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1.
Mol Cell ; 34(2): 223-33, 2009 Apr 24.
Article in English | MEDLINE | ID: mdl-19394299

ABSTRACT

Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)). Based on this structure, we illustrate distinct requirements for PLCgamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCgamma(2), but not the related PLCbeta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPgammaS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.


Subject(s)
Phospholipase C gamma/chemistry , rac GTP-Binding Proteins/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Enzyme Activation , Epidermal Growth Factor/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Phospholipase C gamma/metabolism , Protein Interaction Mapping , Protein Structure, Tertiary , Sequence Alignment , Substrate Specificity , Thermodynamics , rac GTP-Binding Proteins/metabolism , RAC2 GTP-Binding Protein
2.
Methods Mol Biol ; 462: 379-89, 2009.
Article in English | MEDLINE | ID: mdl-19160682

ABSTRACT

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P3]. PLCepsilon is a recently discovered isoform that has been shown to be activated by members of the Ras and Rho families of guanosine trisphosphatases (GTPases) as well as subunits of heterotrimeric G-proteins. We describe a method for expressing a truncated PLCepsilon variant as an MBP fusion protein in E. coli. Subsequently, we describe the methodology necessary to reconstitute this protein with K-Ras-4B and RhoA GTPases and measure its activation.


Subject(s)
Baculoviridae/enzymology , Phosphoinositide Phospholipase C/isolation & purification , Phosphoinositide Phospholipase C/metabolism , ras Proteins/isolation & purification , ras Proteins/pharmacology , rho GTP-Binding Proteins/isolation & purification , rho GTP-Binding Proteins/pharmacology , Animals , Cell Line , Cell Membrane/chemistry , Cell-Free System , Chromatography, Affinity , Chromatography, Gel , Enzyme Activation/drug effects , Escherichia coli/cytology , Isoenzymes/genetics , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Phosphoinositide Phospholipase C/genetics , Rats , Sequence Deletion , Solubility
3.
J Biol Chem ; 283(44): 30351-62, 2008 Oct 31.
Article in English | MEDLINE | ID: mdl-18728011

ABSTRACT

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.


Subject(s)
Blood Proteins/chemistry , Gene Expression Regulation, Enzymologic , Phospholipase C gamma/metabolism , Phosphoproteins/chemistry , Proto-Oncogene Proteins c-akt/metabolism , Animals , COS Cells , Chlorocebus aethiops , Humans , Models, Biological , Models, Molecular , Molecular Conformation , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Signal Transduction
4.
Mol Cell ; 21(4): 495-507, 2006 Feb 17.
Article in English | MEDLINE | ID: mdl-16483931

ABSTRACT

Ras proteins signal to a number of distinct pathways by interacting with diverse effectors. Studies of ras/effector interactions have focused on three classes, Raf kinases, ral guanylnucleotide-exchange factors, and phosphatidylinositol-3-kinases. Here we describe ras interactions with another effector, the recently identified phospholipase C epsilon (PLCepsilon). We solved structures of PLCepsilon RA domains (RA1 and RA2) by NMR and the structure of the RA2/ras complex by X-ray crystallography. Although the similarity between ubiquitin-like folds of RA1 and RA2 proves that they are homologs, only RA2 can bind ras. Some of the features of the RA2/ras interface are unique to PLCepsilon, while the ability to make contacts with both switch I and II regions of ras is shared only with phosphatidylinositol-3-kinase. Studies of PLCepsilon regulation suggest that, in a cellular context, the RA2 domain, in a mode specific to PLCepsilon, has a role in membrane targeting with further regulatory impact on PLC activity.


Subject(s)
Protein Structure, Tertiary , Type C Phospholipases/chemistry , Type C Phospholipases/metabolism , ras Proteins/chemistry , ras Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Crystallography, X-Ray , Enzyme Activation , Humans , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Phosphoinositide Phospholipase C , Protein Binding , Protein Structure, Secondary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Signal Transduction/physiology , Thermodynamics , Type C Phospholipases/genetics , ras Proteins/genetics
5.
Immunity ; 22(4): 451-65, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15845450

ABSTRACT

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.


Subject(s)
Autoimmunity , Calcium/metabolism , Inflammation/genetics , Point Mutation , Type C Phospholipases/genetics , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , B-Lymphocytes/metabolism , Base Sequence , Bone Marrow Cells/cytology , Dermatitis/genetics , Dermatitis/immunology , Male , Mice , Molecular Sequence Data , Phospholipase C gamma , Type C Phospholipases/metabolism , Up-Regulation
6.
FEBS Lett ; 531(2): 329-34, 2002 Nov 06.
Article in English | MEDLINE | ID: mdl-12417336

ABSTRACT

Previous results have indicated that the generation of ceramide by hydrolysis of sphingomyelin by magnesium-dependent neutral sphingomyelinase 1 (NSM1) is reversibly inhibited by hydrogen peroxide (H2O2) and oxidized glutathione (GSSG). This redox-dependent reversible regulation of NSM1 activity has been shown to involve the reversible formation and breakage of disulfide bonds. In this paper, we show that peroxynitrite, a nitric oxide-derived oxidant generated by SIN1, inactivates dose-dependently the NSM1 activity in an irreversible manner. In addition, we show that, in contrast to the reversible inhibition of NSM1 by H2O2 or GSSG which involves the formation of disulfide bonds, irreversible inactivation of this enzyme by peroxynitrite generated from SIN1 is likely due to definitive oxidative thiol modification. These results suggest that depending on the nature of the oxidative stress, the enzymatic activity of NSM1 could be reversibly or irreversibly inactivated.


Subject(s)
Enzyme Inhibitors/pharmacology , Molsidomine/analogs & derivatives , Oxidants/pharmacology , Peroxynitrous Acid/pharmacology , Sphingomyelin Phosphodiesterase/metabolism , Animals , Cysteine/metabolism , Dose-Response Relationship, Drug , Hydrogen Peroxide/pharmacology , Magnesium/physiology , Mice , Molsidomine/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Donors/pharmacology , Oxidative Stress , Peroxynitrous Acid/biosynthesis , Reducing Agents/pharmacology , Sphingomyelin Phosphodiesterase/chemistry
7.
Biochem J ; 365(Pt 1): 69-77, 2002 Jul 01.
Article in English | MEDLINE | ID: mdl-12071841

ABSTRACT

DT40 cells have approx. 10-fold higher Mg2+-dependent neutral sphingomyelinase (NSM) activity in comparison with other B-cell lines and contain very low acidic sphingomyelinase activity. Purification of this activity from DT40 cell membranes suggested the presence of one major NSM isoform. Although complete purification of this isoform could not be achieved, partially purified fractions were examined further with regard to the known characteristics of previously partially purified NSMs and the two cloned enzymes exhibiting in vitro NSM activity (NSM1 and NSM2). For a direct comparative study, highly purified brain preparations, purified NSM1 protein and Bacillus cereus enzyme were used. Analysis of the enzymic properties of the partially purified DT40 NSM, such as cation dependence, substrate specificity, redox regulation and stimulation by phosphatidylserine, together with the localization of this enzyme to the endoplasmic reticulum (ER), suggested that this NSM from DT40 cells corresponds to NSM1. Further studies aimed to correlate presence of the high levels of this NSM1-like activity in DT40 cells with the ability of these cells to accumulate ceramide and undergo apoptosis. When DT40 cells were stimulated to apoptose by a variety of agents, including the ER stress, an increase in endogenous ceramide levels was observed. However, these responses were not enhanced compared with another B-cell line (Nalm-6), characterized by low sphingomyelinase activity. In addition, DT40 cells were not more susceptible to ceramide accumulation and apoptosis when exposed to the ER stress compared with other apoptotic agents. Inhibition of de novo synthesis of ceramide partially inhibited its accumulation, indicating that the ceramide production in DT40 cells could be complex and, under some conditions, could involve both sphingomyelin hydrolysis and ceramide synthesis.


Subject(s)
Apoptosis/physiology , B-Lymphocytes/enzymology , Endoplasmic Reticulum/enzymology , Sphingomyelin Phosphodiesterase/isolation & purification , Sphingomyelin Phosphodiesterase/metabolism , Animals , B-Lymphocytes/cytology , Brain/enzymology , Cell Line , Ceramides/metabolism , Chickens , Rats , Stress, Physiological/enzymology , Subcellular Fractions/enzymology
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