Phosphatidylinositol Turnover Research Articles

Abstract 6266: Neurotensin receptor 1 regulates dimerization of HER2-HER4 and proliferation of non-small cell lung cancer cells

Abstract The neurotensin receptor 1 (NTSR1) is a 418 amino acid type A G protein-coupled receptor which causes phosphatidylinositol turnover and proliferation of non-small cell lung cancer (NSCLC) cells (Moody et al., Peptides 2021; 137: 710480). Adding the agonist NTS to NSCLC cells increases transactivation of the receptor tyrosine kinases (RTK) EGFR, HER2 and HER3 (Moody et al., Biology 2023 12: 957). The RTK tyrosine phosphorylation and growth caused by NTS is impaired by SR48692, a small molecule NTSR1 antagonist. The effects of NTS were investigated on HER4 using NSCLC cells. By Western blot, cell lines NCI-H522 and H661 had high levels of NTSR1, HER 4 and neuregulin 1 (NRG1), a ligand for HER4 but not NTSR2, HER3 or NRG2. By RT-PCT, high levels of HER4 and its isoforms JM-a, CYT1 and CYT2 were present in NCI-H522 and H661 cells. Adding NTS (0.1 μM) or NRG1 (0.01 μg/ml) to NCI-H522 or H661 cells increased P-Tyr1284-HER4 and P-ERK 3-fold. Using immunoprecipitation techniques, adding NTS to NSCLC cells increased formation of HER4-HER2 heterodimers which leads to cancer proliferation (Lucas et al., Pharmacol Rev 2022; 74:18). The increase in PY1284-HER4 caused by NTS was impaired by SR48692, HER4 siRNA, NRG1 siRNA or ibrutinib (TKI). The NTSR1 regulation of HER4 transactivation is impaired by GM6001 (MMP inhibitor]), PP2 (Src inhibitor) or N-acetylcysteine (antioxidant). The clonal growth of NSCLC cells is increased by NTS or NRG1 but decreased by SR48692 or ibrutinib. The results indicate that NTS increases the formation of HER2/HER4 heterodimers leading to the proliferation of NSCLC cells. Citation Format: Terry W. Moody, Irene Ramos-Alvarez, Robert T. Jensen. Neurotensin receptor 1 regulates dimerization of HER2-HER4 and proliferation of non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6266.

Diacylglycerol kinase ζ interacts with sphingomyelin synthase 1 and sphingomyelin synthase‐related protein

Introduction Mammalian sphingomyelin (SM) synthase (SMS) consists of two isoform (SMS1, SMS2). SMS produces diacylglycerol (DG) by the transfer of phosphocholine from phosphatidylcholine (PC) to ceramide. SMS-related protein (SMSr) also generates DG via synthesis of ceramide phosphoethanolamine, a SM analog, using phosphatidylethanolamine. Mammalian DG kinase (DGK) consists of ten isozymes (α, β, γ, δ, η, ϵ, κ, ι, ζ, θ) and governs a wide range of physiological and pathological events such as type 2 diabetes. Among them, SMS1, SMSr, DGKδ and DGKη contain a steraile a motif domain (SAMD), a protein-protein interaction module. We previously reported that DGKδ interacts with SMSr, but not SMS1, via SAMDs [Murakami et al. J. Biol. Chem. 295, 2932–47, 2020]. Moreover, SMSr provided DG to DGKδ. However, it is still unclear whether other DGK isozymes interact with SMSs. In the present study, we analyzed interaction between DGKs and SMSs by co-immunoprecipitation analysis. Results and Conclusion First, we determined whether SAMD-containing DGKs (DGKδ and DGKη) interact with SAMD-containing SMSs (SMS1 and SMSr). Although DGKη-SAMD co-sedimented with DGKη-SAMD itself, SMS1- and SMSr-SAMD failed to interact with DGKη-SAMD. Furthermore, only the combination of full-length DGKδ and full-length SMSr could form a heteromeric complex. These results suggest that the interaction via heteromerization of SAMDs is specific to DGKδ and SMSr. Next, we analyzed whether other DGKs, which contain no SAMD, interact with SMSs. We found that DGKζ was strongly co-immunoprecipitated with SMSr and SMS1, but not SMS2. These results suggest that DGKζ can interact with SMS1 and SMSr without heteromerization between SAMDs. Taken together, these results suggest that, in addition to DGKδ, DGKζ is a new candidate that acts downstream of SMSr and SMS1. SMSr produces palmitic acid (16:0)-containing DG molecular species and SMS1 generates PC-derived DG species including 16:0-containing DGs. Moreover, DGKζ preferably phosphorylates 16:0-containing DG species during neuronal cell differentiation. Therefore, it is possible that these enzymes compose a new pathway independent of the phosphatidylinositol turnover, which primary utilizes arachidonic acid (20:4)-containing DG species.

New Era of Diacylglycerol Kinase, Phosphatidic Acid and Phosphatidic Acid-Binding Protein.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Mammalian DGK consists of ten isozymes (α–κ) and governs a wide range of physiological and pathological events, including immune responses, neuronal networking, bipolar disorder, obsessive-compulsive disorder, fragile X syndrome, cancer, and type 2 diabetes. DG and PA comprise diverse molecular species that have different acyl chains at the sn-1 and sn-2 positions. Because the DGK activity is essential for phosphatidylinositol turnover, which exclusively produces 1-stearoyl-2-arachidonoyl-DG, it has been generally thought that all DGK isozymes utilize the DG species derived from the turnover. However, it was recently revealed that DGK isozymes, except for DGKε, phosphorylate diverse DG species, which are not derived from phosphatidylinositol turnover. In addition, various PA-binding proteins (PABPs), which have different selectivities for PA species, were recently found. These results suggest that DGK–PA–PABP axes can potentially construct a large and complex signaling network and play physiologically and pathologically important roles in addition to DGK-dependent attenuation of DG–DG-binding protein axes. For example, 1-stearoyl-2-docosahexaenoyl-PA produced by DGKδ interacts with and activates Praja-1, the E3 ubiquitin ligase acting on the serotonin transporter, which is a target of drugs for obsessive-compulsive and major depressive disorders, in the brain. This article reviews recent research progress on PA species produced by DGK isozymes, the selective binding of PABPs to PA species and a phosphatidylinositol turnover-independent DG supply pathway.

LPIAT1/MBOAT7 depletion increases triglyceride synthesis fueled by high phosphatidylinositol turnover

ObjectiveNon-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which...

Functional relationship between sphingomyelin synthase‐related protein and diacylglycerol kinase δ

IntroductionThe δ isozyme of diacyglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). Intriguingly, we recently demonstrated that DGKδ preferably metabolized palmitic acid (16:0)‐ and/or palmitoleic acid (16:1)‐containing DG molecular species in response to high glucose stimulation in myoblasts. However, it is still unclear where 16:0‐ and/or 16:1‐containing DG molecular species come from, although another species arachidonic acid (20:4)‐containing DGs are derived from phosphatidylinositol (PI) turnover. Sphingomyelin synthase (SMS) 1 and SMS‐related protein (SMSr) synthesize the sphingomyelin and ceramide‐phosohoethanolamine by the transfer of phosphocholine/phosphoethanolamine to ceramide, respectively. These are also assumed to function as DG‐generating enzymes. SMS1, SMSr and DGKδ contain a sterile α motif domain (SAM), which is a putative protein interaction module. Interestingly, we found that DGKδ interacted with SMSr via their SAMs directly. In the present study, we investigated the functional relationship between DGKδ and SMSr.Results and ConclusionWe first investigated the changes in the amounts of PA molecular species in COS‐7 cells overexpressing DGKδ and/or SMSr using LC‐MS/MS. We found that the overexpression of SMSr and DGKδ significantly enhanced the production of 16:0‐ and/or 16:1‐containing PA species such as 16:0/16:1‐, 16:0/16:0‐, 16:0/18:1‐ and 16:0/18:2‐PA in COS‐7 cells. It is possible that DGKδ and SMSr are functionally linked and that SMSr acts in an upstream pathway of DGKδ by providing 16:0‐ and/or 16:1‐DG. To support the possibility, we determined DG levels in SMSr‐overexpressing cells. Compared with control cells, total DG levels in SMSr‐overexpressing cells were significantly increased. In particular, 16:0‐ and/or 16:1‐containig DG species, such as 14:0/16:0 (30:0)‐, 16:0/16:1 (32:1)‐, 16:1/18:2 (34:3)‐, and 16:1/18:1 (34:2)‐DG were significantly increased. These results further suggest the functional relationship (SMSr supplies DG to DGKδ) between DGKδ and SMSr. To examine whether forming SMSr‐DGKδ heteromeric complex affects DGKδ activity, we measured purified DGKδ activity in vitro using DG and ATP as substrates. Interestingly, when presence of purified SMSr with purified DGKδ, DGK activity was significantly increased. However, the deletion of SAM of SMSr did not significantly augment the DGKδ activity. Taken together, these results suggest that SMSr is one of the candidates of upstream DG‐providing enzymes of DGKδ and that SMSr enhances DGKδ activity by forming heteromeric complex via their SAMs.Support or Funding InformationThis work was supported in part by grants from MEXT/JSPS KAKENHI (Grant Numbers: JP18J20003 to C.M.); The Chiba Bank (to C.M.); the Chiba University Venture Business Laboratory (to C.M.); Chiba University Open Recruitment for International Exchange Program at the Institute for Global Prominent Research (to C.M.); and the American Society for Biochemistry and Molecular Biology (ASBMB 2019 Graduate/Postdoctoral Travel Award to C.M)

Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway.

Phosphoinositides play crucial roles in intracellular membrane dynamics and cell signaling, with phosphatidylinositol (PI) 3-phosphates being the predominant phosphoinositide lipids at endosomes and lysosomes, whereas PI 4-phosphates, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), are enriched at the cell surface including sites of endocytosis. How PI 4-phosphates and PI 3-phosphates are dynamically interconverted within the endocytic pathway and how this is controlled in space and time remains poorly understood. Here, combining live imaging, genome engineering, and acute chemical and genetic manipulations, we found that local synthesis of PI(3,4)P2 by phosphatidylinositol 3-kinase C2α at plasma membrane clathrin-coated pits is spatially segregated from its hydrolysis by the PI(3,4)P2-specific inositol polyphosphate 4-phosphatase 4A (INPP4A). We observed that INPP4A is dispensable for clathrin-mediated endocytosis and is undetectable in endocytic clathrin-coated pits. Instead, we found that INPP4A partially localizes to endosomes and that loss of INPP4A in HAP1 cancer cells perturbs signaling via AKT kinase and mTOR complex 1. These results reveal a function for INPP4-mediated PI(3,4)P2 hydrolysis in local regulation of growth factor and nutrient signals at endosomes in cancer cells. They further suggest a model whereby synthesis and turnover of PI(3,4)P2 are spatially segregated within the endocytic pathway to couple endocytic membrane traffic to growth factor and nutrient signaling.

Peptide receptors as cancer drug targets

Neuropeptides function as neuromodulators in the brain, whereby they are released in a paracrine manner and activate G protein-coupled receptors (GPCRs) in adjacent cells. Because neuropeptides are made in, and secreted from, cancer cells, then bind to cell surface receptors, they function in an autocrine manner. Bombesin (BB)-like peptides synthesized by neuroendocrine tumor small cell lung cancer (SCLC) bind to BB receptors (BBRs), causing phosphatidylinositol turnover and phosphorylation of extracellular signal-regulated kinase (ERK). Phosphorylated ERK enters the nucleus and alters gene expression of SCLC cells, stimulating growth. Vasoactive intestinal peptide (VIP) addition to SCLC cells increases their release rate of BB-like peptides via activation of VIP receptors (VIPR), leading to activation of adenylyl cyclase and subsequent elevation of cAMP. Protein kinase A is then stimulated, leading to phosphorylation of cyclic AMP response element binding protein (CREB), which alters gene expression and stimulates proliferation. The growth of SCLC is inhibited by BBR and VIPR antagonists. This review will focus on how GPCRs for VIP and BB are molecular targets for early detection and treatment of cancer.

Where do substrates of diacylglycerol kinases come from? Diacylglycerol kinases utilize diacylglycerol species supplied from phosphatidylinositol turnover-independent pathways.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprises ten isozymes (α-κ) and regulates a wide variety of physiological and pathological events, such as cancer, type II diabetes, neuronal disorders and immune responses. DG and PA consist of various molecular species that have different acyl chains at the sn-1 and sn-2 positions, and consequently, mammalian cells contain at least 50 structurally distinct DG/PA species. Because DGK is one of the components of phosphatidylinositol (PI) turnover, the generally accepted dogma is that all DGK isozymes utilize 18:0/20:4-DG derived from PI turnover. We recently established a specific liquid chromatography-mass spectrometry method to analyze which PA species were generated by DGK isozymes in a cell stimulation-dependent manner. Interestingly, we determined that DGKδ, which is closely related to the pathogenesis of type II diabetes, preferentially utilized 14:0/16:0-, 14:0/16:1-, 16:0/16:0-, 16:0/16:1-, 16:0/18:0- and 16:0/18:1-DG species (X:Y=the total number of carbon atoms: the total number of double bonds) supplied from the phosphatidylcholine-specific phospholipase C pathway, but not 18:0/20:4-DG, in high glucose-stimulated C2C12 myoblasts. Moreover, DGKα mainly consumed 14:0/16:0-, 16:0/18:1-, 18:0/18:1- and 18:1/18:1-DG species during cell proliferation in AKI melanoma cells. Furthermore, we found that 16:0/16:0-PA was specifically produced by DGKζ in Neuro-2a cells during retinoic acid- and serum starvation-induced neuronal differentiation. These results indicate that DGK isozymes utilize a variety of DG molecular species derived from PI turnover-independent pathways as substrates in different stimuli and cells. DGK isozymes phosphorylate various DG species to generate various PA species. It was revealed that the modes of activation of conventional and novel protein kinase isoforms by DG molecular species varied considerably. However, PA species-selective binding proteins have not been found to date. Therefore, we next attempted to identify PA species-selective binding proteins from the mouse brain and identified α-synuclein, which has causal links to Parkinson's disease. Intriguingly, we determined that among phospholipids, including several PA species (16:0/16:0-PA, 16:0/18:1-PA, 18:1/18:1-PA, 18:0/18:0-PA and 18:0/20:4-PA); 18:1/18:1-PA was the most strongly bound PA to α-synuclein. Moreover, 18:1/18:1-PA strongly enhanced secondary structural changes from the random coil form to the α-helix form and generated a multimeric and proteinase K-resistant α-synuclein protein. In contrast with the dogma described above, our recent studies strongly suggest that PI turnover-derived DG species and also various DG species derived from PI turnover-independent pathways are utilized by DGK isozymes. DG species supplied from distinct pathways may be utilized by DGK isozymes based on different stimuli present in different types of cells, and individual PA molecular species would have specific targets and exert their own physiological functions.

The PNPLA-family phospholipases involved in glycerophospholipid homeostasis of HeLa cells

Mammalian cells maintain the glycerophospholipid (GPL) compositions of their membranes nearly constant. To achieve this, GPL synthesis and degradation must be coordinated. There is strong evidence that A-type phospholipases (PLAs) are key players in homeostatic degradation of GPLs, but the identities of the PLAs involved have not been established. However, some members of the Patatin-like phospholipase domain-containing proteins (PNPLAs) have been implicated. Accordingly, we knocked down all the PNPLAs significantly expressed in human HeLa cells using RNA interference and then determined whether the turnover of the major glycerophospholipids is affected by using mass spectrometry and metabolic labeling with stable isotope-labeled precursors. Knockdown of PNPLA9, PNPLA6 or PNPLA4 significantly (30–50%) reduced the turnover of phosphatidylcholine, −ethanolamine and –serine. In a notable contrast, turnover of phosphatidylinositol was not significantly affected by the knockdown of any PNPLA. Depletion of PNPLA9 and PNPLA4 also inhibited G0/G1 to S cell cycle progression, which could thus be regulated by GPL turnover. These results strongly suggest that PNPLA9, -6 and -4 play a key role in GPL turnover and homeostasis in human cells. A hypothetical model suggesting how these enzymes could recognize the relative concentration of the different GPLs is proposed.

EGFR Transactivation by Peptide G Protein-Coupled Receptors in Cancer.

Lung cancer kills approximately 1.3 million citizens in the world annually. The tyrosine kinase inhibitors (TKI) erlotinib and gefitinib are effective anti-tumor agents especially in lung cancer patients with epidermal growth factor receptor (EGFR) mutations. The goal is to increase the potency of TKI in lung cancer patients with wild type EGFR. G protein-coupled receptors (GPCR) transactivate the wild type EGFR in lung cancer cells. The GPCR can be activated by peptide agonists causing phosphatidylinositol turnover or stimulation of adenylylcyclase. Recently, nonpeptide antagonists were found to inhibit the EGFR transactivation caused by peptides. Nonpeptide antagonists for bombesin (BB), neurotensin (NTS) and cholecystokinin (CCK) inhibit lung cancer growth and increase the cytotoxicity of gefitinib. The results suggest that GPCR transactivation of the EGFR may play an important role in cancer cell proliferation.

Abstract 2125: G-protein coupled receptors for cholecystokinin regulate transactivation of EGF receptors in lung cancer cells

Abstract Cholecystokinin (CCK) receptors (CCK-AR and CCK-BR) are G-protein coupled receptors (GPCR) which are present on lung cancer cells (Yoder and Moody, Peptides 1987; 8: 103). CCK stimulates the proliferation of NCI-H345 cells, whereas the CCK-B receptor antagonist CI-988 inhibits proliferation (Moody et al., JPET 2001; 299: 1154). Peptide receptors for some gastrointestinal hormones/neurotransmitters mediate lung cancer growth by causing EGFR transactivation, but it is unknown if this occurs with CCK-BR. CCK-BR mRNA was detected in NCI-H345 and H727 lung cancer cells. Addition of CCK-8 or gastrin I (100 nM) to NCI-H345 or H727 cells, which have wild type EGFR, increased EGFR Tyr1068 phosphorylation after 5 min. The ability of CCK-8 to cause EGFR or ERK tyrosine phosphorylation was blocked by CI-988 (CCK-BR antagonist), gefitinib (EGFR tyrosine kinase inhibitor), PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), tiron (superoxide scavenger) and U-73122 (phospholipase C inhibitor) but not H89 (protein kinase A inhibitor). U-73122 or CI-988 inhibited the ability of CCK to cause phosphatidylinositol turnover and elevate cytosolic Ca2+. CI-988 or gefitinib inhibited the growth of NCI-H345 or H727 cells. CI-988 and gefitinib synergistically inhibited the growth of lung cancer cells. The results indicate that the CCK-BR regulates lung cancer proliferation in an EGFR-dependent mechanism through activation of Src, MMP and reactive oxygen species. Citation Format: Terry W. Moody, Suk Hee Lee, Paola Moreno Perez, Robert T Jensen. G-protein coupled receptors for cholecystokinin regulate transactivation of EGF receptors in lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2125. doi:10.1158/1538-7445.AM2014-2125

Diacylglycerol Kinase δ Phosphorylates Phosphatidylcholine-specific Phospholipase C-dependent, Palmitic Acid-containing Diacylglycerol Species in Response to High Glucose Levels

Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.

A Rapidly Reversible Chemical Dimerizer System to Study Lipid Signaling in Living Cells

AbstractChemical dimerizers are powerful tools for non‐invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small‐molecule‐based dimerization system and demonstrate a sufficiently fast switch‐off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3‐kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5‐trisphosphate (PIP3) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.

A rapidly reversible chemical dimerizer system to study lipid signaling in living cells.

Chemical dimerizers are powerful tools for non-invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small-molecule-based dimerization system and demonstrate a sufficiently fast switch-off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3-kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.

The turnover of glycerol and acyl moieties of cardiolipin

The dynamical behavior of mitochondria has attracted much attention, but little is known about the dynamics of mitochondrial lipids, specifically cardiolipin (CL). Here, we estimated the turnover of select molecular species of CL in mammalian cell cultures and compared it to the turnover of other lipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol. Cells were labeled with myristic acid, 9,10-2H2-oleic acid, or d-[U-13C6]-glucose and analyzed by mass spectrometry at different time points of pulse-chase experiments. The turnover of glycerol groups was monitored by specific isotopologues that carried 13C primarily in the glycerol carbons, whereas the turnover of acyl groups was monitored by molecular species that carried myristoyl or 2H2-oleoyl groups. We found that the turnover of CL, but not of mitochondrial PC and PE, was substantially slower than the turnover of other cellular phospholipids. In dioleoyl-PC and dioleoyl-PE, the acyl turnover was faster than the glycerol turnover, indicating continuous deacylation and reacylation of the oleoyl residues. In contrast, the acyl turnover was similar to the glycerol turnover in tetraoleoyl-CL, suggesting that oleoyl remodeling did not take place continuously in endogenous CL. We conclude that CL, once assembled in mitochondrial membranes, remains largely inert to degradation and acyl remodeling.

Abstract 1306: SR48692 inhibits EGF receptor transactivation and proliferation of lung cancer cells.

Abstract Neurotensin (NTS) is an autocrine growth factor for some lung cancer cells (Moody et al., 1985;Life Sci 36:1727). NTS binds with high affinity to the GPCR NTSR1 causing phosphatidyl inositol turnover and proliferation of lung cancer cells; SR48692 is a NTSR1 antagonist (Moody et al., 2001; Peptides 22:109). NTSR1 expression is associated with poor survival of non-small cell lung cancer (NSCLC) patients (Alfano et al., 2010; Clin Cancer Res 16:4401). NSCLC cells express the epidermal growth factor receptor (EGFR) whose tyrosine kinase activity is inhibited by gefitinib. Here the abillity of SR48692 to block EGFR transactivation caused by NTS was investigated. NTS and NTS8-13 but not NTS1-8 addition to NSCLC cell lines NCI-H1299 or A549 cells increased phosphorylation of Tyr1068 of the EGFR after 2 min which was blocked by 10 uM SR48692 or 10 ug/ml gefitinib. The ability of NTS to regulate EGF receptor transactivation was blocked by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant) and transforming growth factor TGF) α antibody. By ELISA, NTS significantly increased secretion of TGFα from lung cancer cells. NTS but not levocabastine (NTSR2 agonist) increased cytosolic Ca2+ within seconds after addition to NCI-H1299 cells which was antagonized by SR48692. NTS addition to NCI-H1299 cells increased tyrosine phosphorylation of beta-catenin, ERK, FAK, PYK-2 and Src which was antagonized by SR48692. SR48692 inhibited the proliferation of NCI-H1299 and A549 cells which have wild type EGFR. Also, SR48692 potentiated the ability of gefitinib to inhibit growth of NCI-H1299 and A549 cells. These results indicate that SR48692 is a NTSR1 antagonist which increases the potency of gefitinib in lung cancer cells. Citation Format: Terry W. Moody, Robert T. Jensen. SR48692 inhibits EGF receptor transactivation and proliferation of lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1306. doi:10.1158/1538-7445.AM2013-1306

Reprint of “Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system”

A small quantity of unsaturated diacylglycerol (DG) sharply decreased the Ca2+ and phospholipid concentrations needed for full activation of a Ca2+-activated, phospholipid-dependent multifunctional protein kinase described earlier (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979)J.Biol.Chem.254. 3692–3695). In the presence of unsaturated DG and micromolar order of Ca2+, phosphatidylserine (PS) was most relevant with the capacity to activate the enzyme, whereas phosphatidylethanolamine and phosphatidylinositol (PI) were far less effective. Phosphatidylcholine was practically inactive. It is possible, therefore, that unsaturated DG, which may be derived from PI turnover provoked by various extracellular stimulators, acts as a messenger for activating the enzyme, and that Ca2+ and various phospholipids such as PI and PS seem to play a role cooperatively in this unique receptor mechanism.

Long chain polyunsaturated fatty acids alter oxytocin signaling and receptor density in cultured pregnant human myometrial smooth muscle cells.

Epidemiological studies and interventional clinical trials indicate that consumption of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) such as docosahexaenoic acid (DHA) lengthen gestational duration. Although the mechanisms are not well understood, prostaglandins (PG) of the 2-series are known to play a role in the initiation and progress of labor. In animal studies, modest DHA provision has been shown to reduce placental and uterine PGE2 and PGF2α, matrix metalloproteinase (MMP)-2 and MMP-9 expression, and placental collagenase activity. However, modulation of PG biosynthesis may not account for all the effects of LC n-3 PUFAs in labor. We investigated one potential PG-independent mechanism of LC PUFA action using cultured pregnant human myometrial smooth muscle cells. Our goal was to characterize the effect of LC PUFA treatment on oxytocin signaling, a potent uterotonic hormone involved in labor. The addition of 10 µM–100 µM DHA or arachidonic acid (AA) to the culture media for 48 h resulted in dose dependent enrichment of these fatty acids in membrane lipid. DHA and AA significantly inhibited phosphatidylinositol turnover and [Ca2+]i mobilization with oxytocin stimulation compared to bovine serum albumin control and equimolar oleic acid. DHA and AA significantly reduced oxytocin receptor membrane concentration without altering binding affinity or rate of receptor internalization. These findings demonstrate a role for LC n-3 PUFAs in regulation of oxytocin signaling and provide new insight into additional mechanisms pertaining to reports of dietary fish and fish oil consumption prolonging gestation.