Kinetic analysis of Arabidopsis phospholipase Ddelta. Substrate preference and mechanism of activation by Ca2+ and phosphatidylinositol 4,5-biphosphate.

Phospholipase D (PLD) is a major plant phospholipase family involved in many cellular processes such as signal transduction, membrane remodeling, and lipid degradation. Five classes of PLDs have been identified in Arabidopsis thaliana, and Ca(2+) and polyphosphoinositides have been suggested as key regulators for these enzymes. To investigate the catalysis and regulation mechanism of individual PLDs, surface-dilution kinetics studies were carried out on the newly identified PLDdelta from Arabidopsis.

Regulation of phospholipase D activity by actin. Actin exerts bidirectional modulation of Mammalian phospholipase D activity in a polymerization-dependent, isoform-specific manner.

Many critical cellular processes, including proliferation, vesicle trafficking, and secretion, are regulated by both phospholipase D (PLD) and the actin microfilament system. Stimulation of human PLD1 results in its association with the detergent-insoluble actin cytoskeleton, but the molecular mechanisms and functional consequences of PLD-actin interactions remain incompletely defined. Biochemical and pharmacologic modulation of actin polymerization resulted in complex bidirectional effects on PLD activity, both in vitro and in vivo.

Phospholipase D in hormonal and stress signaling.

Phospholipase D (PLD) is a family of diverse enzymes that are differentially regulated by Ca(2+), polyphosphoinositides, free fatty acids, G-proteins, N-acylethanolamines, and membrane lipid environments. Two new types of PLDs were identified in the past year: one is activated by oleic acid and the other requires no cation for activity. The oleate-stimulated PLD is associated with the plasma membrane and binds to microtubules.

Evidence that the dephosphorylation of Ser(535) in the epsilon-subunit of eukaryotic initiation factor (eIF) 2B is insufficient for the activation of eIF2B by insulin.

Eukaryotic initiation factor (eIF) 2B is a guanine-nucleotide exchange factor that plays a key role in the regulation of protein synthesis. It is activated by insulin, serum and other agents that stimulate general protein synthesis. The largest (epsilon) subunit of eIF2B is a substrate for glycogen synthase kinase (GSK)-3 in vitro, and phosphorylation by GSK3 inhibits the activity of eIF2B.

Networking of phospholipases in plant signal transduction.

Phospholipases are activated in response to various cellular and environmental cues. Their activation can affect many cellular processes through their roles in signal transduction. Recent advances in the biochemical and molecular understanding of phospholipase D (PLD) have provided insights into potential networks of PLDs and other phospholipases in plants.

Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis.

A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids of Arabidopsis deficient in the most abundant phospholipase D, PLD alpha, were analyzed.

Cellular stresses profoundly inhibit protein synthesis and modulate the states of phosphorylation of multiple translation factors.

We have examined the effects of widely used stress-inducing agents on protein synthesis and on regulatory components of the translational machinery. The three stresses chosen, arsenite, hydrogen peroxide and sorbitol, exert their effects in quite different ways. Nonetheless, all three rapidly ( approximately 30 min) caused a profound inhibition of protein synthesis.

Activation of plant phospholipase Dbeta by phosphatidylinositol 4,5-bisphosphate: characterization of binding site and mode of action.

Hydrolysis of phospholipids by plant phospholipase Dbeta (PLDbeta) requires phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here we show that PLDbeta is stimulated by different polyphosphoinositides, among which PI(4,5)P2 is most effective. On the basis of amino acid sequence analysis, PI(4,5)P2 binding assay, and protein engineering studies, we have identified in the catalytic region of PLDbeta a new PI(4,5)P2 binding region (PBR1), which is conserved in eukaryotic PLDs.

The Arabidopsis phospholipase D family. Characterization of a calcium-independent and phosphatidylcholine-selective PLD zeta 1 with distinct regulatory domains.

Four types of phospholipase D (PLD), PLD alpha, beta, gamma, and delta, have been characterized in Arabidopsis, and they display different requirements for Ca(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), substrate vesicle composition, and/or free fatty acids. However, all previously cloned plant PLDs contain a Ca(2+)-dependent phospholipid-binding C2 domain and require Ca(2+) for activity. This study documents a new type of PLD, PLD zeta 1, which is distinctively different from previously characterized PLDs.

PLANT PHOSPHOLIPASES.

Phospholipases are a diverse series of enzymes that hydrolyze phospholipids. Multiple forms of phospholipases D, C, and A have been characterized in plants. These enzymes are involved in a broad range of functions in cellular regulation, lipid metabolism, and membrane remodeling.