Tyrosine Phosphorylation Of c-Cbl Research Articles

Histidine-domain-containing protein tyrosine phosphatase regulates platelet-derived growth factor receptor intracellular sorting and degradation

Histidine domain-containing protein tyrosine phosphatase (HD-PTP) is a putative phosphatase that has been shown to affect the signaling and downregulation of certain receptor tyrosine kinases. To investigate if HD-PTP affects platelet-derived growth factor receptor β (PDGFRβ) signaling, we employed the overexpression of HA-tagged HD-PTP, as well as siRNA-mediated and lentivirus shRNA-mediated silencing of HD-PTP in NIH3T3 cells. We found that HD-PTP was recruited to the PDGFRβ in a ligand-dependent manner. Depletion of HD-PTP resulted in an inability of PDGF-BB to promote tyrosine phosphorylation of the ubiquitin ligases c-Cbl and Cbl-b, with a concomitant missorting and reduction of the degradation of activated PDGFRβ. In contrast, ligand-induced internalization of PDGFRβ was unaffected by HD-PTP silencing. Furthermore, the levels of STAM and Hrs of the ESCRT0 machinery were decreased, and immunofluorescence staining showed that in HD-PTP-depleted cells, PDGFRβ accumulated in large aberrant intracellular structures. After the reduction of HD-PTP expression, an NIH3T3-derived cell line that has autocrine PDGF-BB signaling (sis-3T3) showed increased ability of anchorage-independent growth. However, exogenously added PDGF-BB promoted efficient additional colony formation in control cells, but was not able to do so in HD-PTP-depleted cells. Furthermore, cells depleted of HD-PTP migrated faster than control cells. In summary, HD-PTP affects the intracellular sorting of activated PDGFRβ and the migration, proliferation and tumorigenicity of cells stimulated by PDGF.

The proline‐rich tyrosine kinase Pyk2 regulates platelet integrin αIIbβ3 outside‐in signaling

The proline-rich tyrosine kinase Pyk2 is a focal adhesion kinase expressed in blood platelets, and is activated downstream of G-protein coupled receptors as well as integrin α2β1. In this study we have investigated the involvement of Pyk2 in integrin αIIbβ3 outside-in signaling in human and murine platelets. We analyzed the stimulation of intracellular signaling pathways in platelets from Pyk2 knockout mice adherent to immobilized fibrinogen. Pyk2 was rapidly phosphorylated and activated in human and murine platelets adherent to fibrinogen through integrin αIIbβ3. Activation of Pyk2 was Src-dependent, but did not require phospholipase Cγ2 activity. Platelets from Pyk2 knockout mice showed a defective ability to adhere and spread on fibrinogen, in association with a dramatic reduction of phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation. Pharmacological and genetic analysis demonstrated that integrin αIIbβ3 engagement selectively stimulated the β-isoform of PI3K (PI3Kβ), and that, as for Pyk2, PI3Kβ activation required Src family kinases activity, but not phospholipase Cγ2. In fibrinogen-adherent platelets, both Pyk2 and PI3Kβ were necessary for stimulation of the small GTPase Rap1b, a regulator of cell adhesion and spreading. Integrin αIIbβ3 engagement triggered the association of the PI3Kβ regulatory subunit p85 with the adaptor protein c-Cbl, which was mediated by the p85 SH3 domain, and was independent of c-Cbl tyrosine phosphorylation. However, p85-associated c-Cbl was tyrosine phosphorylated by activated Pyk2 in fibrinogen adherent platelets. These results identify a novel pathway of integrin αIIbβ3 outside-in signaling and recognize the tyrosine kinase Pyk2 as a major regulator of platelet adhesion and spreading on fibrinogen.

Down-regulation of the NKG2D receptor is differentially controlled by MICA and ULBP2 ligands

Event Abstract Back to Event Down-regulation of the NKG2D receptor is differentially controlled by MICA and ULBP2 ligands Linda Quatrini1*, Rosa Molfetta1, Francesca Gasparrini1, Cristina Capuano2, Alessandra Zingoni1, Ricciarda Galandrini2, Angela Santoni1 and Rossella Paolini1 1 "Sapienza" University of Rome, Molecular Medicine, Italy 2 "Sapienza" University of Rome, Experimental Medicine, Italy The activating NKG2D receptor on human NK cells mediates “induced self recognition” in that its ligands are up-regulated by stressed or diseased cells. Evidence collected in the past years demonstrated that chronic exposure to NKG2DLs induces receptor down-modulation. The aim of this study was to evaluate whether different NKG2D ligands (NKG2DLs), namely MICA and ULBP2, are equivalent in their capacities to down-modulate the surface receptor expression on human NK cells. We analyzed the rate and kinetics of NKG2D down-modulation in primary cultured NK cells and in the NKL NK cell line upon stimulation with the Ba/F3 cell line stably overexpressing comparable levels of MICA or ULBP2 by FACS and fluorescence microscopic analysis. Although both ligands were able to reduce NKG2D expression, exposure to MICA over-expressing target cells resulted in a more rapid and efficient receptor down-modulation and in a more pronounced impairment of NKG2D-dependent cytotoxicity. MICA-experienced NK cells also showed a higher tyrosine phosphorylation of the ubiquitin ligase c-Cbl. Furthermore, ligand-induced receptor down-modulation affected the total NKG2D cellular levels, suggesting that the internalized receptor complexes were mainly subjected to degradation. All together these results demonstrate that NKG2D down-regulation is influenced by the nature of its ligand and suggest a different contribution of the ubiquitin pathway in the control of NKG2D internalization and degradation in MICA- versus ULBP2-experienced cells. Understanding the mechanisms of ligand-induced NKG2D down-modulation will be helpful to prevent evasion from NK cell-mediated immune response. Keywords: innate immunity, NK cells, NK receptor, Endocytosis, degradation Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Quatrini L, Molfetta R, Gasparrini F, Capuano C, Zingoni A, Galandrini R, Santoni A and Paolini R (2013). Down-regulation of the NKG2D receptor is differentially controlled by MICA and ULBP2 ligands. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00013 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Linda Quatrini, "Sapienza" University of Rome, Molecular Medicine, Rome, Italy, Italy, linda.quatrini@opbg.net Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Linda Quatrini Rosa Molfetta Francesca Gasparrini Cristina Capuano Alessandra Zingoni Ricciarda Galandrini Angela Santoni Rossella Paolini Google Linda Quatrini Rosa Molfetta Francesca Gasparrini Cristina Capuano Alessandra Zingoni Ricciarda Galandrini Angela Santoni Rossella Paolini Google Scholar Linda Quatrini Rosa Molfetta Francesca Gasparrini Cristina Capuano Alessandra Zingoni Ricciarda Galandrini Angela Santoni Rossella Paolini PubMed Linda Quatrini Rosa Molfetta Francesca Gasparrini Cristina Capuano Alessandra Zingoni Ricciarda Galandrini Angela Santoni Rossella Paolini Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Decreased pulmonary c-Cbl expression and tyrosine phosphorylation in the nitrofen-induced rat model of congenital diaphragmatic hernia

The high morbidity of newborn infants with congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH), which is characterized by a failure of alveolar development. The nitrofen-induced CDH model has been widely used to investigate the pathogenesis of PH in CDH. It has previously been shown that the fibroblast growth factor receptor (FGFR) pathway, which is essential for a proper lung development, is disrupted during late gestation of nitrofen-induced CDH. Casitas B-lineage lymphoma (c-Cbl) proteins are known regulators of signal transduction through FGFRs, indicating their important role during alveolarization in developing lungs. Furthermore, it has been demonstrated that tyrosine phosphorylation of c-Cbl proteins has a pivotal role for their physiological function and activity during fetal lung development. We designed this study to test the hypothesis that pulmonary c-Cbl expression and tyrosine phosphorylation status are decreased in the nitrofen-induced CDH model. Timed-pregnant rats received either 100mg nitrofen or vehicle on gestation day 9 (D9). Fetuses were harvested on D18 and D21, and lungs were divided into two groups: control and hypoplastic lungs with CDH (CDH(+)) (n=10 at each time-point, respectively). Pulmonary gene expression levels of c-Cbl were analyzed by quantitative real-time polymerase chain reaction. Western blotting combined with densitometry analysis was used for semi-quantification of protein levels of pulmonary c-Cbl and tyrosine phosphorylation status. Confocal-immunofluorescence staining was performed to evaluate c-Cbl protein expression and distribution. Relative mRNA expression levels of pulmonary c-Cbl were significantly decreased in CDH(+) on D18 and D21 compared to controls. Western blotting showed markedly decreased protein levels of pulmonary c-Cbl and tyrosine phosphorylation status in CDH(+) on D18 and D21. Confocal-immunofluorescence analysis confirmed decreased c-Cbl expression in CDH(+) on D18 and D21 mainly in the distal alveolar epithelium compared to controls. Decreased pulmonary c-Cbl gene and protein expression accompanied by a decreased tyrosine phosphorylation status during the late stages of fetal lung development may result in reduced c-Cbl activity, and thus interfere with the FGFR-mediated alveolarization in the nitrofen-induced CDH model.

CD43 regulates the threshold for T cell activation by targeting Cbl functions

T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and coreceptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Coreceptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of T cells by regulating TCR signals. In this report, we show that TC prestimulation by the CD43 coreceptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ-dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced mitogen-activated protein kinase activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function.

A CSF-1 Receptor Phosphotyrosine 559 Signaling Pathway Regulates Receptor Ubiquitination and Tyrosine Phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.

Interaction of c-Cbl with Myosin IIA Regulates Bleb Associated Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus

KSHV is etiologically associated with Kaposi's sarcoma (KS), an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d) cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s) involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA), in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex containing c-Cbl and myosin IIA plays a crucial role in blebbing and macropinocytosis during viral infection and suggests that targeting c-Cbl could lead to a block in KSHV infection.

Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia

Notch3 and pTalpha signaling events are essential for T-cell leukemogenesis and characterize murine and human T-cell acute lymphoblastic leukemia. Genetic ablation of pTalpha expression in Notch3 transgenic mice abrogates tumor development, indicating that pTalpha signaling is crucial to the Notch3-mediated leukemogenesis. Here we report a novel direct interaction between Notch3 and pTalpha. This interaction leads to the recruitment and persistence of the E3 ligase protein c-Cbl to the lipid rafts in Notch3-IC transgenic thymocytes. Conversely, deletion of pTalpha in Notch3 transgenic mice leads to cytoplasmic retention of c-Cbl that targets Notch3 protein to the proteasomal-degradative pathway. It appears that protein kinase C theta (PKCtheta), by regulating tyrosine and serine phosphorylation of Cbl, is able to control its function. We report here that the increased Notch3-IC degradation correlates with higher levels of c-Cbl tyrosine phosphorylation in Notch3-IC/pTalpha(-/-) double-mutant thymocytes, which also display a decreased PKCtheta activity. Our data indicate that pTalpha/pre-T-cell receptor is able to regulate the different subcellular localization of c-Cbl and, by regulating PKCtheta activity, is also able to influence its ubiquitin ligase activity upon Notch3 protein.

Cbl negatively regulates JNK activation and cell death

Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apoptosis – nerve growth factor (NGF) deprivation and DNA damage – cellular levels of c-Cbl and Cbl-b fell well before onset of death. NGF deprivation also induced rapid loss of tyrosine phosphorylation (and most likely, activation) of c-Cbl. Targeting c-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuron death is by regulation of apoptotic JNK signaling. We demonstrate that Cbl proteins interact with the JNK pathway components MLK3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK activation and on cell death.

C-Cbl Regulates Neutrophil Integrin-dependent Respiratory Burst (135.67)

Abstract Signaling through integrin receptors is required for optimal reactive oxygen species (ROS) production by polymorphonuclear neutrophils (PMN). The E3 ubiquitin ligase and adapter protein C-Cbl has been implicated in integrin-mediated adhesion, and is a substrate for integrin-stimulated protein tyrosine kinases. We hypothesized that c-Cbl regulates integrin-driven PMN functions. We observed tyrosine phosphorylation of c-Cbl after beta-2 integrin cross-linking in the NB4 myeloid cell line. To address the requirement for c-Cbl in primary PMN signaling, we utilized c-Cbl deficient mice. We found that while c-Cbl is dispensable for PMN maturation, signaling through both immunoreceptors and integrins on PMNs is modulated by c-Cbl. Upon stimulation of c-Cbl-deficient bone marrow-derived PMNs with beta-2 integrin ligands (poly-RGD or fibrinogen plus TNF-alpha), we observed an 80% decrease in ROS generation compared to stimulated control cells. Phorbol ester-driven ROS generation is unaffected by c-Cbl deficiency. We next addressed the biochemical mechanism of c-Cbl action, investigating MAPK, PI3K, and Ca2+dependent pathways downstream of integrins. We observed no defect in either ERK or Akt phosphorylation after integrin engagement in c-Cbl deficient PMNs; furthermore, we observed that poly-RGD stimulated ROS production is unimpaired in PMNs lacking the PI3K p110gamma subunit. However, c-Cbl-null PMNs displayed decreased phospholipase C phosphorylation. Together, these data strongly suggest that c-Cbl selectively regulates Ca2+driven signaling downstream of beta2 integrins and upstream of ROS generation in PMNs. Supported by the Minnesota Medical Foundation

Activation of atypical protein kinase C ζ toward TC10 is regulated by high-fat diet and aerobic exercise in skeletal muscle

We determined whether sustained aerobic exercise reverses high-fat diet–induced impairments in the c-Cbl associated protein (CAP)/Casitas b-lineage lymphoma (c-Cbl) signaling cascade in rodent skeletal muscle. Sprague-Dawley rats were placed into either control (n = 16) or high-fat–fed (n = 32) diet groups for 4 weeks. During a subsequent 4-week experimental period, 16 high-fat–fed rats remained sedentary, 16 high-fat–fed rats completed 4 weeks of exercise training, and control animals were sedentary and remained on the control diet. After the intervention period, animals were subjected to hind limb perfusions in the presence (n = 8 per group) or absence (n = 8 per group) of insulin. In the plasma membrane fractions, neither high-fat feeding nor exercise training altered adaptor protein with PH and SH2 domains, (APS), c-Cbl, or TC10 protein concentrations. In contrast, CAP protein concentration and insulin-stimulated plasma membrane c-Cbl tyrosine phosphorylation were reduced by high-fat feeding; but exercise training reversed these impairments. Of note was that insulin-stimulated atypical protein kinase C ζ kinase activity toward TC10 was reduced by high-fat feeding but normalized by exercise training. We conclude that sustained (4 weeks) exercise training can reverse high-fat diet–induced impairments on the CAP/c-Cbl pathway in high-fat–fed rodent skeletal muscle. We also provide the first evidence that the CAP/c-Cbl insulin signaling cascade in skeletal muscle may directly interact with components of the classic (phosphoinositide 3-kinase dependent) insulin signaling cascade.

Suppression of c-Cbl Tyrosine Phosphorylation Inhibits Neointimal Formation in Balloon-Injured Rat Arteries

c-Cbl, a ubiquitously expressed protooncogene, is tyrosine phosphorylated in response to a variety of stimuli, including growth factors such as platelet-derived growth factor (PDGF), and consequently activates signaling proteins such as phosphatidylinositol-3 kinase (PI3K) and Akt. In the present study, we examined the role of c-Cbl tyrosine phosphorylation in vascular injury. Western blotting showed that the tyrosine phosphorylation of c-Cbl was increased in balloon-injured rat carotid arteries and in cultured smooth muscle cells on stimulation by PDGF-BB, followed by the activations of Akt and the mammalian target of rapamycin. Adenovirus-mediated overexpression of a c-Cbl mutant that ablates the major tyrosine phosphorylation sites attenuated the Akt and the mammalian target of rapamycin activation and decreased the proliferation and migration of smooth muscle cells in response to PDGF-BB or fibroblast growth factor. These effects could be reversed by constitutively active PI3K or Akt, suggesting that c-Cbl phosphorylation promotes the PDGF-BB-induced proliferation and migration of smooth muscle cells through the PI3K/Akt pathways. In addition, overexpression of c-Cbl-m increased the ubiquitination of the PDGF and fibroblast growth factor receptors. Importantly, in balloon-injured rat carotid arteries, local delivery of c-Cbl-m reduced the phosphorylation of Akt and the mammalian target of rapamycin, inhibited the migration and proliferation of smooth muscle cells, and prevented neointimal hyperplasia. Our results demonstrate a novel role of c-Cbl in vascular remodeling after injury and suggest that modulation of c-Cbl tyrosine phosphorylation may be a therapeutic approach to treat vascular neointimal hyperplasia such as restenosis after angioplasty.

Lyn regulates BCR-ABL and Gab2 tyrosine phosphorylation and c-Cbl protein stability in imatinib-resistant chronic myelogenous leukemia cells

AbstractLyn kinase functions as a regulator of imatinib sensitivity in chronic myelogenous leukemia (CML) cells through an unknown mechanism. In patients who fail imatinib therapy but have no detectable BCR-ABL kinase mutation, we detected persistently activated Lyn kinase. In imatinib-resistant CML cells and patients, Lyn activation is BCR-ABL independent, it is complexed with the Gab2 and c-Cbl adapter/scaffold proteins, and it mediates persistent Gab2 and BCR-ABL tyrosine phosphorylation in the presence or absence of imatinib. Lyn silencing or inhibition is necessary to suppress Gab2 and BCR-ABL phosphorylation and to recover imatinib activity. Lyn also negatively regulates c-Cbl stability, whereas c-Cbl tyrosine phosphorylation is mediated by BCR-ABL. These results suggest that Lyn exists as a component of the BCR-ABL signaling complex and, in cells with high Lyn expression or activation, BCR-ABL kinase inhibition alone (imatinib) is not sufficient to fully disengage BCR-ABL–mediated signaling and suggests that BCR-ABL and Lyn kinase inhibition are needed to prevent or treat this form of imatinib resistance.

Structural Basis for UBA-mediated Dimerization of c-Cbl Ubiquitin Ligase

Ligand-induced down-regulation by the ubiquitin-protein ligases, c-Cbl and Cbl-b, controls signaling downstream from many receptor-tyrosine kinases (RTK). Cbl proteins bind to phosphotyrosine residues on activated RTKs to affect ligand-dependent ubiquitylation of these receptors targeting them for degradation in the lysosome. Both c-Cbl and Cbl-b contain a ubiquitin-associated (UBA) domain, which is important for Cbl dimerization and tyrosine phosphorylation; however, the mechanism of UBA-mediated dimerization and its requirement for Cbl biological activity is unclear. Here, we report the crystal structure of the UBA domain of c-Cbl refined to 2.1-A resolution. The structure reveals the protein is a symmetric dimer tightly packed along a large hydrophobic surface formed by helices 2 and 3. NMR chemical shift mapping reveals heterodimerization can occur with the related Cbl-b UBA domain via the same surface employed for homodimerization. Disruption of c-Cbl dimerization by site-directed mutagenesis impairs c-Cbl phosphorylation following activation of the Met/hepatocyte growth factor RTK and c-Cbl-dependent ubiquitination of Met. This provides direct evidence for a role of Cbl dimerization in terminating signaling following activation of RTKs.

Lyn Kinase Alters Gab2 Phosphorylation and c-Cbl Protein Levels To Regulate Imatinib Sensitivity and Survival of Chronic Myelogenous Leukemia Cells.

The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in some early phase patients while it is more common in advanced disease. Resistance has been generally associated with mutations in the BCR-ABL kinase that effect drug affinity. However patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. Our previous studies suggested a role for Lyn, a Src-related kinase, in imatinib resistance. K562 cells selected for imatinib resistance (K562R) overexpress Lyn kinase and its targeted silencing overcomes imatinib resistance and engages apoptosis. Overexpression of Lyn in K562 cells reduces imatinib sensitivity (3-fold) and patients that fail imatinib therapy in the absence of BCR-ABL mutations express a highly activated Lyn kinase that is not suppressed by imatinib. Silencing Lyn expression in patient specimens induces changes in cell survival that are proportional to the level of Lyn protein reduction. To understand the role of Lyn kinase in imatinib resistance and apoptosis we examined proteins associated with this kinase in imatinib resistant cell lines, leukemic cells overexpressing Lyn and specimens derived from imatinib resistant patients. Lyn overexpression blocked complete suppression of BCR-ABL tyrosine phosphorylation by imatinib and affected BCR-ABL signaling adaptors. Although BCR-ABL forms a stable complex with the leukemogenic-critical adaptor protein Gab2 in imatinib sensitive cells, Lyn overexpression resulted in the formation of Lyn:Gab2 complexed in resistant cells. BCR-ABL kinase inhibition failed to reduce tyrosine phosphorylation of Gab2 in these cells while Lyn silencing or kinase inhibition (with dasatinib) completely suppressed Gab2 tyrosine phosphorylation and correlated with the induction of apoptosis. Lyn silencing in K562R cells also lead to a reciprocal increase in the tyrosine phosphorylation and association with a protein of ~120kDa, identified as the E3 ligase, c-Cbl. Lyn overexpression in K562 cells reduced their imatinib sensitivity and reduced c-Cbl protein levels. Kinase inhibitor and co-transfection studies demonstrated that tyrosine phosphorylation of c-Cbl at a critical signaling site (Y774) is primarily controlled by BCR-ABL and deletion or mutation of the c-Cbl RING domain altered its BCR-ABL phosphorylation. These results suggest that c-Cbl complexes are regulated at both the protein and phosphorylation level by Lyn and BCR-ABL kinase activities, respectively. Overexpression and/or activation of Lyn may disrupt the balance and regulation of critical regulators of leukemogenic signaling (Gab2) or protein trafficking and stability (c-Cbl), resulting in increased cell survival and reduced responsiveness to BCR-ABL kinase inhibition. We conclude that Lyn alters the level and function of critical signaling adaptor proteins in CML cells.

High-fat feeding effects on components of the CAP/Cbl signaling cascade in Sprague-Dawley rat skeletal muscle

The aim of this investigation was to determine whether the CAP (Cbl-associated protein)/Cbl signaling cascade is present and responsive to insulin in skeletal muscle and if high-fat feeding impairs insulin-stimulated activation of this signaling cascade. Sprague-Dawley rats were assigned to either control (n = 16) or high fat–fed (n = 16) dietary groups. After a 12-week dietary period, animals were subjected to hind limb perfusions in the presence (n = 8 per group) or absence (n = 8 per group) of insulin. High-fat feeding reduced rates of insulin-stimulated skeletal muscle phosphatidylinositol 3-kinase activity and 3- O-methylglucose transport. In plasma membrane fractions, neither the high-fat diet nor insulin altered the insulin receptor β subunit (IR- β), APS (adaptor protein containing PH and SH2 domains), c-Cbl, or TC10 protein concentration, but high-fat feeding did decrease CAP protein concentration. APS, c-Cbl, CAP, and TC10 messenger RNA were present in the skeletal muscle and reflected the protein concentration of experimental groups. Despite insulin-stimulated plasma membrane IR- β tyrosine phosphorylation being unaffected by high-fat feeding, c-Cbl tyrosine phosphorylation, the kinase activity of IR- β toward APS, and glucose transporter 4 protein concentration were all significantly reduced in insulin-stimulated plasma membrane prepared from the skeletal muscle of high fat–fed animals. These findings suggest that the CAP/Cbl signaling cascade is present in skeletal muscle, activated by insulin, and impaired by high-fat feeding.

Cross-talk between the Two Divergent Insulin Signaling Pathways Is Revealed by the Protein Kinase B (Akt)-mediated Phosphorylation of Adapter Protein APS on Serine 588

The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.

Requirement of Src-Family Tyrosine Kinases in Fat Accumulation

Src-family tyrosine kinases mediate many receptor signals to various biological responses. Here we investigate the requirement of Src-family tyrosine kinases in adipogenesis. The biochemical mechanism by which insulin induces adipogenesis, converting fibroblast cells to adipocytes, is not clear. We show that fibroblast cells deficient of three ubiquitously expressed Src-family members (Src, Yes, and Fyn), SYF cells, are refractory to hormonally induced fat accumulation. The defect is rescued by reintroduction of c-Src into SYF cells. Furthermore, Src-family tyrosine kinases are required in the early steps of insulin signaling; it is responsible for the tyrosine phosphorylation of adaptor protein c-Cbl. Deficiency of c-Cbl blocked adipogenesis. These genetic and biochemical data clearly demonstrate that Src-family tyrosine kinases serve as a critical signal relay, via phosphorylation of c-Cbl, for fat accumulation, and provide potential new strategies for treating obesity.

Nef enhances c-Cbl phosphorylation in HIV-infected CD4 + T lymphocytes

The multifunctional HIV-1 protein Nef possesses several motifs that interact with signaling molecules in infected T cells. In order to determine whether Nef influences T cell activation, cells were infected with Nef-positive and Nef-negative clones of HIV. CD28 expression and changes in tyrosine phosphorylation were monitored. We observed no Nef-dependent changes in CD28 expression or function. However, infection with Nef-positive virus led to changes in tyrosine phosphorylation. This Nef-induced phosphorylation was observed in unstimulated cells, and c-Cbl was identified as one of the proteins whose phosphorylation was upregulated by Nef. Furthermore, Lck is required for Nef-mediated c-Cbl tyrosine phosphorylation. These results suggest that Nef modifies T cell signaling in the absence of T cell receptor engagement and co-stimulation.

C-Cbl Mediates Ubiquitination, Degradation, and Down-regulation of Human Protease-activated Receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR(2)), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR(2) is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR(2) was mono-ubiquitinated. Immunofluorescence indicated that activated PAR(2) translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR(2) lacking intracellular lysine residues (PAR(2)Delta14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR(2) Delta14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR(2) stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR(2) at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR(2) ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR(2) was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR(2) to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR(2) to irrevocably terminate signaling by this and perhaps other GPCRs.