PLD2, as a GEF or as a Lipase, is Central to Leukocyte Chemotaxis

Summary

Principal Investigator: JULIAN G CAMBRONERO
Abstract: DESCRIPTION (provided by applicant): Blood leukocytes migrate towards the sites of inflammation (a beneficial effect) or towards compromised tissues after an ischemic attack (a deleterious effect), such as in the case of ischemia/reperfusion injury (I/RI) in the heart. In I/R, when the supply of blood is restored to the affected area, neutrophils cause damage to healthy tissue via the massive release of reactive oxygen species (ROS). We have discovered that the enzyme Phospholipase D2 (PLD2) is a chemoattractant for neutrophils. In addition to this, we present three major lines of evidence that have allowed us to propose three Specific Aims. (1) First, novel data in our lab has revealed an unexpected GEF activity that exists in the lipase PLD2. Since the target, Rac2 Rho GTPase is responsible for cell movement PLD2 might be a major GEF mediating chemotaxis. Because GEFs are not constitutively active but are kept under tight regulation, we propose in AIM 1 to investigate the regulation of PLD2 novel GEF activity. The hypothesis is that PLD2 GEF activity is regulated by tyrosine phosphorylation and by interaction with PDZ domain proteins, which lead to PX and PH domains of PLD2 and phosphatidic acid (PA) combined action to ensure a GTP/GDP exchange activity. The candidate kinases are VEGFR2 and JAK3, while the PDZ-Domain proteins are MUPP1 and Mda9/Syntenin. (2) Second, we present preliminary evidence of PLD2 as the center of a protein network during cell signaling. We propose in AIM 2 to study a new pathway through which PLD2 and 14-3-3 interact to prevent chemotaxis in ischemia reperfusion injury conditions via GDF-15. We will study the molecular interactions involving PLD2, Rac2, WASp, and the adaptors Grb2 and 14-3-3 through biochemical, genetic (Rac2-/-, WASp-/- and PLD2-/- KO mice) and fluorescence microscopy experiments. Through visualization of protein complex formation in real time by FRET, we will quantify the spatial organization of these signaling molecules during chemotaxis and in I/RI-like conditions. (3) Third, PLD inhibitors protect the heart from injury caused by ischemia/reperfusion in a Murine myocardial/reperfusion injury model, placing PLD at the center of this disease. In AIM 3 we hypothesize that PLD from leukocytes that infiltrate an ischemia/infarct area will have a deleterious effect on the heart conducive to the exacerbation of the injury in vivo. We will test this hypothesis in wild type and PLD2- /- KO mice, as well as in osmotic pump-implanted mice with specific PLD inhibitors. We will also ascertain the PLD regulatory mechanisms of signal transduction that operate in vivo. We expect to demonstrate that depriving PLD confers myocardial protection, with therapeutic potential in conjunction with currently used thrombolytic therapy. This grant will uncover targets that can be exploited pharmacologically to diminish the harmful presence of neutrophils in inflammation-mediated heart injury. If we can avoid the untimely presence of these cells in ischemia, heart failure after myocardial infarction would then be diminished.
Funding Period: 1998-04-01 - 2017-03-31
more information: NIH RePORT

Top Publications

  1. pmc The elucidation of novel SH2 binding sites on PLD2
    M Di Fulvio
    Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, OH 45435, USA
    Oncogene 25:3032-40. 2006
  2. pmc Serum deprivation confers the MDA-MB-231 breast cancer line with an EGFR/JAK3/PLD2 system that maximizes cancer cell invasion
    Qing Ye
    Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA
    J Mol Biol 425:755-66. 2013
  3. pmc Increased cell growth due to a new lipase-GEF (Phospholipase D2) fastly acting on Ras
    Karen M Henkels
    Wright State University School of Medicine, Department of Biochemistry and Molecular Biology, OH 45435, USA
    Cell Signal 25:198-205. 2013
  4. pmc The exquisite regulation of PLD2 by a wealth of interacting proteins: S6K, Grb2, Sos, WASp and Rac2 (and a surprise discovery: PLD2 is a GEF)
    Julian Gomez-Cambronero
    Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA
    Cell Signal 23:1885-95. 2011
  5. pmc The mechanism of cell membrane ruffling relies on a phospholipase D2 (PLD2), Grb2 and Rac2 association
    Madhu Mahankali
    Dept Biochemistry and Molecular Biology, Wright State University School Medicine, Dayton, OH 45435, USA
    Cell Signal 23:1291-8. 2011
  6. pmc Cell invasion of highly metastatic MTLn3 cancer cells is dependent on phospholipase D2 (PLD2) and Janus kinase 3 (JAK3)
    Karen M Henkels
    Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA
    J Mol Biol 408:850-62. 2011
  7. pmc IL-8-induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3)
    Karen M Henkels
    Department of Biochemistry and Molecular Biology, Wright State University School Medicine, Dayton, OH 45435, USA
    FEBS Lett 585:159-66. 2011
  8. pmc New concepts in phospholipase D signaling in inflammation and cancer
    Julian Gomez-Cambronero
    Department of Biochemistry and Molecular Biology, Wright State University School Medicine, Dayton, OH, USA
    ScientificWorldJournal 10:1356-69. 2010
  9. pmc PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation
    Karen M Henkels
    Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA
    Biochem Biophys Res Commun 389:224-8. 2009
  10. pmc Mutation of Y179 on phospholipase D2 (PLD2) upregulates DNA synthesis in a PI3K-and Akt-dependent manner
    Mauricio Di Fulvio
    Cell Biology and Physiology, Wright State University, School of Medicine, Dayton, OH 45435, USA
    Cell Signal 20:176-85. 2008

Detail Information

Publications13

  1. pmc The elucidation of novel SH2 binding sites on PLD2
    M Di Fulvio
    Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, OH 45435, USA
    Oncogene 25:3032-40. 2006
    ..When this is released, Y(169) mediates cellular proliferation through the Ras/MAPK pathway...
  2. pmc Serum deprivation confers the MDA-MB-231 breast cancer line with an EGFR/JAK3/PLD2 system that maximizes cancer cell invasion
    Qing Ye
    Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA
    J Mol Biol 425:755-66. 2013
    ..This is especially important in the absence of growth factors in serum, coincidental with migration of these cells to new locations...
  3. pmc Increased cell growth due to a new lipase-GEF (Phospholipase D2) fastly acting on Ras
    Karen M Henkels
    Wright State University School of Medicine, Department of Biochemistry and Molecular Biology, OH 45435, USA
    Cell Signal 25:198-205. 2013
    ..This can be crucial to cancer biology in that not only Ras mutations explain abnormal growth, but the existence of a new GEF for Ras: a GEF molecule that happens to be a phospholipase...
  4. pmc The exquisite regulation of PLD2 by a wealth of interacting proteins: S6K, Grb2, Sos, WASp and Rac2 (and a surprise discovery: PLD2 is a GEF)
    Julian Gomez-Cambronero
    Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA
    Cell Signal 23:1885-95. 2011
    ..This provides only the latest level of PLD2 regulation in a field that promises newer and exciting advances in the next few years...
  5. pmc The mechanism of cell membrane ruffling relies on a phospholipase D2 (PLD2), Grb2 and Rac2 association
    Madhu Mahankali
    Dept Biochemistry and Molecular Biology, Wright State University School Medicine, Dayton, OH 45435, USA
    Cell Signal 23:1291-8. 2011
    ..Since membrane ruffling precedes cell migration, the results herein provide a novel mechanism for control of membrane dynamics, crucial for the physiology of leukocytes...
  6. pmc Cell invasion of highly metastatic MTLn3 cancer cells is dependent on phospholipase D2 (PLD2) and Janus kinase 3 (JAK3)
    Karen M Henkels
    Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA
    J Mol Biol 408:850-62. 2011
    ....
  7. pmc IL-8-induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3)
    Karen M Henkels
    Department of Biochemistry and Molecular Biology, Wright State University School Medicine, Dayton, OH 45435, USA
    FEBS Lett 585:159-66. 2011
    ..These new findings lay the basis for understanding the molecular mechanism of cell migration as it relates to neutrophil-mediated chronic inflammatory processes...
  8. pmc New concepts in phospholipase D signaling in inflammation and cancer
    Julian Gomez-Cambronero
    Department of Biochemistry and Molecular Biology, Wright State University School Medicine, Dayton, OH, USA
    ScientificWorldJournal 10:1356-69. 2010
    ..In summary, the involvement of PLD2 in cell signaling continues to expand geometrically. It involves gene transcription, mitogenic and cell migration effects as seen in normal growth, tumor development, and inflammation...
  9. pmc PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation
    Karen M Henkels
    Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA
    Biochem Biophys Res Commun 389:224-8. 2009
    ....
  10. pmc Mutation of Y179 on phospholipase D2 (PLD2) upregulates DNA synthesis in a PI3K-and Akt-dependent manner
    Mauricio Di Fulvio
    Cell Biology and Physiology, Wright State University, School of Medicine, Dayton, OH 45435, USA
    Cell Signal 20:176-85. 2008
    ..This might be critical for the maintenance of the PLD2-regulated proliferative status...
  11. pmc Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis
    Mauricio Di Fulvio
    Cell Biology and Physiology, Wright State University, School of Medicine, Dayton, OH 45435, USA
    Biochem Biophys Res Commun 357:737-42. 2007
    ..Thus, a viable knock-down and rescue protocol has demonstrated that Grb2 is crucial for cell proliferation...
  12. pmc The Grb2/PLD2 interaction is essential for lipase activity, intracellular localization and signaling in response to EGF
    Mauricio Di Fulvio
    Cell Biology and Physiology, Wright State University, School of Medicine, Dayton, OH 45435, USA
    J Mol Biol 367:814-24. 2007
    ..These results demonstrate for the first time that the presence of Grb2 and its interaction with localized intracellular structures is essential for PLD2 activity and signaling in vivo...
  13. pmc Phosphatidic acid, phospholipase D and tumorigenesis
    Julian Gomez-Cambronero
    Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University School Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA Electronic address
    Adv Biol Regul 54:197-206. 2014
    ....

Research Grants30

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
    ..Using novel and state-of-the-art techniques, floxed mice and molecular approaches to gene regulation, we are well-positioned to lead the study of RAGE in the next cycle of this Program. ..
  2. Surgical Revascularization's Impact on Mitochondria in Hibernating Myocardium
    ROSEMARY FRANCES KELLY; Fiscal Year: 2013
    ..By performing a surgical operation to resupply blood to the heart, our research will test whether these adaptations to ischemia are reversible and allow the heart to return to normal function, or if the adaptations are permanent. ..
  3. CYTOPROTECTIVE EFFECTS OF INFLAMMATION MEDIATED MEMBRANE REPAIR
    DOUGLAS LOWELL MANN; Fiscal Year: 2013
    ..abstract_text> ..
  4. Mechanisms of Atherogenesis in Insulin Resistance
    IRA A TABAS; Fiscal Year: 2013
    ..End of Abstract) ..
  5. Neutralizing Antibody &AAV FIX Gene Therapy
    Richard J Samulski; Fiscal Year: 2013
    ..The long-term objective of this PPG is to advance basic understanding of vector-cell-animal model interactions for safe gene delivery. ..
  6. Immune-Based Interventions Against Infectious Diseases
    Alan L Rothman; Fiscal Year: 2013
    ..3. Recruit promising junior investigators and provide mentoring by established NIH-funded researchers. 4. Support a multidisciplinary research program led by junior investigators in translational infectious diseases immunology. ..
  7. Signaling of Endothelial Permeability and Lung Vascular Injury
    Asrar B Malik; Fiscal Year: 2013
    ..Moreover the depth of understanding to be gained of the signaling pathways mediating the increase in lung vascular permeability will provide novel insights into the mechanisms of protein-rich pulmonary edema and ARDS. ..