RESUMO
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.
Assuntos
Baculoviridae/enzimologia , Fosfoinositídeo Fosfolipase C/isolamento & purificação , Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas ras/isolamento & purificação , Proteínas ras/farmacologia , Proteínas rho de Ligação ao GTP/isolamento & purificação , Proteínas rho de Ligação ao GTP/farmacologia , Animais , Linhagem Celular , Membrana Celular/química , Sistema Livre de Células , Cromatografia de Afinidade , Cromatografia em Gel , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/citologia , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Fosfoinositídeo Fosfolipase C/genética , Ratos , Deleção de Sequência , SolubilidadeRESUMO
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.
Assuntos
Proteínas Sanguíneas/química , Regulação Enzimológica da Expressão Gênica , Fosfolipase C gama/metabolismo , Fosfoproteínas/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Células COS , Chlorocebus aethiops , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
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.