Dual Regulation of Phospholipase C-beta by G betagamma

Agonist-bound G protein coupled receptors (GPCRs) activate G protein heterotrimers by catalyzing release of GDP and binding of GTP to the G alpha subunit (Ga), releasing active Ga and G betagamma (Gbg) subunits. Activated alpha subunits of the Gq family and betagamma subunits of the Gi family stimulate phospholipase C-beta (PLC-b) isoforms to catalyze hydrolysis of phosphatidylinositol-1,2-bisphosphate (PIP2) generating the second messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG). PLC-b isoforms are also GTPase activating proteins (GAPs) for Gaq, and Gbg subunits inhibit the GAP activity of PLC-b. Coordinated regulation of these activities is essential for sustained signaling at steady state. Regulation of PLC-b by Gaq and Gbg is well studied but details of the mechanism are still lacking. Activation of PLC-b simultaneously by G protein pathways has been suggested based on observations in cells, but it is not known if scaffolding proteins or other factors are necessary for simultaneous stimulation of PLC-b by G protein subunits. The binding interface between Gbg and PLC-b is unclear, and so is the mechanism of PLC-b GAP inhibition by Gbg. To enable the study of these mechanisms in vitro, I developed a new method to purify Gaq subunits based on observations from the Tall group. This method combined Ric8A-mediated enhancement of Gaq expression and the traditional method of using detergents to isolate functional Ga from membrane bound G protein heterotrimers, resulting in 3- to 4-fold increase in yields of Gaq. Using purified proteins and working with other members of the lab, I showed that the PLC-b3 isoform is synergistically activated by Gaq and Gbg subunits. The observed synergism is up to 10-fold, quantitatively consistent with cellular observations, thus establishing that no additional proteins or pathways are required. Next, I developed a FRET-based binding assay between Gbg and PLC-b and identified the pleckstrin homology (PH) domain in PLC-b as the Gbg binding site. Using structural and biochemical analyses, I showed that Gbg-PLC-b requires intrinsic motion of the PH domain. This led to the proposal for a new conformation of PLC-b not observed in crystal structures and a new model for Gbg-PLC-b binding. Subsequent studies suggested that Gbg inhibits PLC-b GAP activity by a mechanism that does not require Gbg-PLC-b binding

The effects of mutant Ras proteins on the cell signalome

The genetic alterations in cancer cells are tightly linked to signaling pathway dysregulation. Ras is a key molecule that controls several tumorigenesis-related processes, and mutations in RAS genes often lead to unbiased intensification of signaling networks that fuel cancer progression. In this article, we review recent studies that describe mutant Ras-regulated signaling routes and their cross-talk. In addition to the two main Ras-driven signaling pathways, i.e., the RAF/MEK/ERK and PI3K/AKT/mTOR pathways, we have also collected emerging data showing the importance of Ras in other signaling pathways, including the RAC/PAK, RalGDS/Ral, and PKC/PLC signaling pathways. Moreover, microRNA-regulated Ras-associated signaling pathways are also discussed to highlight the importance of Ras regulation in cancer. Finally, emerging data show that the signal alterations in specific cell types, such as cancer stem cells, could promote cancer development. Therefore, we also cover the up-to-date findings related to Ras-regulated signal transduction in cancer stem cells. © 2020, The Author(s)

FGFR3 – a Central Player in Bladder Cancer Pathogenesis?

The identification of mutations in FGFR3 in bladder tumors in 1999 led to major interest in this receptor and during the subsequent 20 years much has been learnt about the mutational profiles found in bladder cancer, the phenotypes associated with these and the potential of this mutated protein as a target for therapy. Based on mutational and expression data, it is estimated that >80% of non-muscle-invasive bladder cancers (NMIBC) and ∼40% of muscle-invasive bladder cancers (MIBC) have upregulated FGFR3 signalling, and these frequencies are likely to be even higher if alternative splicing of the receptor, expression of ligands and changes in regulatory mechanisms are taken into account. Major efforts by the pharmaceutical industry have led to development of a range of agents targeting FGFR3 and other FGF receptors. Several of these have entered clinical trials, and some have presented very encouraging early results in advanced bladder cancer. Recent reviews have summarised the drugs and related clinical trials in this area. This review will summarise what is known about the effects of FGFR3 and its mutant forms in normal urothelium and bladder tumors, will suggest when and how this protein contributes to urothelial cancer pathogenesis and will highlight areas that may benefit from further study

Synergistic Activation of Phospholipase C-β3 by Gαq and Gβγ Describes a Simple Two-State Coincidence Detector

SummaryBackgroundReceptors that couple to Gi and Gq often interact synergistically in cells to elicit cytosolic Ca2+ transients that are several-fold higher than the sum of those driven by each receptor alone. Such synergism is commonly assumed to be complex, requiring regulatory interaction between components, multiple pathways, or multiple states of the target protein.ResultsWe show that cellular Gi-Gq synergism derives from direct supra-additive stimulation of phospholipase C-β3 (PLC-β3) by G protein subunits Gβγ and Gαq, the relevant components of the Gi and Gq signaling pathways. No additional pathway or proteins are required. Synergism is quantitatively explained by the classical and simple two-state (inactive↔active) allosteric mechanism. We show generally that synergistic activation of a two-state enzyme reflects enhanced conversion to the active state when both ligands are bound, not merely the enhancement of ligand affinity predicted by positive cooperativity. The two-state mechanism also explains why synergism is unique to PLC-β3 among the four PLC-β isoforms and, in general, why one enzyme may respond synergistically to two activators while another does not. Expression of synergism demands that an enzyme display low basal activity in the absence of ligand and becomes significant only when basal activity is ≤ 0.1% of maximal.ConclusionsSynergism can be explained by a simple and general mechanism, and such a mechanism sets parameters for its occurrence. Any two-state enzyme is predicted to respond synergistically to multiple activating ligands if, but only if, its basal activity is strongly suppressed

Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program

AbstractMethionine, through S-adenosylmethionine, activates multifaceted growth programs where ribosome biogenesis, carbon metabolism, amino acid and nucleotide biosynthesis are induced. This growth program requires activity of the Gcn4 transcription factor (called ATF4 in mammals), which enables metabolic precursor supply essential for anabolism. Here, we discover how the Gcn4 protein is induced by methionine, despite conditions of high translation and anabolism. This induction mechanism is independent of transcription, as well as the conventional Gcn2/eIF2α mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 ubiqitination and therefore degradation is reduced, due to the decreased phosphorylation of this protein. This Gcn4 stabilization is mediated by the activity of the conserved methyltransferase, Ppm1, which specifically methylates the catalytic subunit of protein phosphatase PP2A when methionine is abundant. This methylation of PP2A shifts the balance of Gcn4 to a dephosphorylated state, which stabilizes the protein. The loss of Ppm1, or PP2A-methylation destabilizes Gcn4 when methionine is abundant, and the Gcn4-dependent anabolic program collapses. These findings reveal a novel signaling and regulatory axis, where methionine directs a conserved methyltransferase Ppm1, via its target phosphatase PP2A, to selectively stabilize Gcn4. Thereby, when methionine is abundant, cells conditionally modify a major phosphatase in order to stabilize a metabolic master-regulator and drive anabolism.</jats:p

Article Synergistic Activation of Phospholipase C-b3 by Ga q and Gbg Describes a Simple Two-State Coincidence Detector

Summary Background: Receptors that couple to G i and G q often interact synergistically in cells to elicit cytosolic Ca 2+ transients that are several-fold higher than the sum of those driven by each receptor alone. Such synergism is commonly assumed to be complex, requiring regulatory interaction between components, multiple pathways, or multiple states of the target protein. Results: We show that cellular G i -G q synergism derives from direct supra-additive stimulation of phospholipase C-b3 (PLC-b3) by G protein subunits Gbg and Ga q , the relevant components of the G i and G q signaling pathways. No additional pathway or proteins are required. Synergism is quantitatively explained by the classical and simple two-state (inactive4active) allosteric mechanism. We show generally that synergistic activation of a two-state enzyme reflects enhanced conversion to the active state when both ligands are bound, not merely the enhancement of ligand affinity predicted by positive cooperativity. The two-state mechanism also explains why synergism is unique to PLC-b3 among the four PLC-b isoforms and, in general, why one enzyme may respond synergistically to two activators while another does not. Expression of synergism demands that an enzyme display low basal activity in the absence of ligand and becomes significant only when basal activity is % 0.1% of maximal. Conclusions: Synergism can be explained by a simple and general mechanism, and such a mechanism sets parameters for its occurrence. Any two-state enzyme is predicted to respond synergistically to multiple activating ligands if, but only if, its basal activity is strongly suppressed