The Oncogene HER2/neu (ERBB2) Requires the Hypoxia-inducible Factor HIF-1 for Mammary Tumor Growth and Anoikis Resistance

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.     

Identification of caspase 3 motifs and critical aspartate residues in human phospholipase D1b and phospholipase D2a

Stimulation of mammalian cells frequently initiates phospholipase D-catalyzed hydrolysis of phosphatidylcholine in the plasma membrane to yield phosphatidic acid (PA) a novel lipid messenger. PA plays a regulatory role in important cellular processes such as secretion, cellular shape change, and movement. A number of studies have highlighted that PLD-based signaling also plays a pro-mitogenic and pro-survival role in cells and therefore anti-apoptotic. We show that human PLD1b and PLD2a contain functional caspase 3 cleavage sites and identify the critical aspartate residues within PLD1b that affect its activation by phorbol esters and attenuate phosphatidylcholine hydrolysis during apoptosis.     

Biophysics and function of phosphatidic acid: A molecular perspective

Phosphatidic acid is the simplest (diacyl)glycerophospholipid present in cells and is now a well established second messenger with direct biological functions. It is specifically recognized by diverse proteins and plays an important role in cellular signaling and membrane dynamics in all eukaryotes. An important determinant of the biological functions of phosphatidic acid is its anionic headgroup. In this review we will focus on the peculiar ionization properties of phosphatidic acid and their crucial role in lipid–protein interactions. We will take a molecular approach focusing entirely on the physical chemistry of the lipid and develop a model explaining the ionization properties of phosphatidic acid, termed the electrostatic-hydrogen bond switch model. Diverse examples from recent literature in support of this model will be presented and the broader implications of our findings will be discussed.     

Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase,hSPP2

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.     

Mitochondria: signaling with phosphatidic acid

Mitochondria, once viewed as functioning relatively autonomously in the cell, have increasingly been recognized to be involved in numerous signaling networks that impact on a wide range of cell biological processes. In addition to the many types of proteins that mediate these pathways, the importance of signaling functions regulated via lipids and lipid second messengers generated on the mitochondrial surface is also becoming well appreciated. We focus here on phosphatidic acid, a lipid second messenger produced via several different pathways that can in turn stimulate the formation of multiple other bioactive lipids. Taken together, fascinating roles for phosphatidic acid and the connected lipids in mitochondrial function and interaction with other organelles are being uncovered. These pathways present new opportunities for the development of therapeutic approaches relevant to reproduction, metabolism, and neurodegenerative disease.     

Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1-enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.     

Proliferative signaling by ERBB proteins and RAF/MEK/ERK effectors in polycystic kidney disease

A primary pathological feature of polycystic kidney disease (PKD) is the hyperproliferation of epithelial cells in renal tubules, resulting in formation of fluid-filled cysts. The proliferative aspects of the two major forms of PKD—autosomal dominant PKD (ADPKD), which arises from mutations in the polycystins PKD1 and PKD2, and autosomal recessive PKD (ARPKD), which arises from mutations in PKHD1—has encouraged investigation into protein components of the core cell proliferative machinery as potential drivers of PKD pathogenesis. In this review, we examine the role of signaling by ERBB proteins and their effectors, with a primary focus on ADPKD. The ERBB family of receptor tyrosine kinases (EGFR/ERBB1, HER2/ERBB2, ERBB3, and ERBB4) are activated by extracellular ligands, inducing multiple pro-growth signaling cascades; among these, activation of signaling through the RAS GTPase, and the RAF, MEK1/2, and ERK1/2 kinases enhance cell proliferation and restrict apoptosis during renal tubuloepithelial cyst formation. Characteristics of PKD include overexpression and mislocalization of the ERBB receptors and ligands, leading to enhanced activation and increased activity of downstream signaling proteins. The altered regulation of ERBBs and their effectors in PKD is influenced by enhanced activity of SRC kinase, which is promoted by the loss of cytoplasmic Ca²⁺ and an increase in cAMP-dependent PKA kinase activity that stimulates CFTR, driving the secretory phenotype of ADPKD. We discuss the interplay between ERBB/SRC signaling, and polycystins and their depending signaling, with emphasis on thes changes that affect cell proliferation in cyst expansion, as well as the inflammation-associated fibrogenesis, which characterizes progressive disease. We summarize the current progress of preclinical and clinical trials directed at inhibiting this signaling axis, and discuss potential future strategies that may be productive for controlling PKD.     

A lipid-signaled myosin phosphatase surge disperses cortical contractile force early in cell spreading

When cells cease migrating through the vasculature, adhere to extracellular matrix, and begin to spread, they exhibit rapid changes in contraction and relaxation at peripheral regions newly contacting the underlying substrata. We describe here a requirement in this process for myosin II disassembly at the cell cortex via the action of myosin phosphatase (MP), which in turn is regulated by a plasma membrane signaling lipid. Cells in suspension exhibit high levels of activity of the signaling enzyme phospholipase D2 (PLD2), elevating production of the lipid second messenger phosphatidic acid (PA) at the plasma membrane, which in turn recruits MP and stores it there in a presumed inactive state. On cell attachment, down-regulation of PLD2 activity decreases PA production, leading to MP release, myosin dephosphorylation, and actomyosin disassembly. This novel model for recruitment and restraint of MP provides a means to effect a rapid cytoskeletal reorganization at the cell cortex upon demand.     

A novel protein kinase target for the lipid second messenger phosphatidic acid

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.     

Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.     

Role of the ANKMY2-FKBP38 Axis in Regulation of the Sonic Hedgehog (Shh) Signaling Pathway

Sonic hedgehog (Shh) is a secreted morphogen that controls the patterning and growth of various tissues in the developing vertebrate embryo, including the central nervous system. Ablation of the FK506-binding protein 38 (FKBP38) gene results in activation of the Shh signaling pathway in mouse embryos, but the molecular mechanism by which FKBP38 suppresses Shh signaling has remained unclear. With the use of a proteomics approach, we have now identified ANKMY2, a protein with three ankyrin repeats and a MYND (myeloid, Nervy, and DEAF-1)-type Zn²⁺ finger domain, as a molecule that interacts with FKBP38. Co-immunoprecipitation analysis confirmed that endogenous FKBP38 and ANKMY2 interact in the mouse brain. Depletion or overexpression of ANKMY2 resulted in down- and up-regulation of Shh signaling, respectively, in mouse embryonic fibroblasts. Furthermore, combined depletion of both FKBP38 and ANKMY2 attenuated Shh signaling in these cells, suggesting that ANKMY2 acts downstream of FKBP38 to activate the Shh signaling pathway. Targeting of the zebrafish ortholog of mouse Ankmy2 (ankmy2a) in fish embryos with an antisense morpholino oligonucleotide conferred a phenotype reflecting loss of function of the Shh pathway, suggesting that the regulation of Shh signaling by ANKMY2 is conserved between mammals and fish. Our findings thus indicate that the FKBP38-ANKMY2 axis plays a key role in regulation of Shh signaling in vivo.     

Trigalactosyldiacylglycerol 3 protein orthologs are required for basal disease resistance in Nicotiana benthamiana

Phosphatidic acid plays an important role in Nicotiana benthamiana immune responses against phytopathogenic bacteria. We analyzed the contributions of endoplasmic reticulum-derived chloroplast phospholipids, including phosphatidic acid, to the resistance of N. benthamiana against Ralstonia solanacearum. Here, we focused on trigalactosyldiacylglycerol 3 (TGD3) protein as a candidate required for phosphatidic acid signaling. On the basis of Arabidopsis thaliana TGD3 sequences, we identified two putative TGD3 orthologs in the N. benthamiana genome, NbTGD3-1 and NbTGD3-2. To address the role of TGD3s in plant defense responses, we created double NbTGD3-silenced plants using virus-induced gene silencing. The NbTGD3-silenced plants showed a moderately reduced growth phenotype. Bacterial growth and the appearance of bacterial wilt disease were accelerated in NbTGD3-silenced plants, compared with control plants, challenged with R. solanacearum. The NbTGD3-silenced plants showed reduced both expression of allene oxide synthase that encoded jasmonic acid biosynthetic enzyme and NbPR-4, a marker gene for jasmonic acid signaling, after inoculation with R. solanacearum. Thus, NbTGD3-mediated endoplasmic reticulum—chloroplast lipid transport might be required for jasmonic acid signaling-mediated basal disease resistance in N. benthamiana.     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

Rapid attenuation of receptor-induced diacylglycerol and phosphatidic acid by phospholipase D-mediated transphosphatidylation: formation of bisphosphatidic acid.

Generation and attenuation of lipid second messengers are key processes in cellular signalling. Receptor-mediated increase in 1,2-diacylglycerol (DG) levels is attenuated by DG kinase and DG lipase. We here report a novel mechanism of DG attenuation by phospholipase D (PLD), which also precludes the production of another (putative) second messenger, phosphatidic acid (PA). In the presence of an alcohol, PLD converts phosphatidylcholine (PC) into a phosphatidylalcohol (by transphosphatidylation) rather than into PA. We found in bradykinin-stimulated human fibroblasts that PLD mediates transphosphatidylation from PC (donor) to the endogenous 'alcohol' DG (acceptor), yielding bis(1,2-diacylglycero)-3-sn-phosphate (bisphosphatidic acid; bisPA). This uncommon phospholipid is thus a condensation product of the phospholipase C (PLC) and PLD signalling pathways, where PLC produces DG and PLD couples this DG to a phosphatidyl moiety. Long-term phorbol ester treatment blocks bradykinin-induced activation of PLD and consequent bisPA formation, thereby unveiling rapid formation of DG. BisPA formation is rapid (15 s) and transient (peaks at 2-10 min) and is also induced by other stimuli capable of raising DG and activating PLD simultaneously, e.g. endothelin, lysophosphatidic acid, fetal calf serum, phorbol ester, dioctanoylglycerol or bacterial PLC. This novel metabolic route counteracts rapid accumulation of receptor-induced DG and PA, and assigns for the first time a physiological role to the transphosphatidylation activity of PLD, that is signal attenuation.     

A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: application in human myocardium

Phosphatidic acid phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol, the second messenger responsible for activation of protein kinase C. Despite the crucial role of PAP lipid signaling, there are no data on PAP signaling function in the human heart. Here we present a nonradioactive assay for the investigation of PAP activity in human myocardium using a fluorescent derivative of PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate (BODIPY-PA), as substrate in an in vitro PAP-catalyzed reaction. Unreacted BODIPY-PA was resolved from the PAP products by a binary gradient HPLC system and BODIPY-diacylglycerol was detected by fluorimetry. The reaction proceeded at a linear rate for up to 60 min and increased linearly with increasing amounts of cardiac protein in a range of 0.25 to 8.0 μg. This assay proved to be sensitive for accurate quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human atrial tissue and right ventricular endomyocardial biopsies. Total PAP activity was approximately fourfold higher in ventricular myocardium than in atrial tissue. There was negligible PAP-1 activity in atrial myocardium compared with ventricular myocardium, indicating regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart.     

Production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidic acid in platelet rafts: evidence for a critical role of cholesterol-enriched domains in human platelet activation.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.     

Biochemical Characterization of Two Wheat Phosphoethanolamine N-Methyltransferase Isoforms with Different Sensitivities to Inhibition by Phosphatidic Acid

In plants the triple methylation of phosphoethanolamine to phosphocholine catalyzed by phosphoethanolamine N-methyltransferase (PEAMT) is considered a rate-limiting step in the de novo synthesis of phosphatidylcholine. Besides being a major membrane phospholipid, phosphatidylcholine can be hydrolyzed into choline and phosphatidic acid. Phosphatidic acid is widely recognized as a second messenger in stress signaling, and choline can be oxidized within the chloroplast to yield the putative osmoprotectant glycine betaine. Here we describe the cloning and biochemical characterization of a second wheat PEAMT isoform that has a four times higher specific activity than the previously described WPEAMT/TaPEAMT1 enzyme and is less sensitive to product inhibition by S-adenosyl homocysteine, but more sensitive to inhibition by phosphocholine. Both enzymes follow a sequential random Bi Bi mechanism and show mixed-type product inhibition patterns with partial inhibition for TaPEAMT1 and a strong non-competitive component for TaPEAMT2. An induction of TaPEAMT protein expression and activity is observed after cold exposure, ahead of an increase in gene expression. Our results demonstrate direct repression of in vitro enzymatic activities by phosphatidic acid for both enzymes, with TaPEAMT1 being more sensitive than TaPEAMT2 in the physiological concentration range. Other lipid ligands identified in protein-lipid overlays are phosphoinositide mono- as well as some di-phosphates and cardiolipin. These results provide new insights into the complex regulatory circuits of phospholipid biosynthesis in plants and underline the importance of head group biosynthesis in adaptive stress responses.