Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes

AbstractTyr phosphorylation of the multifunctional transmembrane protein band 3 has been implicated in several erythrocyte functions and disorders. We previously demonstrated that pervanadate treatment of human erythrocytes induces band-3 Tyr phosphorylation, which is catalyzed by the sequential action of tyrosine kinase Syk and tyrosine kinase(s) belonging to the Src family. In this study, we show that Tyr phosphorylation of band 3, elicited by pervanadate, N-ethylmaleimide, or diamide, greatly increases band-3 interaction with the tyrosine phosphatase SHP-2 in parallel with the translocation of SHP-2 to erythrocyte membranes. These events seem to be mediated by Src-like catalyzed phosphorylation of band 3 because both SHP-2 translocation to cellular membranes and its interaction with Tyr-phosphorylated protein are greatly counteracted by PP2, a specific inhibitor of Src kinases. Binding-competition experiments demonstrate that SHP-2 recruitment to band 3 occurs via its SH2 domain(s). In particular, our data support the view that SHP-2 docks specifically with P-Y359 of band 3. Experiments performed with intact erythrocytes in the presence of the SHP-2 inhibitor calpeptin suggest that, once recruited to Tyr-phosphorylated band 3, the tyrosine phosphatase dephosphorylates the protein. P-Y8, 21, and 904 are the residues affected by SHP-2, as judged by 32P-peptide mapping of band 3 digested with trypsin. These results indicate that in treated erythrocytes, recruitment of cytosolic SHP-2 to band 3 is a prerequisite for the subsequent dephosphorylation of the transmembrane protein

The effect of timing and composition of gestational weight gain in obese pregnant women on infant birth weight: A prospective cohort study.

Introduction: CK2 is a protein kinase implicated in several essential cellular processes, over-expressed in cancer and described to regulate insulin signaling cascade. Recently CK2 has been described to negatively regulate thermogenesis (Shinoda K et al, 2015, Cell Metabolism) and to inhibit insulin release (Rossi M et al, 2015, PNAS). Nevertheless, the role of CK2 in adipose tissue (AT) and its involvement in human obesity development and therapy has been poorly investigated. Methods: Our multi-disciplinary team performed biochemical analysis of signaling pathways by WB and in vitro kinase activity assays, and glucose handling studies using glucose uptake assay and IF in adipocyte cultures and glucose and insulin tolerance test in mice. Moreover we quantify CK2 expression/activity in human AT specimens of 27 obese patients, clinically characterized, in 12 obese patients underwent relevant weight loss and 11 normal-weight controls. Results: We proved that CK2 amount and activity were not influenced by insulin stimulation and that CK2 activity was efficiently inhibited by specific inhibitors, structurally unrelated. We worked with CX-4945, a CK2 inhibitor currently used in cancer clinical trials, using the minimal concentration (2.5 \u192 dM) and pre-treatment time (1hr) able to efficiently inhibit CK2 activity, avoiding any cytotoxic effect. Pharmacological inhibition of CK2 did not significantly affect in vitro adipogenic differentiation or expression profiling of mature adipocytes. Conversely, we showed that in human and murine adipocytes CK2-inhibition decreases the insulin-induced glucose uptake by counteracting Akt-signaling and GLUT4-translocation to the plasma membrane. We compared CK2 expression and activity in different mouse tissues highlighted that white skeletal muscle fibres and liver contained the highest quantity of this kinase. CK2 was expressed more in brown AT than in white AT depots. We show that CK2 promotes insulin-signaling in mouse AT, liver and skeletal muscle and that in vivo acute treatment with CX-4945 impairs glucose- tolerance in mice. Studies in tissues of ob/ob and db/db mice highlights an up-regulation of CK2 expression and activity only in WAT. CK2 hyper-activation is strongly evident also in SAT and VAT of obese patients and weight loss obtained by bariatric surgery or hypocaloric diet reverts CK2 up-regulation to normal level. Conclusion: We show that CK2 is involved in insulin sensitivity, glucose handling and remodeling of WAT. Moreover we identify CK2 hyper-activation as a hallmark of human obesity, suggesting a new potential therapeutic target for metabolic diseases

CK2 modulates adipocyte insulin-signaling and is up-regulated in human obesity

Insulin plays a major role in glucose metabolism and insulin-signaling defects are present in obesity and diabetes. CK2 is a pleiotropic protein kinase implicated in fundamental cellular pathways and abnormally elevated in tumors. Here we report that in human and murine adipocytes CK2-inhibition decreases the insulin-induced glucose-uptake by counteracting Akt-signaling and GLUT4-translocation to the plasma membrane. In mice CK2 acts on insulin-signaling in adipose tissue, liver and skeletal muscle and its acute inhibition impairs glucose tolerance. Notably, CK2 protein-level and activity are greatly up-regulated in white adipose tissue from ob/ob and db/db mice as well as from obese patients, regardless the severity of their insulin-resistance and the presence of pre-diabetes or overt type 2 diabetes. Weight loss obtained by both bariatric surgery or hypocaloric diet reverts CK2 hyper-activation to normal level. Our data suggest a central role of CK2 in insulin-sensitivity, glucose homeostasis and adipose tissue remodeling. CK2 up-regulation is identified as a hallmark of adipose tissue pathological expansion, suggesting a new potential therapeutic target for human obesity

Characterization of compound 584, an Abl kinase inhibitor with lasting effects

Background: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosomepositive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). Design and Methods: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotransplanted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. Results: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC50: 8 nM) and in cell-based assays (IC50: 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC50: 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. Conclusions: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former. ©2008 Ferrata Storti Foundation

Molecular targets for the protodynamic action of cis-urocanic acid in human bladder carcinoma cells

<p>Abstract</p> <p>Background</p> <p>cis-urocanic acid (cis-UCA) is an endogenous amino acid metabolite capable of transporting protons from the mildly acidic extracellular medium into the cell cytosol. The resulting intracellular acidification suppresses many cellular activities. The current study was aimed at characterizing the molecular mechanisms underlying cis-UCA-mediated cytotoxicity in cultured cancer cells.</p> <p>Methods</p> <p>5367 bladder carcinoma cells were left untreated or treated with cis-UCA. Cell death was assessed by measuring caspase-3 activity, mitochondrial membrane polarization, formation and release of cytoplasmic histone-associated DNA fragments, and cellular permeabilization. Cell viability and metabolic activity were monitored by colorimetric assays. Nuclear labelling was used to quantify the effects of cis-UCA on cell cycle. The activity of the ERK and JNK signalling pathways was studied by immunoblotting with specific antibodies. Phosphatase activity in cis-UCA-treated cells was determined by assay kits measuring absorbance resulting from the dephosphorylation of an artificial substrate. All statistical analyses were performed using the two-way Student's t-test (p < 0.05).</p> <p>Results</p> <p>Here we report that treatment of the 5637 human bladder carcinoma cells with 2% cis-UCA induces both apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells is rapidly impaired, and the cells arrest in cell cycle in response to cis-UCA. Importantly, we show that cis-UCA promotes the ERK and JNK signalling pathways by efficiently inhibiting the activity of serine/threonine and tyrosine phosphatases.</p> <p>Conclusions</p> <p>Our studies elucidate how cis-UCA modulates several cellular processes, thereby inhibiting the proliferation and survival of bladder carcinoma cells. These anti-cancer effects make cis-UCA a potential candidate for the treatment of non-muscle invasive bladder carcinoma.</p

The Legionella effector WipB is a translocated Ser/Thr phosphatase that targets the host lysosomal nutrient sensing machinery

Legionella pneumophila infects human alveolar macrophages and is responsible for Legionnaire’s disease, a severe form of pneumonia. L. pneumophila encodes more than 300 putative effectors, which are translocated into the host cell via the Dot/Icm type IV secretion system. These effectors highjack the host’s cellular processes to allow bacterial intracellular growth and replication. Here we adopted a multidisciplinary approach to investigate WipB, a Dot/Icm effector of unknown function. The crystal structure of the N-terminal domain at 1.7 Å resolution comprising residues 25 to 344 revealed that WipB harbours a Ser/Thr phosphatase domain related to the eukaryotic phospho-protein phosphatase (PPP) family. The C-terminal domain (residues 365–524) is sufficient to pilot the effector to acidified LAMP1-positive lysosomal compartments, where WipB interacts with the v-ATPase and the associated LAMTOR1 phosphoprotein, key components of the lysosomal nutrient sensing (LYNUS) apparatus that controls the mammalian target of rapamycin (mTORC1) kinase complex at the lysosomal surface. We propose that WipB is a lysosome-targeted phosphatase that modulates cellular nutrient sensing and the control of energy metabolism during Legionella infection

Switches, Excitable Responses and Oscillations in the Ring1B/Bmi1 Ubiquitination System

In an active, self-ubiquitinated state, the Ring1B ligase monoubiquitinates histone H2A playing a critical role in Polycomb-mediated gene silencing. Following ubiquitination by external ligases, Ring1B is targeted for proteosomal degradation. Using biochemical data and computational modeling, we show that the Ring1B ligase can exhibit abrupt switches, overshoot transitions and self-perpetuating oscillations between its distinct ubiquitination and activity states. These different Ring1B states display canonical or multiply branched, atypical polyubiquitin chains and involve association with the Polycomb-group protein Bmi1. Bistable switches and oscillations may lead to all-or-none histone H2A monoubiquitination rates and result in discrete periods of gene (in)activity. Switches, overshoots and oscillations in Ring1B catalytic activity and proteosomal degradation are controlled by the abundances of Bmi1 and Ring1B, and the activities and abundances of external ligases and deubiquitinases, such as E6-AP and USP7

Regulation of serotonin transport in human platelets by tyrosine kinase Syk

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the regulation of numerous neuro-physiological processes. The circulating level of 5-HT is regulated by the membrane transporter SERT present both in the presynaptic nerve terminals and blood platelets. 5-HT transport is a process tightly regulated by a variety of factors including protein phosphorylation. Aim of this study was to ascertain if also the SERT Tyr-phosphorylation mediated by Syk-kinase concurs to the regulation of SERT activity. Indeed we found that 5-HT uptake decreased upon platelet exposure to piceatannol or Syk-inhibitor II, two structurally unrelated inhibitors of the tyrosine-kinase Syk. Tyr-phosphorylation of anti-SERT-immuno-stained proteins in membrane extracts and in anti-SERT-immuno-precipitates, decreased upon platelet treatment with piceatannol, in parallel with a reduction of Syk-activity. Syk was immuno-revealed in the anti-SERT immuno-precipitates, which displayed a piceatannol-sensitive kinase activity towards SERT itself and the Syk-substrate \u3b1-sinuclein. Syk inhibitors also caused a decrease of the monensin-induced 5-HT-efflux from platelets and of imipramine binding to them. It is concluded that, in addition to the phosphorylation of SERT mediated by various other kinases, also that catalyzed by Syk might play an important role in the 5-HT transport, likely favoring the transporter conformation exposing the neurotransmitter binding sites