The roles of phospholipase D in EGFR signaling

Epidermal growth factor receptor (EGFR) is a representative model of receptor tyrosine kinases (RTKs), and offers a means of understanding their common principles and fundamental mechanisms. Furthermore, EGFR plays an essential role in cell proliferation and migration, and the disruption of EGFR signaling has been implicated in the development and growth of cancer. Phospholipase D (PLD) is a key mediator of EGFR function, and can be directly regulated by upstream binding partners in an EGF-dependent manner. PLD regulates downstream molecules by generating phosphatidic acid (PA), but it also dynamically interacts with a variety of intracellular molecules and these interactions spatiotemporally regulate EGFR function and serve as a hub that orchestrates signaling flow. This review summarizes the interrelationship between PLD and its binding molecules in the context of EGFR signaling, and addresses the roles of PLD in the mediation and coordination of this signaling.     

Interactome Analysis of Human Phospholipase D and Phosphatidic Acid-Associated Protein Network

Mammalian phospholipase D (PLD) enzyme family consists of six members. Among them, PLD1/2/6 catalyzes phosphatidic acid (PA) production, while PLD3/4/5 has no catalytic activities. Deregulation of the PLD-PA lipid signaling has been associated with various human diseases including cancer. However, a comprehensive analysis of the regulators and effectors for this crucial lipid metabolic pathway has not been fully achieved. Using a proteomic approach, we defined the protein interaction network for the human PLD family of enzymes and PA and revealed diverse cellular signaling events involving them. Through it, we identified PJA2 as a novel E3 ubiquitin ligase for PLD1 involved in control of the PLD1-mediated mammalian target of rapamycin signaling. Additionally, we showed that PA interacted with and positively regulated sphingosine kinase 1. Taken together, our study not only generates a rich interactome resource for further characterizing the human PLD-PA lipid signaling but also connects this important metabolic pathway with numerous biological processes.     

Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases

Phosphatidic acid (PA) has been increasingly recognized as an important signaling lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization. Recent studies indicate that the signaling PA generated from phospholipase D (PLD) and diacylglycerol kinase (DGK) plays critical roles in regulating the activity of some members of Ras superfamily of small guanosine triphosphatases (GTPases), such as Ras, Rac and Arf. Change of PA levels regulates the activity of small GTPases by modulating membrane localization and activity of small GTPase regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In addition, PA also targets some small GTPases to membranes by direct binding. This review summarizes the roles of PLD and DGK in regulating the activity of several Ras superfamily members and cellular processes they control. Some future directions and the implication of PA regulation of Ras small GTPases in pathology are also discussed.     

Use of Mechanistic Models to Integrate and Analyze Multiple Proteomic Datasets

Proteins in cell signaling networks tend to interact promiscuously through low-affinity interactions. Consequently, evaluating the physiological importance of mapped interactions can be difficult. Attempts to do so have tended to focus on single, measurable physicochemical factors, such as affinity or abundance. For example, interaction importance has been assessed on the basis of the relative affinities of binding partners for a protein of interest, such as a receptor. However, multiple factors can be expected to simultaneously influence the recruitment of proteins to a receptor (and the potential of these proteins to contribute to receptor signaling), including affinity, abundance, and competition, which is a network property. Here, we demonstrate that measurements of protein copy numbers and binding affinities can be integrated within the framework of a mechanistic, computational model that accounts for mass action and competition. We use cell line-specific models to rank the relative importance of protein-protein interactions in the epidermal growth factor receptor (EGFR) signaling network for 11 different cell lines. Each model accounts for experimentally characterized interactions of six autophosphorylation sites in EGFR with proteins containing a Src homology 2 and/or phosphotyrosine-binding domain. We measure importance as the predicted maximal extent of recruitment of a protein to EGFR following ligand-stimulated activation of EGFR signaling. We find that interactions ranked highly by this metric include experimentally detected interactions. Proteins with high importance rank in multiple cell lines include proteins with recognized, well-characterized roles in EGFR signaling, such as GRB2 and SHC1, as well as a protein with a less well-defined role, YES1. Our results reveal potential cell line-specific differences in recruitment.     

Click chemistry and optogenetic approaches to visualize and manipulate phosphatidic acid signaling

The simple structure of phosphatidic acid (PA) belies its complex biological functions as both a key phospholipid biosynthetic intermediate and a potent signaling molecule. In the latter role, PA controls processes including vesicle trafficking, actin dynamics, cell growth, and migration. However, experimental methods to decode the pleiotropy of PA are sorely lacking. Because PA metabolism and trafficking are rapid, approaches to accurately visualize and manipulate its levels require high spatiotemporal precision. Here, we describe recent efforts to create a suite of chemical tools that enable imaging and perturbation of PA signaling. First, we describe techniques to visualize PA production by phospholipase D (PLD) enzymes, which are major producers of PA, called Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation (IMPACT). IMPACT harnesses the ability of endogenous PLD enzymes to accept bioorthogonally tagged alcohols in transphosphatidylation reactions to generate functionalized reporter lipids that are subsequently fluorescently tagged via click chemistry. Second, we describe two light-controlled approaches for precisely manipulating PA signaling. Optogenetic PLDs use light-mediated heterodimerization to recruit a bacterial PLD to desired organelle membranes, and photoswitchable PA analogs contain azobenzene photoswitches in their acyl tails, enabling molecular shape and bioactivity to be controlled by light. We highlight select applications of these tools for studying GPCR–G_q signaling, discovering regulators of PLD signaling, tracking intracellular lipid transport pathways, and elucidating new oncogenic signaling roles for PA. We envision that these chemical tools hold promise for revealing many new insights into lipid signaling pathways.     

mTOR signaling: PLD takes center stage

The mammalian target of rapamycin (mTOR) assembles a signaling network that transduces nutrient signals and various other stimuli to regulate a wide range of cellular functions. Of the two distinct mTOR complexes, mTORC1 is under the control of the TSC-Rheb pathway, which serves as an integrator of multiple upstream signals. A lipid signaling cascade involving phospholipase D (PLD) and phosphatidic acid (PA) has also been known to mediate mitogenic signals upstream of mTORC1. A new study now reveals a direct connection between these two regulatory pathways and demonstrates that PLD1 is an effector of Rheb in the activation of mTORC1. A novel role of PLD as a nutrient sensor has also been suggested. In this extra-view, we discuss the emerging importance of PA and PLD in the mTORC1 signaling network and the biological processes it governs. We also consider the implications from several recent findings and propose mechanistic models of PLD-mTOR signaling to be tested in the near future.     

Structural insights into phospholipase D function

Phospholipase D (PLD) and its metabolic active product phosphatidic acid (PA) engage in a wide range of physiopathologic processes in the cell. PLDs have been considered as a potential and promising drug target. Recently, the crystal structures of PLDs in mammalian and plant have been solved at atomic resolution. These achievements allow us to understand the structural differences among different species of PLDs and the functions of their key domains. In this review, we summarize the sequence and structure of different species of PLD isoforms, and discuss the structural mechanisms for PLD interactions with their binding partners and the functions of each key domain in the regulation of PLDs activation and catalytic reaction.     

Mechanism of membrane binding of the phospholipase D1 PX domain

Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphosphate (PtdIns-(3,4,5)P(3)) > phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate > other phosphoinositides. The PtdIns(3,4,5)P(3) binding was facilitated by the cationic residues (Lys(119), Lys(121), and Arg(179)) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P(3) to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P(3) and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1.     

An upstream regulator and downstream target of phospholipase D1 activity during pheromone response in Saccharomyces cerevisiae

Phospholipase D1 (PLD1), which is the product of the SPO14 gene, has been shown to play a role in the process of polarized cell growth (PCG) during the pheromone response in Saccharomyces cerevisiae. PLD1 hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA) and a free choline headgroup. This study investigated the interactions of PLD1 and PA with two proteins known to be involved in the cellular signaling leading to PCG in yeast, the small GTPase Cdc42p and the PAK family kinase Ste20p. Constitutively activated Cdc42p stimulates PLD1 activity. Protein-lipid binding blots confirmed the specific binding of Ste20p to the PLD1 product, PA. Finally, kinase activity assays provided evidence for the stimulation of Ste20p by PA. These findings highlight the important interactions among PLD1, Cdc42p and Ste20p during PCG in S. cerevisiae.     

Doing (F/L)PPPPs: EVH1 domains and their proline-rich partners in cell polarity and migration.

Actin filament assembly is a tightly regulated process that functions in many aspects of cell physiology. Members of the Ena/VASP (Drosophila Enabled/vasodilator-stimulated phosphoprotein) family are key players in regulating actin filament assembly, in many cases through their association with binding partners that display a particular proline-rich motif, FPPPP. Ena/VASP proteins interact with these partners via the highly conserved Ena/VASP homology 1 (EVH1) domain. The diverse array of binding partners for EVH1 domains, including cytoskeletal proteins such as zyxin, transmembrane guidance receptors such as Roundabout, and the T-cell signaling protein Fyb/SLAP, shows that these interactions are likely to be important in a number of cellular processes that require regulated actin filament assembly.     

Functional analysis of phospholipase Dδ family in tobacco pollen tubes

Phosphatidic acid (PA), an important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD), but the isoforms responsible for production of PM PA were not identified yet and their functional roles remain unknown. Following genome‐wide bioinformatic analysis of the PLD family in tobacco, we focused on the pollen‐overrepresented PLDδ class. Combining live‐cell imaging, gene overexpression, lipid‐binding and structural bioinformatics, we characterized five NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane‐bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Overexpression analyses suggested isoform PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. Moreover, only PLDδ3 shows significant constitutive PLD activity in vivo and, in turn, PA promotes binding of PLDδ3 to the PM. This forms a positive feedback loop leading to PA accumulation and the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes that are crucial for plant cell tip growth.     

Phospholipase D in the signaling networks of plant response to abscisic acid and reactive oxygen species

Phospholipase D (PLD) has been implicated in different cellular processes in plant growth, development, and stress responses. Recent results have provided insights into the molecular mechanism by which PLD and its lipid product phosphatidic acid (PA) participate in cell signaling. Effector proteins that have been identified for PLD and PA in plants include a heterotrimeric G protein, protein phosphatase, and protein kinase. Evidence has been presented for a direct link from a PLD, PA, to a target protein in specific physiological processes. PLD and PA play multiple roles in the signaling networks of plant response to abscisic acid and reactive oxygen species.     

The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation.     

Roles for the lipid-signaling enzyme MitoPLD in mitochondrial dynamics, piRNA biogenesis, and spermatogenesis

Phospholipase D (PLD), a superfamily of signaling enzymes that most commonly generate the lipid second messenger Phosphatidic Acid (PA), is found in diverse organisms from bacteria to man and functions in multiple cellular pathways. A fascinating member of the family, MitoPLD, is anchored to the mitochondrial surface and has two reported roles. In the first role, MitoPLD-generated PA regulates mitochondrial shape through facilitating mitochondrial fusion. In the second role, MitoPLD performs a critical function in a pathway that creates a specialized form of RNAi required by developing spermatocytes to suppress transposon mobilization during meiosis. This spermatocyte-specific RNAi, known as piRNA, is generated in the nuage, an electron-dense accumulation of RNA templates and processing proteins that localize adjacent to mitochondria in a structure also called intermitochondrial cement. In this review, we summarize recent findings on these roles for MitoPLD functions, highlighting directions that need to be pursued to define the underlying mechanisms.     

Binding of proteolytically processed phospholipase D from Streptomyces chromofuscus to phosphatidylcholine membranes facilitates vesicle aggregation and fusion.

Ca(2+)-dependent phospholipase D is secreted from Streptomyces chromofuscus as an intact enzyme of 57 kDa (PLD(57)). Under certain growth conditions, PLD is proteolytically cleaved and activated to form PLD(42/20) (named for the apparent size of the peptides). The PLD(42) catalytic core and 20 kDa C-terminal domain remain tightly associated through noncovalent interactions. In the presence of Ba(2+) (to enhance protein binding to zwitterionic vesicles without hydrolysis of substrate), PLD(42/20), but not PLD(57), induces POPC vesicle leakiness as measured by entrapped CF leakage. PLD(42/20) also induces vesicle fusion (as measured by light scattering, fluorescence quenching, and cryo-TEM) under these conditions (1 mM POPC, 5 mM Ba(2+)); neither PLD(42) nor PLD(20) alone can act as a fusogen. For intact PLD(57) to cause CF leakiness, the soluble activator diC(4)PA must be present. However, even with diC(4)PA, PLD(57) does not induce significant vesicle fusion. In the absence of metal ions, all PLD forms bind to PC vesicles doped with 10 mol % PA. Again, only PLD(42/20) is fusogenic and causes aggregation and fusion on a rapid time scale. Taken together, these data suggest that activated PLD(42/20) inserts more readily into the lipid bilayer than other PLD forms and creates structures that allow bilayers to fuse. Cleavage of the PLD(57) by a secreted protease to generate PLD(42/20) occurs in the late stages of S. chromofuscus cell cultures. Production of this more active and fusogenic enzyme may play a role in nutrient scavenging in stationary phase cultures.     

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.     

Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization,Macrophage Phagocytosis,and Cytokine-Stimulated Neutrophil Recruitment

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.     

灵芝细胞中磷脂酸互作蛋白鉴定

灵芝是名贵药用真菌,三萜是灵芝的关键药效成分。前期研究发现,磷脂酶D (Phospholipase D,PLD) 产生的磷脂酸 (Phosphatidic acid,PA) 可调控三萜合成,为进一步阐明PA调控灵芝三萜合成的分子机制,研究采用PA-beads富集结合LC-MS/MS技术,鉴定灵芝细胞中PA互作蛋白,结果共鉴定到了19个PA互作蛋白,主要包括细胞色素P450单加氧酶 (GL22084)、特异性蛋白激酶MAPK (GL23765)、过氧化氢酶和细胞表面疏水性蛋白等。通过基因克隆、原核表达载体构建、蛋白诱导表达和分离纯化,获得了融合GST标签的GL22084和GL23765蛋白,采用GST-pull down实验,验证了灵芝GL22084和GL23765蛋白与PA互作。研究结果揭示了灵芝细胞中PA互作蛋白,为后续解析PLD介导的PA信号分子调控灵芝三萜合成的分子机理奠定了基础;同时,鉴定到的PA互作蛋白也为其他物种的PLD/PA信号通路相关研究提供借鉴。