Purification and G Protein Subunit Regulation of a Phospholipase C-β from Xenopus laevis Oocytes

Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits Galphai, Galphao, Galphaq, Galphas, and Gbetagamma all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-betaX) that exhibits homology to the PLC-beta class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin-sensitive inositol lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-betaX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-betaX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-betaX was activated by both Galphaq and Gbetagamma. PLC-betaX exhibited a higher apparent affinity for Galphaq than Gbetagamma, and Galphaq was more efficacious than Gbetagamma at lower concentrations of PLC-betaX. Relative to other PLC-beta isoenzymes, PLC-betaX was less sensitive to stimulation by Galphaq than PLC-beta1 but similar to PLC-beta2 and PLC-betaT. PLC-betaX was more sensitive to stimulation by Gbetagamma than PLC-beta1 but less sensitive than PLC-beta2 and PLC-betaT. In contrast PLC-betaX was not activated by the pertussis toxin substrate G proteins Galphai1, Galphai2, Galphai3, or Galphao. These results are consistent with the idea that PLC-betaX is regulated by alpha-subunits of the Gq family and by Gbetagamma and do not support the idea that alpha-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein

Protein Kinase C-Promoted Inhibition of G␣ 11 -Stimulated Phospholipase C-␤ Activity

ABSTRACT The effects of protein kinase C (PKC) activation on inositol lipid signaling were examined. Using the turkey erythrocyte model of receptor-regulated phosphoinositide hydrolysis, we developed a membrane reconstitution assay to study directly the effects of activation of PKC on the activities of G␣ 11 , independent of potential effects on the receptor or on PLC-␤. Membranes isolated from erythrocytes pretreated with 4␤-phorbol-12␤-myristate-13␣-acetate (PMA) exhibited a decreased capacity for G␣ 11 -mediated activation of purified, reconstituted PLC-␤1. This inhibitory effect was dependent on both the time and concentration of PMA incubation and occurred as a decrease in the efficacy of GTP␥S for activation of PLC-␤1, both in the presence and absence of agonist; no change in the apparent affinity for the guanine nucleotide occurred. Similar inhibitory effects were observed after treatment with the PKC activator phorbol-12,13-dibutyrate but not after treatment with an inactive phorbol ester. The inhibitory effects of PMA were prevented by coaddition of the PKC inhibitor bisindolylmaleimide. Although the effects of PKC could be localized to the membrane, no phosphorylation of G␣ 11 occurred either in vitro in the presence of purified PKC or in intact erythrocytes after PMA treatment. These results support the hypothesis that a signaling protein other than G␣ 11 is the target for PKC and that PKCpromoted phosphorylation of this protein results in a phosphorylation-dependent suppression of G␣ 11 -mediated PLC-␤1 activation. Various receptors transduce signals through heterotrimeric G proteins of the G q family, resulting in activation of phospholipase C (PLC)-␤ isoenzymes and subsequent cleavage of membrane phosphatidylinositol(4,5)P 2 [PtdIns(4,5)P 2 ] to the second messengers inositol(1,4,5)P 3 [Ins(1,4,5)P 3 ] and diacylglycerol Agonist-induced desensitization is an important regulatory process in inositol lipid signaling The turkey erythrocyte is a well-characterized model of receptor-promoted inositol phospholipid signaling and is particularly useful because the three primary proteins in the pathway, i.e., the turkey P2Y 1 recepto

Regulation of transcription factor activity by interconnected, post-translational modifications

Transcription factors comprise just over 7% of the human proteome and serve as gatekeepers of cellular function, integrating external signal information into gene expression programs that reconfigure cellular physiology at the most basic levels. Surface-initiated cell signaling pathways converge on transcription factors, decorating these proteins with an array of post-translational modifications (PTMs) that are often interdependent, being linked in time, space, and combinatorial function. These PTMs orchestrate every activity of a transcription factor over its entire lifespan—from subcellular localization to protein–protein interactions, sequence-specific DNA binding, transcriptional regulatory activity, and protein stability—and play key roles in the epigenetic regulation of gene expression. The multitude of PTMs of transcription factors also offers numerous potential points of intervention for development of therapeutic agents to treat a wide spectrum of diseases. We review PTMs most commonly targeting transcription factors, focusing on recent reports of sequential and linked PTMs of individual factors

A Guanine Nucleotide-independent Inwardly Rectifying Cation Permeability Is Associated with P2Y 1 Receptor Expression in Xenopus Oocytes

The functional properties of the G protein-coupled P2Y1 receptor were investigated in Xenopus oocytes. Incubation of oocytes expressing either the human or turkey P2Y1 receptor with adenine nucleotide agonists resulted in an increase in Cl- current and activation of a novel cation current with an inwardly rectifying current-voltage relationship. Activation of either the human P2Y2 (P2U-purinergic) or M1 muscarinic receptor expressed in oocytes resulted in an increase in Cl- current similar to that observed in P2Y1 receptor-expressing oocytes but had no effect on cation current. P2 receptor agonists stimulated both the cation current and Cl- current in P2Y1 receptor-expressing oocytes with EC50 values and an order of potency (2-methylthioadenosine diphosphate > 2-methylthioadenosine triphosphate (2MeSATP) > ATP > UTP) that were similar to those previously observed for activation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human or turkey P2Y1 receptor. The P2Y receptor antagonists suramin and pyridoxal phosphate 6-azophenyl-2'-4'-disulfonic acid both shifted to the right the concentration-response relationship for 2MeSATP for stimulation of oocyte currents. Although injection of oocytes with either GDPbetaS (guanyl-5'-yl thiophosphate) or GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) resulted in loss of adenine nucleotide-promoted Cl- channel activation, neither guanine nucleotide altered the 2MeSATP-stimulated cation current. These data are consistent with the view that activation of the novel cation current by the P2Y1 receptor does not involve a G protein. Tail current analysis of the novel P2Y1 receptor-associated cation conductance revealed that the open channel current-voltage relationship was outwardly rectifying with a reversal potential of -38 mV for the turkey P2Y1 receptor and -36 mV for the human P2Y1 receptor. Replacement of Na+ with K+ ions in the bathing solution produced a shift in reversal potential to near zero mV, but significant outward rectification remained. The cation current was not permeable to either Ca2+ or Ba2+ and exhibited steady-state inactivation at holding potentials below -60 mV. These results indicate that the P2Y1 receptor exhibits both metabotropic properties and a novel G protein-independent ionotropic response when expressed in Xenopus oocytes

EVects of phenytoin and carbamazepine on calcium transport in Caco-2 cells

Abstract Adverse eVects of anti-seizure/anti-epileptic medications on bone density have been observed and reported since the early 1960s. Phenytoin and carbamazepine are two commonly prescribed anti-epileptic drugs most frequently associated with osteomalacia including fractures, bone demineralization, and reduced bone formation. The mechanism by which anti-epileptic drugs induce bone loss is not fully explained. We hypothesized that anti-epileptic drugs may impair dietary calcium absorption in the intestine. Using Caco-2 cells, a model transport system for study of the function of the intestinal epithelium, we determined the eVects of several anti-epileptic drugs on intestinal epithelial calcium transport. In our system, phenytoin and carbamazepine dose-dependently inhibit active calcium transport from the apical to basolateral side of Caco-2 cells under physiologic calcium conditions. Vitamin D ameliorates the anti-epileptic drug-induced decrease in calcium permeability

Kinetic Analysis of BCL11B Multisite Phosphorylation–Dephosphorylation and Coupled Sumoylation in Primary Thymocytes by Multiple Reaction Monitoring Mass Spectroscopy

Transcription factors with multiple post-translational modifications (PTMs) are not uncommon, but comprehensive information on site-specific dynamics and interdependence is comparatively rare. Assessing dynamic changes in the extent of PTMs has the potential to link multiple sites both to each other and to biological effects observable on the same time scale. The transcription factor and tumor suppressor BCL11B is critical to three checkpoints in T-cell development and is a target of a T-cell receptor (TCR)-mediated MAP kinase signaling. Multiple reaction monitoring (MRM) mass spectroscopy was used to assess changes in relative phosphorylation on 18 of 23 serine and threonine residues and sumoylation on one of two lysine resides in BCL11B. We have resolved the composite phosphorylation–dephosphorylation and sumoylation changes of BCL11B in response to MAP kinase activation into a complex pattern of site-specific PTM changes in primary mouse thymocytes. The site-specific resolution afforded by MRM analyses revealed four kinetic patterns of phosphorylation and one of sumoylation, including both rapid simultaneous site-specific increases and decreases at putative MAP kinase proline-directed phosphorylation sites, following stimulation. These data additionally revealed a novel spatiotemporal bisphosphorylation motif consisting of two kinetically divergent proline-directed phosphorylation sites spaced five resides apart.Keywords: SRM, T-cell receptor, Post-translational modification, Signal transduction, Sumoylation, MRM, BCL11

Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-β

The potential role of protein kinase C (PKC)-promoted phosphorylation has been examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [32P]Pi-labelled turkey erythrocytes with either the P2Y1 receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA resulted in a marked increase in incorporation of 32P into the G-protein-activated phospholipase C PLC-betaT. Purified PLC-betaT also was phosphorylated by PKC in vitro to a stoichiometry (mean+/-S. E.M.) of 1.06+/-0.2 mol of phosphate/mol of PLC-betaT. Phosphorylation by PKC was isoenzyme-specific because, under identical conditions, mammalian PLC-beta2 also was phosphorylated to a stoichiometry near unity, whereas mammalian PLC-beta1 was not phosphorylated by PKC. The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-betaT with turkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylation resulted in a decrease in basal activity, AlF4(-)-stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5'-[gamma-thio]triphosphate in the reconstituted membranes. The decreases in enzyme activities were proportional to the extent of PKC-promoted phosphorylation. Catalytic activity assessed by using mixed detergent/phospholipid micelles also was decreased by up to 60% by phosphorylation. The effect of phosphorylation on Gqalpha-stimulated PLC-betaT in reconstitution experiments with purified proteins was not greater than that observed on basal activity alone. Taken together, these results illustrate that PKC phosphorylates PLC-betaT in vivo and to a physiologically relevant stoichiometry in vitro. Phosphorylation is accompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-protein-regulated activity

Rescue leadless pacemaker implantation in a pacemaker-dependent patient with congenital heart disease and no alternative routes for pacing

Congenital heart disease patients are considered a unique group of patients regarding their high risk of conduction abnormalities, whether de novo or surgically induced, and the challenges in both implantation and management of device related complications. We present a case of a pacemaker-dependent patient with congenital heart disease who experienced complications of both previous epicardial and transvenous pacing which rendered her a non-suitable candidate of both routes

Nonmuscle myosins II-B and Va are components of detergentresistant membrane skeletons derived from mouse forebrain

Myosins are actin-based molecular motors that may have specialized trafficking and contractile functions in cytoskeletal compartments that lack microtubules. The postsynaptic excitatory synapse is one such specialization, yet little is known about the spatial organization of myosin motor proteins in the mature brain. We used a proteomics approach to determine if class II and class V myosin isoforms are associated with Triton X-100-resistant membranes isolated from mouse forebrain. Two nonmuscle myosin isoforms (II-B and Va), were identified as components of lipid raft fractions that also contained typical membrane skeletal proteins such as non-erythrocyte spectrins, actin, alpha-actinin-2 and tubulin subunits. Other raft-associated proteins included lipid raft markers, proteins involved in cell adhesion and membrane dynamics, receptors and channels including glutamate receptor subunits, scaffolding and regulatory proteins. Myosin II-B and Va were also present in standard postsynaptic density (PSD) fractions, however retention of myosin II-B was strongly influenced by ATP status. If homogenates were supplemented with ATP, myosin II-B could be extracted from PSD I whereas myosin Va and other postsynaptic proteins were resistant to extraction. In summary, both myosin isoforms are components of a raft-associated membrane skeleton and are likely detected in standard PSD fractions as a result of their intrinsic ability to form actomyosin. Myosin IIB, however, is loosely or more peripherally associated with the PSD than myosin Va, which appears to be a core PSD protein.Keywords: lipid raft, mass spectrometry, actin cytoskeleton, proteomics, postsynaptic density, myosin II, myosin