An N-Terminal Polybasic Motif of Gα<sub>q</sub>Is Required for Signaling and Influences Membrane Nanodomain Distribution

Crouthamel, Marykate; Abankwa, Daniel; Zhang, Li; DiLizio, Cherisse; Manning, David R.; Hancock, John F.; Wedegaertner, Philip

doi:10.1124/mol.110.066340

Cited by 17 publications

(12 citation statements)

References 42 publications

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“… 20 This assay allowed us to identify the modulators of nanoclustering 20 as well as the inhibitors of processes upstream of nanoclustering, such as membrane targeting and lipid modifications (for example, statins or farnesylation inhibitors). 21 , 22 , 23 These results illustrate that modulation of the nanoclustering-FRET signal can be due to multiple reasons that may involve direct modulation of the membrane, 20 lateral segregation changes of the FRET-probe, 24 conformational changes 25 or indirect, intracellular processes. 22 …”

Section: Introductionmentioning

confidence: 79%

Cancer stem cell drugs target K-ras signaling in a stemness context

et al. 2016

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Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC.

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Section: Introductionmentioning

confidence: 79%

Cancer stem cell drugs target K-ras signaling in a stemness context

et al. 2016

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“…7B), G␣ q does not seem to function by relocating p63RhoGEF to specific locales on the membrane or by reorienting the catalytic core. However, because both p63RhoGEF and G␣ q are palmitoylated, it is possible that they are already found in the same lipid microdomains (26,45,46), and we also cannot rule out the possibility that locale-specific recruitment may be important for endogenous p63RhoGEF.…”

Section: Discussionmentioning

confidence: 96%

“…Thus, it seems that p63RhoGEF and, by extension, the homologous domains in Trio and Duet are regulated by G␣ q primarily through an allosteric mechanism that involves release of PH domain-mediated autoinhibition (25). However, in cells, it is also possible that G␣ q recruits p63RhoGEF to specific locales on the PM (26) or repositions the RhoGEF at the membrane so that it can more productively interact with its substrate RhoA.…”

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confidence: 99%

Plasma Membrane Association of p63 Rho Guanine Nucleotide Exchange Factor (p63RhoGEF) Is Mediated by Palmitoylation and Is Required for Basal Activity in Cells

Aittaleb

Nishimura

Linder

et al. 2011

Journal of Biological Chemistry

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Activation of G protein-coupled receptors at the cell surface leads to the activation or inhibition of intracellular effector enzymes, which include various Rho guanine nucleotide exchange factors (RhoGEFs). RhoGEFs activate small molecular weight GTPases at the plasma membrane (PM). Many of the known G protein-coupled receptor-regulated RhoGEFs are found in the cytoplasm of unstimulated cells, and PM recruitment is a critical aspect of their regulation. In contrast, p63RhoGEF, a G␣ q -regulated RhoGEF, appears to be constitutively localized to the PM. The objective of this study was to determine the molecular basis for the localization of p63RhoGEF and the impact of its subcellular localization on its regulation by G␣ q . Herein, we show that the pleckstrin homology domain of p63RhoGEF is not involved in its PM targeting. Instead, a conserved string of cysteines (Cys-23/25/26) at the N terminus of the enzyme is palmitoylated and required for membrane localization and full basal activity in cells. Conversion of these residues to serine relocates p63RhoGEF from the PM to the cytoplasm, diminishes its basal activity, and eliminates palmitoylation. The activity of palmitoylation-deficient p63RhoGEF can be rescued by targeting to the PM by fusion with tandem phospholipase C-␦1 pleckstrin homology domains or by co-expression with wild-type G␣ q but not with palmitoylation-deficient G␣ q . Our data suggest that p63RhoGEF is regulated chiefly through allosteric control by G␣ q , as opposed to other known G␣-regulated RhoGEFs, which are instead sequestered in the cytoplasm, perhaps because of their high basal activity.Rho GTPases act as molecular switches that mediate a wide variety of cellular processes, such as actin cytoskeletal organization, cell migration, cell cycle progression, and transcriptional control (1-3). They are activated by Rho guanine nucleotide exchange factors (RhoGEFs), 3 which catalyze the exchange of GDP for GTP on the G protein (4, 5). The largest family of RhoGEFs is characterized by a Dbl homology (DH) domain that is almost always found immediately N-terminal to a pleckstrin homology (PH) domain. The DH domain forms the primary binding site for the GTPase, whereas the PH domain either modulates nucleotide exchange by the DH domain or has other functions. Heterotrimeric G-protein ␣ subunits are known to directly interact with and activate at least two classes of Dbl family RhoGEFs (6 -8). G␣ 12/13 subunits bind to the regulator of G protein signaling (RGS) homology (RH) domain of RH domaincontaining RhoGEFs (RH-RhoGEFs) such as leukemia-associated RhoGEF (LARG), PDZ-RhoGEF, and p115-RhoGEF (9 -12). G␣ q/11 subunits bind to the DH/PH core of another subfamily of RhoGEFs that includes p63RhoGEF and the C-terminal DH/PH domains of Trio and Duet (13,14). Because small GTPases are tethered to the plasma membrane (PM) via posttranslational prenylation of their C-terminal CAAX motifs (15), RhoGEFs must also be targeted to the PM in order to efficiently activate their substrates in living cells. Because...

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“…For example Gα q contains 10 basic residues, both arginines and lysines, within amino acids 16–38. Most importantly, helical predictions indicate that most of the positively charged N-terminal amino acids align on one face of the helix, grouped together to form basic patches in position to interact with negatively charged surfaces of cellular membranes (Kosloff et al 2002; Crouthamel et al 2008, 2010; Pedone and Hepler 2007). Indeed, replacement of select N-terminal basic amino acids in Gα s and Gα q with non-charged alanines or glutamines results in decreased membrane localization of the mutant Gα s and Gα q compared to wild type (Crouthamel et al 2008).…”

Section: 2 Mechanisms Of Membrane Binding By G Proteinsmentioning

confidence: 99%

G Protein Trafficking

Wedegaertner

2012

Subcellular Biochemistry

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The classical view of heterotrimeric G protein signaling places G proteins at the cytoplasmic surface of the cell’s plasma membrane where they are activated by an appropriate G protein-coupled receptor. Once activated, the GTP-bound Gα and the free Gβγ are able to regulate plasma membrane-localized effectors, such as adenylyl cyclase, phospholipase C-β, RhoGEFs and ion channels. Hydrolysis of GTP by the Gα subunit returns the G protein to the inactive Gαβγ heterotrimer. Although all of these events in the G protein cycle can be restricted to the cytoplasmic surface of the plasma membrane, G protein localization is dynamic. Thus, it has become increasingly clear that G proteins are able to move to diverse subcellular locations where they perform non-canonical signaling functions. This chapter will highlight our current understanding of trafficking pathways that target newly synthesized G proteins to the plasma membrane, activation-induced and reversible translocation of G proteins from the plasma membrane to intracellular locations, and constitutive trafficking of G proteins.

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An N-Terminal Polybasic Motif of Gα_qIs Required for Signaling and Influences Membrane Nanodomain Distribution

Cited by 17 publications

References 42 publications

Cancer stem cell drugs target K-ras signaling in a stemness context

Cancer stem cell drugs target K-ras signaling in a stemness context

Plasma Membrane Association of p63 Rho Guanine Nucleotide Exchange Factor (p63RhoGEF) Is Mediated by Palmitoylation and Is Required for Basal Activity in Cells

G Protein Trafficking

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An N-Terminal Polybasic Motif of GαqIs Required for Signaling and Influences Membrane Nanodomain Distribution

Cited by 17 publications

References 42 publications

Cancer stem cell drugs target K-ras signaling in a stemness context

Cancer stem cell drugs target K-ras signaling in a stemness context

Plasma Membrane Association of p63 Rho Guanine Nucleotide Exchange Factor (p63RhoGEF) Is Mediated by Palmitoylation and Is Required for Basal Activity in Cells

G Protein Trafficking

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An N-Terminal Polybasic Motif of Gα_qIs Required for Signaling and Influences Membrane Nanodomain Distribution