Stephanie N. Hicks

Kinetic Scaffolding Mediated by a Phospholipase C–β and Gq Signaling Complex

Reciprocal Regulation An essential step in many signaling cascades is inositol lipid hydrolysis catalyzed by phospholipase C–β. The latter is activated by the α subunit of the heterotrimeric G protein Gq, and it in turn inactivates Gαq, thus sharpening the signal. Waldo et al. (p. 974, published online 21 October) report structural and biochemical data that explain the basis of this reciprocal regulation. One domain of phospholipase C–β binds to activated Gαq. Though the phospholipase C–β active site remains occluded in the structure, the plug is probably removed upon G protein–dependent orientation of the lipase at the membrane. A second domain of phospholipase C–β accelerates guanosine triphosphate hydrolysis by Gαq causing the signaling complex to dissociate. A crystal structure shows how the two components of a central signaling complex regulate each other. Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)–dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide–binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gαq reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein–dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gαq, causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.

General and Versatile Autoinhibition of PLC Isozymes

Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-beta2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-beta2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli.

Mechanism of Phosphorylation-induced Activation of Phospholipase C-γ Isozymes

The lipase activity of most phospholipases C (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y linker. In contrast, PLC-γ isozymes (PLC-γ1 and -γ2) are structurally distinct from other PLCs because multiple domains are present in their X/Y linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-γ isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-γ isozymes by their X/Y linker. Specifically, we identify the C-terminal SH2 domain within the X/Y linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y linker mediates high affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs.

Acireductone Dioxygenase 1 (ARD1) Is an Effector of the Heterotrimeric G Protein β Subunit in Arabidopsis

Heterotrimeric G protein complexes are conserved from plants to mammals, but the complexity of each system varies. Arabidopsis thaliana contains one Gα, one Gβ (AGB1), and at least three Gγ subunits, allowing it to form three versions of the heterotrimer. This plant model is ideal for genetic studies because mammalian systems contain hundreds of unique heterotrimers. The activation of these complexes promotes interactions between both the Gα subunit and the Gβγ dimer with enzymes and scaffolds to propagate signaling to the cytoplasm. However, although effectors of Gα and Gβ are known in mammals, no Gβ effectors were previously known in plants. Toward identifying AGB1 effectors, we genetically screened for dominant mutations that suppress Gβ-null mutant (agb1-2) phenotypes. We found that overexpression of acireductone dioxygenase 1 (ARD1) suppresses the 2-day-old etiolated phenotype of agb1-2. ARD1 is homologous to prokaryotic and eukaryotic ARD proteins; one function of ARDs is to operate in the methionine salvage pathway. We show here that ARD1 is an active metalloenzyme, and AGB1 and ARD1 both control embryonic hypocotyl length by modulating cell division; they also may contribute to the production of ethylene, a product of the methionine salvage pathway. ARD1 physically interacts with AGB1, and ARD enzymatic activity is stimulated by AGB1 in vitro. The binding interface on AGB1 was deduced using a comparative evolutionary approach and tested using recombinant AGB1 mutants. A possible mechanism for AGB1 activation of ARD1 activity was tested using directed mutations in a loop near the substrate-binding site.

Dual Activation of Phospholipase C-ε by Rho and Ras GTPases

Phospholipase C-ϵ (PLC-ϵ) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-ϵ to stimulate its phospholipase activity. Purified PLC-ϵ was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P2-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-ϵ prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-ϵ eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-ϵ markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383–394), but PLC-ϵ lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-ϵ by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-ϵ was quantified with PtdIns(4,5)P2-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-ϵ to activation by K-Ras. These results indicate that PLC-ϵ can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-ϵ.

Mechanism of Activation and Inactivation of Gq/Phospholipase C-β Signaling Nodes

The phospholipase C (PLC) isozymes catalyze conversion of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into the Ca2+-mobilizing second messenger inositol 1,4,5-trisphosphate (inositol (1,4,5)P3) and the protein kinase-activating second messenger diacylglycerol (DAG).1 Many PLC-dependent cellular responses occur in addition to those mediated by these classical second messengers since the activities of a broad range of membrane, cytosolic, and cytoskeletal proteins are regulated by PtdIns(4,5)P2 binding.2–4 Mammals express 13 different PLCs (Figure 1), which differ markedly in their modes of upstream regulation and physiological functions.5 Many growth factors, antigens, and other extracellular stimuli signal through tyrosine phosphorylation of the PLC-γ isozymes, whereas an even larger group of hormones, neurotransmitters, chemoattractant and chemosensory molecules, and other extracellular stimuli promote physiological effects through heterotrimeric G protein-dependent activation of the PLC-β isozymes. These and other forms of receptor-promoted signaling pathways also communicate to Ras superfamily GTPases, which in turn directly activate certain PLC isoyzmes. Thus, PLC-dependent signal transduction provides one of the major fabrics for communication of cells, and delineation of its function at all levels from the intact animal to the atomic resolution of mechanism is fundamental to understanding mammalian biology. Figure 1 Conserved domain structure of the mammalian PLC isozymes. The 13 functional human PLC isozymes were aligned on the basis of the conservation of the protein sequence, and a dendrogram was constructed to cluster similar sequences into shared branches. The ... Many excellent reviews on PLC-dependent signaling are available that focus, for example, on early aspects of the discovery and function of receptor-promoted formation of Ins(1,4,5)P3/diacylglycerol and mobilization of Ca2+,1,6,7 classification and regulation of the PLC isozymes,8–10 tyrosine phosphorylation-dependent activation,11,12 activation through Ras superfamily GTPases,5,13,14 physiology,15,16 and structure/function.17–19 We have limited this review to a general introduction of the domain and structural features of the PLC isozymes and then consider in detail recent advances made in understanding the mechanisms through which Gα-subunits of the Gq family bind to and activate PLC-β isozymes and how the PLC-β isozymes in turn promote inactivation of this signaling complex by stimulating GTP hydrolysis by the GTP-activated G protein.

A Fluorogenic, Small Molecule Reporter for Mammalian Phospholipase C Isozymes

Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimers disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable K(m) and V(max) with the endogenous PIP(2). This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high-throughput screening of PLC inhibitors.

Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

Background: Phospholipase C (PLC) isozymes are increasingly attractive therapeutic targets; however, pharmacological modulators are lacking. Results: A facile fluorescent high-throughput screen was developed and used to identify small molecule inhibitors of PLC activity. Conclusion: The new assay is robust and suitable for the rapid discovery of novel PLC modulators. Significance: This new methodology eliminates the major roadblock hampering the discovery of small molecule PLC inhibitors. Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.

Prediction of Protein-Protein Interfaces on G-Protein β Subunits Reveals a Novel Phospholipase C β2 Binding Domain

Gbeta subunits from heterotrimeric G-proteins (guanine nucleotide-binding proteins) directly bind diverse proteins, including effectors and regulators, to modulate a wide array of signaling cascades. These numerous interactions constrained the evolution of the molecular surface of Gbeta. Although mammals contain five Gbeta genes comprising two classes (Gbeta1-like and Gbeta5-like), plants and fungi have a single ortholog, and organisms such as Caenorhabditis elegans and Drosophila melanogaster contain one copy from each class. A limited number of crystal structures of complexes containing Gbeta subunits and complementary biochemical data highlight specific sites within Gbetas needed for protein interactions. It is difficult to determine from these interaction sites what, if any, additional regions of the Gbeta molecular surface comprise interaction interfaces essential to Gbetas role as a nexus in numerous signaling cascades. We used a comparative evolutionary approach to identify five known and eight previously unknown putative interfaces on the surface of Gbeta. We show that one such novel interface occurs between Gbeta and phospholipase C beta2 (PLC-beta2), a mammalian Gbeta interacting protein. Substitutions of residues within this Gbeta-PLC-beta2 interface reduce the activation of PLC-beta2 by Gbeta1, confirming that our de novo comparative evolutionary approach predicts previously unknown Gbeta-protein interfaces. Similarly, we hypothesize that the seven remaining untested novel regions contribute to putative interfaces for other Gbeta interacting proteins. Finally, this comparative evolutionary approach is suitable for application to any protein involved in a significant number of protein-protein interactions.