/metabolism , Proteins/physiology , Proto-Oncogene Proteins c-cbl , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Tyrosine/metabolism , U937 Cells
12.
Exp Cell Res ; 245(2): 330-42, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9851874

ABSTRACT

Fc receptors modulate inflammatory processes, including phagocytosis, serotonin and histamine release, superoxide production, and secretion of cytokines. Aggregation of FcgammaRIIa, the low-affinity receptor for monomeric IgG, activates nonreceptor protein tyrosine kinases such as Lyn, Hck, and Syk, potentially driving the phosphorylation of the downstream adaptor proteins, including Cbl and/or Nck. Previous work from our laboratory using interferon-gamma-differentiated U937 (U937IF) myeloid cells investigated mechanisms which regulate Fcgamma receptor-induced assembly of adaptor complexes. Herein we report that FcgammaRII receptor signaling in U937IF and HEL cells involves Cbl and Nck, suggesting that Cbl-Nck interactions may link FcgammaRII to downstream activation of Pak kinase. FcgammaRII crosslinking induced the phosphorylation of Cbl and Nck on tyrosine. The alphaCbl immunoprecipitations revealed constitutive binding of Nck and Grb2 to Cbl and FcgammaRII-inducible binding of CrkL to Cbl. The interactions of Cbl with Nck and CrkL were phosphorylation dependent since dephosphorylation of cellular proteins with potato acid phosphatase abrogated binding. GST-Nck fusion protein pulldown experiments show that Cbl and Pak1 bind to the second SH3 domain of Nck. A specific Src inhibitor, PP1, was shown to completely abrogate the FcgammaR-induced superoxide response, correlating with a decrease in Cbl and Nck tyrosine phosphorylation. Our results provide the first evidence that Src is required for FcgammaR activation of the respiratory burst in myeloid cells and suggest that Cbl-Nck, Cbl-Pak1, and Nck-Pak1 interactions may regulate this response.


Subject(s)
Adaptor Proteins, Signal Transducing , Antigens, CD/metabolism , Oncogene Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, IgG/metabolism , Retroviridae Proteins, Oncogenic/metabolism , Signal Transduction , Acid Phosphatase/metabolism , Cell Line , GRB2 Adaptor Protein , Humans , Nuclear Proteins/metabolism , Oncogene Protein v-cbl , Oncogene Proteins/chemistry , Phosphorylation , Phosphotyrosine/metabolism , Precipitin Tests , Protein Binding , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/pharmacology , Proteins/metabolism , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , Proto-Oncogene Proteins pp60(c-src)/physiology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Recombinant Fusion Proteins/metabolism , Respiratory Burst/drug effects , U937 Cells , p21-Activated Kinases , src Homology Domains
13.
J Immunol ; 161(10): 5555-63, 1998 Nov 15.
Article in English | MEDLINE | ID: mdl-9820532

ABSTRACT

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.


Subject(s)
Adaptor Proteins, Signal Transducing , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, IgG/metabolism , Receptors, Immunologic/metabolism , Signal Transduction/immunology , Tyrosine/metabolism , Ubiquitin-Protein Ligases , Amino Acid Sequence , Guanine Nucleotide Exchange Factors , Humans , Lymphocyte Activation , Molecular Sequence Data , Nuclear Proteins/immunology , Phosphoproteins/immunology , Phosphorylation , Proteins/immunology , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-cbl , Receptor Aggregation/immunology , Receptors, IgG/physiology , U937 Cells , src Homology Domains/immunology
14.
Blood ; 92(5): 1697-706, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9716598

ABSTRACT

SLP-76 and Cbl are complex adapter proteins that have the capacity to bind to smaller adapter proteins, such as Grb2, which subsequently binds the nucleotide exchange protein Sos in the transmission of intracellular signals. SLP-76, Cbl, Shc, and Grb2 have been implicated in immunoreceptor tyrosine-based activation motif (ITAM) signaling, leading to activation of Ras. However, their mechanism of action has not been determined. To date, there have been no reports of SLP-76 involvement in FcgammaRI-receptor signaling and no data exist for an interaction between Cbl, Shc, and SLP-76 in vivo. We provide evidence that SLP-76, Cbl, and Shc are tyrosine phosphorylated on FcgammaRI-receptor stimulation and are associated with the adapter protein Grb2 in gamma-interferon-differentiated U937 cells (U937IF). The interactions between SLP-76 and Cbl and SLP-76 and Grb2 are present in resting U937IF cells. However, the interaction between SLP-76 and Grb2 becomes augmented twofold on FcgammaRI-receptor aggregation. Our results provide the first evidence for a phosphorylation-dependent interaction between SLP-76 and Shc, induced at least 10-fold on FcgammaRI receptor stimulation. Our data indicate that a significant portion of a multimolecular complex containing Cbl, SLP-76, Shc, and Grb2 is distinct from a trimolecular complex containing the Ras guanine nucleotide exchanger Sos, Shc, and Grb2. FcgammaRI-induced tyrosine phosphorylation of SLP-76, Cbl, Shc, and the highly induced SLP-76-Shc interaction provide the first evidence that SLP-76 and Cbl are involved in FcgammaRI signaling and suggest a functional significance for these interactions in FcgammaRI signal relay in the control of Ras in myeloid cells.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Phosphoproteins/metabolism , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, IgG/metabolism , Signal Transduction , Ubiquitin-Protein Ligases , Antibodies, Monoclonal/pharmacology , Binding Sites , Cell Line , GRB2 Adaptor Protein , Granulocytes/metabolism , Immunoblotting , Immunoglobulin Fab Fragments/pharmacology , Immunoglobulin G/pharmacology , Immunosorbent Techniques , Kinetics , Phosphoproteins/genetics , Phosphorylation , Phosphotyrosine/metabolism , Proto-Oncogene Proteins c-cbl , Receptors, IgG/antagonists & inhibitors , Recombinant Fusion Proteins/metabolism , Shc Signaling Adaptor Proteins
15.
J Immunol ; 160(10): 5018-27, 1998 May 15.
Article in English | MEDLINE | ID: mdl-9590251

ABSTRACT

In this study, we provide the first evidence for role of the CBL adapter protein interaction in Fc gammaRI receptor signal transduction. We study the Fc gammaRI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-gamma-differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, Fc gammaRIgamma of Fc gammaRI, providing direct evidence that CBL functions in myeloid ITAM signaling. Fc gammaRI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon Fc gammaRI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461-670) binds to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671-906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL-GRB2 and GRB2-SOS complexes function in separate pathways for myeloid Fc gammaRI signaling.


Subject(s)
Adaptor Proteins, Signal Transducing , Proteins/physiology , Proto-Oncogene Proteins/physiology , Receptors, IgG/physiology , Tyrosine/metabolism , Ubiquitin-Protein Ligases , Amino Acid Sequence , GRB2 Adaptor Protein , Humans , Membrane Proteins/physiology , Molecular Sequence Data , Nuclear Proteins/physiology , Phosphorylation , Proto-Oncogene Proteins c-cbl , Son of Sevenless Proteins , src Homology Domains
16.
Blood ; 89(2): 388-96, 1997 Jan 15.
Article in English | MEDLINE | ID: mdl-9002939

ABSTRACT

Engagement of the high-affinity IgG Fc receptor (Fc gamma RI) activates a signal transduction pathway involving tyrosine phosphorylation of associated kinases. We compared the activation of the related protein tyrosine kinases (PTKs), Syk and ZAP-70, in Fc gamma RI-mediated signaling. Cross-linking of the Fc gamma RI multimeric receptor in monocytic cells results in tyrosine phosphorylation of the Fc epsilon RI gamma subunit and association of Syk with this complex. We stably introduced ZAP-70 via a retroviral vector into two monocytic cell lines, U937 and THP-1, which normally do not express ZAP-70. Neither Syk nor MAP kinase activation was affected by the presence of ZAP-70. Although transduced ZAP-70 had in vitro kinase activity and associated with Fc epsilon RI gamma after receptor aggregation, it was not tyrosine phosphorylated. In contrast, both ZAP-70 and Syk were phosphorylated in a T-cell line in which their respective levels of expression were similar to those detected in U937/ZAP-70 cells. Therefore, these results suggest that requirements for Syk and ZAP-70 phosphorylation are distinct in a monocytic cell context.


Subject(s)
Enzyme Precursors/metabolism , Monocytes/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, IgG/metabolism , Signal Transduction , Cell Line , Humans , Intracellular Signaling Peptides and Proteins , Phosphorylation , Second Messenger Systems , Syk Kinase , ZAP-70 Protein-Tyrosine Kinase
17.
Exp Cell Res ; 237(2): 288-95, 1997 Dec 15.
Article in English | MEDLINE | ID: mdl-9434624

ABSTRACT

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the Fc gamma RI-induced respiratory burst in interferon-gamma-differentiated U937 cells (U937IF) while augmenting the Fc gamma RI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in Fc gamma RI signaling. We show that orthovanadate and PAO augmented the Fc gamma RI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to Fc gamma RI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to Fc gamma RI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the Fc gamma RI signal to result in activation of the myeloid respiratory burst response.


Subject(s)
Leukocytes/enzymology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Proto-Oncogene Proteins/physiology , Receptors, IgG/physiology , Respiratory Burst , Ubiquitin-Protein Ligases , Arsenicals/pharmacology , Cell Line , Enzyme Inhibitors/pharmacology , Humans , Phosphotyrosine/metabolism , Proto-Oncogene Proteins c-cbl , Signal Transduction , Vanadates/pharmacology
18.
J Biol Chem ; 271(23): 13342-8, 1996 Jun 07.
Article in English | MEDLINE | ID: mdl-8662746

ABSTRACT

The activation of the serine/threonine kinase, Raf-1, serves to connect upstream protein tyrosine kinases to downstream signaling events. We previously reported that FcgammaRI stimulation of interferon gamma-differentiated U937 cells (termed U937IF cells) induces a mobility shift in Erk2. Herein, we report that cross-linking of FcgammaRI receptor in U937IF cells induces a marked tyrosine phosphorylation of Raf-1 (10-fold increase). Tyrosine phosphorylation of Raf-1 is induced by FcgammaRI activation and not by PMA (1 microg/ml), N-formyl-Met-Leu-Phe (1 microM), calcium ionophore (1 microM), thrombin (0.05 unit/ml), FcgammaRII, or FcgammaRIII stimulation. The kinetics of Raf-1 tyrosine phosphorylation is rapid, reaching peak levels 1-2 min after FcgammaRI activation, and the tyrosine phosphorylation of Raf-1 precedes the activation of the respiratory burst. FcgammaRI cross-linking induces the tyrosine phosphorylation of Shc; tyrosine-phosphorylated Shc binds to Grb2 forming a Shc-Grb2 complex. The data provide evidence that the FcgammaRI receptor signals via the upstream activation of nonreceptor protein tyrosine kinases, which leads to the subsequent activation of Ras family GTPases and serine/threonine kinases, Raf-1 and mitogen-activated protein kinase.


Subject(s)
Adaptor Proteins, Signal Transducing , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, IgG/metabolism , Signal Transduction/physiology , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , GRB2 Adaptor Protein , Humans , Kinetics , Mice , Mitogen-Activated Protein Kinase 1 , Molecular Sequence Data , Phosphorylation , Proto-Oncogene Proteins c-raf , Receptors, IgG/genetics , Respiratory Burst , Tyrosine/metabolism
19.
J Immunol ; 155(8): 3994-4003, 1995 Oct 15.
Article in English | MEDLINE | ID: mdl-7561108

ABSTRACT

Vascular endothelial cell (EC) injury or activation by LPS plays a critical role in the pathogenesis of Gram-negative meningitis and endotoxic shock. EC do not express membrane CD14, but respond to LPS in a soluble CD14-dependent manner. The signal transduction mechanisms involved in LPS-induced EC responses are largely unknown. We used bovine and human brain microvessel EC (BBMEC, and HBMEC) to study LPS-induced protein tyrosine phosphorylation. LPS rapidly induced the tyrosine phosphorylation of several proteins in BBMEC and HBMEC, which was detectable by 5 to 15 min, reached a maximum by 30 min, and declined by 60 to 90 min. The increase in tyrosine phosphorylation was apparent following stimulation with LPS at 0.1 ng/ml and was dose dependent up to 100 ng/ml. Similar changes in tyrosine phosphorylation were induced by smooth and rough LPS as well as lipid A, but not by the inactive lipid A analogue, Rhodopseudomonas sphaeroides diphosphoryl lipid A. Pretreatment of EC with the tyrosine kinase inhibitor, herbimycin A, inhibited LPS-stimulated protein tyrosine phosphorylation and LPS-mediated lactic dehydrogenase release from BBMEC and IL-6 release from HBMEC in a dose-dependent manner. Three proteins with apparent m.w. of 44, 42, and 41 kDa were predominant among the LPS-induced tyrosine phosphoproteins, and they were identified as mitogen-activated protein kinase isoforms ERK1, ERK2, and p38, respectively. LPS-induced protein tyrosine phosphorylation in HBMEC and BBMEC was soluble CD14 dependent, since pretreatment of these cells with anti-hCD14 mAb inhibited the LPS-induced tyrosine phosphorylation of p44, p42, and p41. Additionally, LPS induced a mobility shift in p44 and p42 mitogen-activated protein kinase isozymes, which was inhibited by herbimycin A pretreatment of the EC. These findings demonstrate for the first time that increased protein tyrosine phosphorylation and activation of mitogen-activated protein kinases occur rapidly after LPS stimulation of EC in the presence of soluble CD14. Our data also suggest that a herbimycin-sensitive step, presumably a tyrosine kinase, is involved in mediating LPS-induced human EC activation and IL-6 secretion.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Endothelium, Vascular/enzymology , Lipopolysaccharide Receptors/physiology , Lipopolysaccharides/pharmacology , Protein-Tyrosine Kinases/metabolism , Tyrosine/metabolism , Amino Acid Sequence , Animals , Benzoquinones , Brain/blood supply , Calcium-Calmodulin-Dependent Protein Kinases/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/immunology , Cattle , Cells, Cultured , Child , Child, Preschool , Dose-Response Relationship, Immunologic , Endothelium, Vascular/drug effects , Endothelium, Vascular/immunology , Humans , Lactams, Macrocyclic , Lipopolysaccharides/antagonists & inhibitors , Mice , Microcirculation/enzymology , Microcirculation/immunology , Molecular Sequence Data , Phosphorylation/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/drug effects , Quinones/pharmacology , Rifabutin/analogs & derivatives
20.
J Immunol ; 154(8): 4039-47, 1995 Apr 15.
Article in English | MEDLINE | ID: mdl-7535819

ABSTRACT

U937 cells differentiated with IFN-gamma (termed U937IF cells) were used to study Fc gamma RI signaling. IFN induces a functional Fc gamma RI receptor signaling pathway in U937 cells, leading to the activation of the respiratory burst. IFN induces the expression of the nonreceptor protein tyrosine kinase, hck, and cross-linking the Fc gamma RI receptor in U937IF cells results in the activation of hck kinase as evidenced by the three- to fivefold increased tyrosine phosphorylation of hck. In vitro kinase assays demonstrate that the specific kinase activity of hck is increased 10-fold after Fc gamma RI stimulation. hck is observed to associate with two prominent tyrosine-phosphorylated proteins, p72 and p95, after Fc gamma RI-activation. Fc gamma RI cross-linking also results in mobility shift in MAP kinase in U937IF cells, suggesting that the Fc gamma RI receptor signals through the activation of MAP kinase. The data suggest that hck, p72, p95, and MAP kinase are involved in signal transduction through the Fc gamma RI receptor.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, IgG/physiology , Amino Acid Sequence , Cells, Cultured , Enzyme Activation , Humans , In Vitro Techniques , Interferons/pharmacology , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Molecular Sequence Data , Phosphotyrosine , Proto-Oncogene Proteins c-hck , Signal Transduction , Tyrosine/analogs & derivatives , Tyrosine/metabolism
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