Identification and characterization of a diamine exporter in colon epithelial cells

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N(1)-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.     

Mitochondrial metabolism of valproic acid

The beta-oxidation of valproic acid (2-propylpentanoic acid), an anticonvulsant drug with hepatotoxic side effects, was studied with subcellular fractions of rat liver and with purified enzymes of beta-oxidation. 2-Propyl-2-pentenoyl-CoA, a presumed intermediate in the beta-oxidation of valproic acid, was chemically synthesized and used to demonstrate that enoyl-CoA hydratase or crotonase catalyzes its hydration to 3-hydroxy-2-propylpentanoyl-CoA. The latter compound was not acted upon by soluble L-3-hydroxyacyl-CoA dehydrogenases from mitochondria or peroxisomes but was dehydrogenated by an NAD(+)-dependent dehydrogenase associated with a mitochondrial membrane fraction. The product of the dehydrogenation, presumably 3-keto-2-propylpentanoyl-CoA, was further characterized by fast bombardment mass spectrometry. 3-Keto-2-propylpentanoyl-CoA was not cleaved thiolytically by 3-ketoacyl-CoA thiolase or a mitochondrial extract but was slowly degraded, most likely by hydrolysis. The availability of 2-propylpentanoyl-CoA (valproyl-CoA) and its beta-oxidation metabolites facilitated a study of valproate metabolism in coupled rat liver mitochondria. Mitochondrial metabolites identified by high-performance liquid chromatography were 2-propylpentanoyl-CoA, 3-keto-2-propylpentanoyl-CoA, 2-propyl-2-pentenoyl- CoA, and trace amounts of 3-hydroxy-2-propylpentanoyl-CoA. It is concluded that valproic acid enters mitochondria where it is converted to 2-propylpentanoyl-CoA, dehydrogenated to 2-propyl-2-pentenoyl-CoA by 2-methyl-branched chain acyl-CoA dehydrogenase, and hydrated by enoyl-CoA hydratase to 3-hydroxy-2-propylpentanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hybrid pyruvate dehydrogenase complexes reconstituted from components of the complexes from Escherichia coli and Azotobacter vinelandii

The pyruvate dehydrogenase complex of Escherichia coli was isolated in a simple three-step procedure. Its chain stoichiometry, determined by trinitrobenzoate modification was found to be 1.4 E1:1 E2:0.6 E3. It was reproducible within 10% from preparation to preparation. The E. coli complex was resolved by chromatography on activated thiol Sepharose. Reconstitution of activity yielded a stoichiometry of 1.0 E1:1 E2:0.5 E3. The optimum binding stoichiometry of E1E2 and E2E3 subcomplexes was determined by sedimentation experiments and found to be 2.0 E1:1 E2 and 2.5 E3:1 E2, respectively. Competition between E1 and E3 was observed in the binding experiments, but not in the kinetic experiments. Hybrid active complexes could be reconstituted from either an E1E2 subcomplex from Azotobacter vinelandii and the E3 component from E. coli or from E2E3 subcomplex from E. coli and the E1 component from A. vinelandii. Low activity and weak binding was observed when E1 from E. coli was recombined with an E2E3 subcomplex from A. vinelandii or when E3 from A. vinelandii was recombined with an E1E2 subcomplex from E. coli. The association behaviour and stoichiometry of the reconstituted complexes is determined by the nature of the E2 component. The formation of hybrid complexes indicates a considerable structural similarity between the complexes from both sources, despite the differences in size and stoichiometry.     

GTP-sensitivity of the energy-dependent Ca2+ storage pool in permeabilized pancreatic acinar cells

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 3.3 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Part of this energy-dependent pool could be released by GTP in a polyethylene glycol-dependent manner. The kinetics of GTP-induced release of Ca2+ showed a biphasic pattern with an initial rapid phase followed by a sustained slower phase. In contrast, IP3-induced release of Ca2+ was completed within 30 s following addition of IP3. No reuptake of Ca2+ was observed following GTP- or IP3-induced release of Ca2+. The GTP effect was independent of IP3 and not inhibited by Ca2+, indicating that the IP3-operated Ca2+ channel is not involved in GTP-induced release of Ca2+. The size of the IP3-releasable pool was not affected by GTP, indicating that GTP, when added to permeabilized acinar cells, does not promote the coupling between IP3-insensitive and IP3-sensitive Ca2+ accumulating organelles. Thus, in permeabilized acinar cells, GTP and IP3 act on different Ca2+ sequestering pools. Interestingly, however, comparison of the size of the GTP-releasable pool with that of the IP3-releasable pool for the cell preparations used in the present study, revealed an inversed relationship, indicating that at the time of permeabilization the GTP-releasable pool can be coupled to a greater or lesser extent to the IP3-releasable pool. This suggests that, in the intact cell, a GTP-dependent mechanism may exist that controls the size of the IP3-releasable pool by coupling IP3-insensitive to IP3-sensitive organelles. Moreover, this suggests that the extent of coupling is preserved during permeabilization.     

Ca(2+)-induced Ca2+ release amplifies the Ca2+ response elicited by inositol trisphosphate in macrophages.

We have studied the rise in intracellular calcium concentration ([Ca2+]i) elicited in macrophages stimulated by platelet-activating factor (PAF) by using fura-2 measurements in individual cells. The [Ca2+]i increase begins with a massive and rapid release of Ca2+ from intracellular stores. We have examined the mechanism of this Ca2+ release, which has been generally assumed to be triggered by inositol trisphosphate (IP3). First, we confirmed that IP3 plays an important role in the initiation of the PAF-induced [Ca2+]i rise. The arguments are 1) an increase in IP3 concentration is observed after PAF stimulation; 2) injection of IP3 mimics the response to PAF; and 3) after introduction of heparin in the cell with a patch-clamp electrode, the PAF response is abolished. Second, we investigated the possibility of an involvement of Ca(2+)-induced Ca2+ release (CICR) in the development of the Ca2+ response. Ionomycin was found to elicit a massive Ca2+ response that was inhibited by ruthenium red or octanol and potentiated by caffeine. The PAF response was also inhibited by ruthenium red or octanol and potentiated by caffeine, suggesting that CICR plays a physiological role in these cells. Because our results indicate that in this preparation IP3 production is not sensitive to [Ca2+]i, CICR appears as a primary mechanism of positive feedback in the Ca2+ response. Taken together, the results suggest that the response to PAF involves an IP3-induced [Ca2+]i rise followed by CICR.     

Inositol 1,4,5-trisphosphate and the endoplasmic reticulum Ca2+ cycle of a rat insulinoma cell line

Regulation of endoplasmic reticulum (ER) Ca2+ cycling by inositol 1,4,5-trisphosphate (IP3) was studied in saponin-permeabilized RINm5F insulinoma cells. Cells were incubated with mitochondrial inhibitors, and medium Ca2+ concentration established by nonmitochondrial pool(s) (presumably the ER) was monitored with a Ca2+ electrode. IP3 degradation accounted for the transience of the Ca2+ response induced by pulse additions of the molecule. To compensate for degradation, IP3 was infused into the medium. This resulted in elevation of [Ca2+] from about 0.2 microM to a new steady state between 0.3 and 1.0 microM, depending on both the rate of IP3 infusion and the ER Ca2+ content. The elevated steady state represented a bidirectional buffering of [Ca2+] by the ER, as slight displacements in [Ca2+], by small aliquots of Ca2+ or the Ca2+ chelator quin 2, resulted in net uptake or efflux of Ca2+ to restore the previous steady state. When IP3 infusion was stopped, [Ca2+] returned to its original low level. Ninety per cent of the Ca2+ accumulated by the ER was released by IP3 when the total Ca2+ content did not exceed 15 nmol/mg of cell protein. Above this high Ca2+ content, Ca2+ was accumulated in an IP3-insensitive, A23187-releasable pool. The maximal amount of Ca2+ that could be released from the ER by IP3 was 13 nmol/mg of cell protein. The data support the concept that in the physiological range of Ca2+ contents, almost all the ER is an IP3-sensitive Ca2+ store that is capable of finely regulating [Ca2+] through independent influx (Ca2+-ATPase) and efflux (IP3-modulated component) pathways of Ca2+ transport. IP3 may continuously modulate Ca2+ cycling across the ER and play an important role in determining the ER Ca2+ content and in regulating cytosolic Ca2+ under both stimulated and possibly basal conditions.     

Purification and characterization of active component and active fragment of colicin E3.

1. Two components of colicin E3, namely proteins A and B, were prepared by means of an improved method. 2. Protein A thus obtained was more than a thousand times as active as native colicin E3 when they were assayed in terms of activity for ribosome inactivation. 3. Protein A was reconstituted to colicin E3 simply by mixing with protein B. 4. Trypsin digestion of colicin E3 yielded two fragments, T1 and T2, probably by cleaving one specific bond of the A moiety of colicin E3. 5. T2 was a complex of T2A and B proteins. T2A showed an activity equivalent to that of protein A when assayed in the in vitro system, and its activity was neutralized by protein B. Thus T2A was assigned as an active fragment of protein A. 6. T2A has a characteristic amino acid composition rich in the basic amino acid, lysine. 7. The structure and function of the colicin E3 molecule is discussed based on the results obtained with its components as well as with fragments of the components.     

The cholecystokinin-induced Ca2+ shuttle from the inositol trisphosphate-sensitive and ATP-dependent pool, and initial pepsinogen release connected with cytoskeleton of the chief cell

In guinea pig chief cells, inositol 1,4,5-trisphosphate (IP3) caused release of Ca2+, which was accumulated by ATP, from an endoplasmic reticulum-enriched fraction in both the permeable system and the cell-free system. This was mimicked with the Ca2+ ionophores A23187 and ionomycin on a large scale since an IP3-sensitive Ca2+ pool might be a subset of the Ca2+ ionophore-sensitive Ca2+ pool. The permeable chief cells, but not the cell-free system, retained the ability to react to synthetic cholecystokinin octapeptide (CCK-OP) with Ca2+ release from an IP3-sensitive pool due to of the non-additive but constant effect in exerting Ca2+ release from the store(s) induced by the combination with IP3 and CCK-OP. The increase in the cytosolic free Ca2+ concentration of intact chief cells responding to CCK-OP or the Ca2+ ionophore, ionomycin, comprised two components, namely, that by the Ca2+ entry from the extracellular space, and that by the Ca2+ release from the intracellular space(s) (as measured by fura-2). When CCK-OP or ionomycin was added, there was a biphasic response of pepsinogen secretion. An initial but transient response reaching a peak in 5 min was followed by a sustained response reaching a peak in 30 min. The initial pepsinogen release was independent of medium Ca2+, whereas the sustained one was dependent on medium Ca2+. The results suggest that the intracellular Ca2+ release from the store(s), presumably endoplasmic reticulum, may trigger the initial pepsinogen release, whereas the sustained pepsinogen secretion may be caused by acting in concert with the initial response and external Ca2+ entry. On the other hand, the disruption of the microtubular-microfilamentous system by colchicine or cytochalasin D failed to cause the Ca2+ release evoked by either IP3, CCK-OP or Ca2+ ionophores and to cause the CCK-OP- or ionomycin-induced initial pepsinogen release. These findings suggest that the IP3-sensitive pool is the same Ca2+ store which is completely or partially sensitive to CCK-OP and Ca2+ ionophores, respectively, and that the assembly of the cytoskeletal system is involved in initial intracellular Ca2+ metabolism and the following initial pepsinogen release. The assembly of the cytoskeletal system may be an early event in mediating the CCK-OP-induced initial pepsinogen release, perhaps by causing the Ca2+ release from an IP3-sensitive pool of the chief cell. The translocation or attachment of the IP3-sensitive pool brought about by cytoskeletal system might be necessary to cause Ca2+ release after the cell stimulation with CCK-OP.     

Recombinant complement receptor 2 radiolabeled with [99mTc(CO)3]+: a potential new radiopharmaceutical for imaging activated complement

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [(99m)Tc(CO)(3)(OH(2))(3)](+)). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d(+) red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d(+), in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a K(D)≈500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [(99m)Tc(CO)(3)(OH(2))(3)](+) at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d(+) RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d(+) RBCs. We conclude that rCR2-Tc(99m) has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent.     

Interleukin-2-collagen chimeric protein which liberates interleukin-2 upon collagenolysis

Interleukin-2 (IL-2) is a potent activator of cellular immunity and has been utilized as an immunotherapeutic agent. We stably immobilized human IL-2 to collagen by covalently binding it to the N-terminus of human type III collagen (3A1) as IL2-3A1 chimeric protein using recombinant technology. The present study was aimed at liberating IL-2 from the immobilized chimeric protein by treating the chimera with bacterial collagenase. These IL2-3A1 chimeras were synthesized in insect cells which had been infected with baculovirus vectors carrying IL2-3A1 cDNA. The IL2-3A1 protein produced was shown to be in a pepsin-resistant triple helical structure and exhibited IL-2 activity to a similar extent as IL-2 itself. IL2-3A1 could be immobilized on the surface of plastic dishes by incubating it in the dishes. The IL-2 region of the immobilized IL2-3A1 was liberated to culture media by collagenase treatment and this freed IL-2 stimulated the growth of lined T cells. Thus, IL2-3A1 chimeric protein could be utilized as an IL-2 deliverer whose T cell mitogenic activity can be liberated by a collagenolytic environment.     

Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic beta-cells.

Pancreatic beta-cells isolated from obese-hyperglycaemic mice released intracellular Ca2+ in response to carbamoylcholine, an effect dependent on the presence of glucose. The effective Ca2+ concentration reached was sufficient to evoke a transient release of insulin. When the cells were deficient in Ca2+, the Ca2+ pool sensitive to carbamoylcholine stimulation was equivalent to that released by ionomycin. Unlike intact cells, cells permeabilized by high-voltage discharges failed to generate either inositol 1,4,5-triphosphate (InsP3) or to release Ca2+ after exposure to carbamoylcholine. However, the permeabilized cells released insulin sigmoidally in response to increasing concentrations of Ca2+. Also in the absence of functional mitochondria these cells exhibited a large ATP-dependent buffering of Ca2+, enabling the maintenance of an ambient Ca2+ concentration corresponding to about 150 nM even after several additional pulses of Ca2+. InsP3, maximally effective at 6 microM, promoted a rapid and pronounced release of Ca2+. The InsP3-sensitive Ca2+ pool was rapidly filled and lost its Ca2+ late after ATP depletion. The transient nature of the Ca2+ signal was not overcome by repetitive additions of InsP3. It was possible to restore the response to InsP3 after a delay of approx. 20 min, an effect which had less latency after the addition of Ca2+. These latter findings argue against degradation and/or desensitization as factors responsible for the transiency in InsP3 response. It is suggested that Ca2+ released by InsP3 is taken up by a part of the endoplasmic reticulum (ER) not sensitive to InsP3. On metabolism of InsP3, Ca2+ recycles to the InsP3-sensitive pool, implying that this pool indeed has a very high affinity for the ion. The presence of functional mitochondria did not interfere with the recycling process. The ER in pancreatic beta-cells is of major importance in buffering Ca2+, but InsP3 only modulates Ca2+ transport for a restricted period of time following immediately upon its formation. Thereafter the non-sensitive part of the ER takes over the continuous regulation of Ca2+ cycling.     

Immunoaffinity Purification of Epitope-Tagged Human β2-Adrenergic Receptor to Homogeneity

To obtain large quantities of pure human β₂-adrenergic receptor (β₂-AR) needed for structural studies, an efficient method for β₂-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged β₂-AR was expressed in Sf9 cells with a specific activity of 5–20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-β-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60–70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of >30%. The purified receptor was concentrated to >1 mg/ml without loss of ligand binding activity.     

Analysis of calcium responses mediated by the A3 adenosine receptor in cultured newborn rat cardiac myocytes

Intracellular calcium signaling cascade induced by adenosine A(3) receptor activation was studied in this work. It was found that adenosine A(3) receptor activation (and not A(1) or A(2A) adenosine receptors activation) leads to an increase in cytosolic calcium and its further extrusion. A selective A(3) agonist Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) induced an increase in cytoplasmic calcium in a dose-dependent manner, and was independent on extracellular calcium. The Ca(2+) signal in newborn cardiomyocytes, induced by A(3) receptor activation, is dependent on a pertussis toxin-sensitive G-protein. The action of Cl-IB-MECA was not inhibited by an inhibitor of phospholipase C (PLC), and by antagonists to inositol 1,4,5-trisphosphate (IP(3)) receptor. In contrast, inhibition of ryanodine receptor prevented calcium elevation induced by this agonist. It was shown that extrusion of the elevated cytosolic Ca(2+) was achieved via activation of sarcoplasmic reticulum (SR) Ca(2+)-reuptake and of sarcolemmal Na(+)/Ca(2+) exchanger (NCX). The increase in the SR Ca(2+)-uptake and NCX Ca(2+) efflux were sufficient not only for compensation of Ca(2+) release from SR after A(3) receptor activation, but also for an effective prevention of extensive increase in intracellular Ca(2+) and may provide mechanism against cellular Ca(2+) overload. In cells with elevated [Ca(2+)](i) (due to increase of [Ca(2+)](o)), adenosine or Cl-IB-MECA decreased the [Ca(2+)](i) toward diastolic control level, whereas agonist of A(1) receptor was ineffective. The protective effect of A(3) receptor agonist was abolished in the presence of selective A(3) receptor antagonist MRS1523.     

Mobilization of intracellular calcium by endothelin in Swiss 3T3 cells

When intracellular free Ca2+ concentration [( Ca2+]i) was monitored in fura2-loaded Swiss 3T3 cells, endothelin increased [Ca2+]i in a dose-dependent manner; after the addition of endothelin, an initial transient peak was observed immediately and was followed by a sustained increase in [Ca2+]i lasting at least 5 min. 45Ca2+ efflux and influx experiments in endothelin-stimulated Swiss 3T3 cells revealed that the change in [Ca2+]i could be explained by a dual mechanism; an initial transient peak induced mainly by the release of Ca2+ from intracellular stores and the sustained increase by an influx of extracellular Ca2+. Cellular generation of inositol 1,4,5-trisphosphate and cyclic AMP were not induced by endothelin, suggesting that other cellular mediators with the capacity to release Ca2+ from intracellular stores play a significant role in the signal transduction pathway of endothelin in Swiss 3T3 cells.     

Convenient synthesis of a propargylated cyclic (3'-5') diguanylic acid and its "click" conjugation to a biotinylated azide

The ribonucleoside building block, N2-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine, was prepared from commercial N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-propargyl guanosine in a yield of 91%. The propargylated guanylyl(3'-5')guanosine phosphotriester was synthesized from the reaction of N2-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine with N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl] guanosine and isolated in a yield of 88% after P(III) oxidation, 3'-/5'-deprotection, and purification. The propargylated guanylyl(3'-5')guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester, which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups from the propargylated c-di-GMP triester were performed by treatment with aqueous ammonia at ambient temperature. The final 2'-desilylation reaction was effected by exposure to triethylammonium trihydrofluoride affording the desired propargylated c-di-GMP diester, the purity of which exceeded 95%. Biotinylation of the propargylated c-di-GMP diester was easily accomplished through its cycloaddition reaction with a biotinylated azide derivative under click conditions to produce the biotinylated c-di-GMP conjugate of interest in an isolated yield of 62%.     

Enzymatic synthesis of unlabeled and beta-(32)P-labeled beta-L-2', 3'-dideoxyadenosine-5'-triphosphate as a potent inhibitor of adenylyl cyclases and its use as reversible binding ligand

beta-L-2',3'-Dideoxyadenosine-5'-triphosphate (beta-L-2', 3'-dd-5'-ATP) was prepared enzymatically from the corresponding monophosphate by the use of adenylate kinase, creatine phosphate, and creatine kinase in a single step. The beta-(32)P-labeled analog was prepared similarly, but in a two step reaction. beta-L-2', 3'-dd-5'-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5'-ATP.Mn(2+)) and exhibited an IC(50) approximately 24 nM. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of beta-L-2',3'-dd-[beta-(32)P]5'-ATP was saturable with increasing concentrations of ligand and increased in proportion to membrane protein, and was enhanced by Mn(2+) to a greater extent than by Mg(2+). Binding was displaced with adenine nucleotides known to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatine phosphate, or creatine kinase. Binding was rapid, with a half-time for the on-rate <1.8 min and for the off-rate <0.8 min. The potency and mechanism of the inhibition of this ligand and the pattern of agents that displace binding suggest an interaction with adenylyl cyclase per se and to a configuration of the enzyme consistent with an interaction at the catalytic active site. The data suggest that this is a pretransition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state noncompetitive inhibitor.     

The role of the L3T4 molecule in mitogen and antigen-activated signal transduction

We investigated the role of the L3T4 molecule in mitogen and antigen-initiated signal transduction in the L3T4(+) murine T cell hybridoma, 3DT52.5.9 and an L3T4(-) variant, 3DT52.5.24. Both Concanavalin A (Con A) and specific antigen stimulated increases in cytosolic-free calcium ([Ca2+]i), phosphatidylinositol turnover, and interleukin-2 (IL-2) production in both cell lines. About 85% of the stimulated rise in [Ca2+]i was from an extracellular source. Anti-L3T4 monoclonal antibody (MAb) inhibited 90% of antigen- and 50% of Con A-stimulated increases in [Ca2+]i and IL-2 production but had no effect on the ability of either activation signal to stimulate phosphatidylinositol turnover in the parent L3T4(+) cells. Stimulus-response coupling in the L3T4(-) cells was unaffected by the MAb. The anti-L3T4-insensitive increase in [Ca2+]i induced by Con A was inhibited by EGTA, suggesting that this mitogen also stimulated an influx of Ca2+ via an additional transport mechanism distinct from that stimulated by antigen. The fact that anti-L3T4 antibodies inhibit antigen and Con A-stimulated Ca2+ transport and IL-2 production without affecting phosphatidylinositol turnover suggests that L3T4 may play a critical role in modulating the activation of the T cell receptor-associated Ca2+ transporter in T cell stimulus-response coupling.     

Notch3 Interactome Analysis Identified WWP2 as a Negative Regulator of Notch3 Signaling in Ovarian Cancer

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.     

Extracellular signal-regulated kinases in T cells. Anti-CD3 and 4 beta-phorbol 12-myristate 13-acetate-induced phosphorylation and activation.

Extracellular signal-regulated kinases (ERK) 1 and 2 are growth factor- and cytokine-sensitive serine/threonine kinases that are known to phosphorylate microtubule-associated protein 2 and myelin basic protein. The current studies examined whether ERK1 and/or ERK2 was present in T cells and whether they were phosphorylated and activated as a consequence of T cell activation. The data demonstrated that both ERK1 and ERK2 were present in Jurkat cells and peripheral blood T cells. In T cells, ERK2 was more prevalent than ERK1. The concentrations of ERK1 and ERK2 were not altered by stimulating the cells for 16 h with immobilized anti-CD3 mAb or anti-CD3 mAb and phorbol myristate acetate. mAb to CD3 and phorbol myristate acetate stimulated an increase in ERK1 and ERK2 MBP kinase activity. Anti-CD3 mAb triggered an increase their phosphate content which was detectable at 2 min but reached a maximum at 5 min. A portion of the increase in phosphate was caused by an increase in phosphotyrosine. We also examined the rate of ERK2 degradation. ERK2 was stable for up to 36 h, and its degradation was unaffected by the activation state of the cells. The data demonstrate that ERK1 and ERK2 are part of an anti-CD3 mAb-stimulated signal transduction cascade that is downstream of protein kinase C and, therefore, suggest that these kinases play an important role in T cell activation.