Phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). DAG and IP(3) each control diverse cellular processes and are also substrates for synthesis of other important signaling molecules. PLC is thus central to many important interlocking regulatory networks. Mammals express six families of PLCs, each with both unique and overlapping controls over expression and subcellular distribution. Each PLC also responds acutely to its own spectrum of activators that includes heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca(2+), and phospholipids. Mammalian PLCs are autoinhibited by a region in the catalytic TIM barrel domain that is the target of much of their acute regulation. In combination, the PLCs act as a signaling nexus that integrates numerous signaling inputs, critically governs PIP(2) levels, and regulates production of important second messengers to determine cell behavior over the millisecond to hour timescale.

Summary Background Receptors 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. Results We 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. 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.

Mammalian phospholipase C-␤ (PLC-␤) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how G␣ q and Rac1 bind PLC-␤, there is a lack of consensus regarding the G␤␥ binding site in PLC-␤. Using FRET between cerulean fluorescent protein-labeled G␤␥ and the Alexa Fluor 594-labeled PLC-␤ pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal G␤␥ binding region in PLC-␤3. We show that the isolated PH domain can compete with full-length PLC-␤3 for binding G␤␥ but not G␣ q , Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-␤ PH domain as the G␤␥ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-␤, necessitating conformational rearrangement to allow G␤␥ binding. Blocking PH domain motion in PLC-␤ by crosslinking it to the EF hand domain inhibits stimulation by G␤␥ without altering basal activity or G␣ q response. The fraction of PLC-␤ cross-linked is proportional to the fractional loss of G␤␥ response. Cross-linked PLC-␤ does not bind G␤␥ in a FRETbased G␤␥-PLC-␤ binding assay. We propose that unliganded PLC-␤ exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-␤ modulates G␤␥ access to its binding site. Phospholipase C (PLC)2 isozymes integrate signaling inputs downstream of diverse receptors to catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in the inner leaflet of the plasma membrane. This reaction generates two products, inositol 1,4,5-trisphosphate and diacylglycerol, important second messengers and second messenger precursors that regulate multiple cellular processes (1). The mammalian genome encodes six families of PLCs (␤, ␦, ␥, ⑀, , and ) that share a conserved core architecture composed of a pleckstrin homology (PH) domain, four EF hands, a split TIM barrel, and a C2 domain. The TIM barrel contains the active site, and its two halves are separated by the so-called X-Y linker that is thought to occlude the active site and whose motion is central to the regulation of activity. In the PLC-␤ isozymes, the linker is highly cationic, flanked by a highly anionic region, and disordered. Sondek and co-workers (2) speculated that PLC recruitment to the plasma membrane by protein activators causes electrostatic repulsion of the linker by negatively charged phospholipids that moves it away from the active site to relieve autoinhibition. Proteolysis or genetic removal of the linker elevates basal enzyme activity across many PLC isozymes (3, 4), supporting the generality of this mechanism. However, mutant PLC-␤s that lack this linker are activated further by their physiological ligands (2), suggesting the existence of additional regulatory mechanisms that are yet to be discovered.The four mammalian PLC-␤ ...

Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (where the roles of Gcn4 are not well studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase PP2A; our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 towards a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs a conserved methyltransferase Ppm1, via its target phosphatase PP2A, to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master-regulator and drive anabolism.

Methionine, 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.

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herein required the expression and purification of TMD1 in yeast cells and the isolation and purification of C3 from bovine blood plasma. A protein pull-down assay was used to verify that the two proteins interact. To aid in specific immobilization of the TMD1, two lysine residues were converted to methionine by site-directed mutagenesis via polymerase chain reaction leaving only one free amine available for future reactions. The wild type and mutant TMD1 proteins were characterized by mass spectrometry and urea-induced unfolding. The proteins were also both used in pull-down assays with C3. Once interactions are confirmed, hydrogen/deuterium exchange followed by matrix assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF MS) will be performed to determine which regions of each protein are involved in binding. Additionally, interactions between the two proteins will be characterized using fluorescence resonance energy transfer (FRET) experiments. 2596-Pos Board B26The Escherichia coli protein RecA catalyzes the strand exchange reaction used in DNA repair and genetic recombination. Previous studies in our lab have shown buffer-specific changes in RecA stability and unfolding transitions. However, these studies suggest only minimal buffer dependent changes in nucleotide binding and secondary structure that did not explain the large buffer dependent differences in RecA stability and unfolding profiles. These observations led to further investigations of how the four common biological buffers Tris, HEPES, MES, and PO 4 alter RecA structure and nucleotide binding.Here we have employed circular dichroism (CD) and infrared (IR) spectroscopy to further discern if buffers influence nucleotide binding to RecA. CD spectra of RecA were obtained in the presence and absence of ADP in each buffer condition. Laser-induced photolysis of caged nucleotides was used in conjunction with difference IR to generate RecA-ADP minus RecA difference infrared spectra in each of the four buffers. These studies detected bufferspecific changes in nucleotide binding to RecA including possible perturbations in Gln, Glu, Asp, Asn, Tyr, and Lys residues and unique secondary structural transitions. These differences between RecA-ADP minus RecA difference spectra will be discussed. The lac repressor protein (lac) is an allosterically regulated transcription factor which controls expression of the lac operon in bacteria. Binding of a small molecule inducer to a site 40Å away from the DNA-binding domain relieves repression through what is thought to be local unfolding of the hinge helix. Despite decades of characterization, our understanding of this allosteric transition remains incomplete_mostly inferred from partial crystal structures. In principle, high-resolution solution NMR could provide detailed structural and dynamical information unobtainable by crystallography. However, due to lac's high molecular weight (70 kDa free, 85 kDa operator-bound), low solubility, and transient stability, such studies have been limite...