title: Analysis of Phosphatidic Acid Binding and Regulation of PIPKI In Vitro and in Intact Cells.
abstract: Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a lipid second messenger that regulates a wide array of essential cellular events, such as signal transduction, vesicle trafficking, actin cytoskeleton dynamics, adhesion, and motility. To control the spatiotemporal production of PI(4,5)P2, the activity of type 1 phosphotidylinositol-4-phosphate-5-kinases (PIPKIs) is tightly regulated by small GTPases and another signaling lipid, phosphatidic acid (PA). It is of interest that PI(4,5)P2 is also a critical cofactor for the activation of the PA-generating enzyme, phospholipase D (PLD). It has been proposed that the reciprocal stimulation of PLD and PIPKI enzymes enables a rapid feedforward stimulation loop for the localized and acute generation of signaling lipids that are critical for the regulation of actin cytoskeletal reorganization and membrane trafficking. Here, we outline the methods for the expression and purification of PIPKIγ from bacteria, determination of direct PA binding, and activation of PIPKIγ using in vitro liposomes assays, and examination of actin cytoskeletal reorganization promoted by the PA-PIPKIγ signaling in intact cells using fluorescent microscopy.
authors.affiliation: University of Texas Health Science Center at Houston, Houston, TX, United States.
authors.forename: L W R
authors.initials: LW
authors.lastname: Tay
authors.affiliation: University of Texas Health Science Center at Houston, Houston, TX, United States.
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.affiliation: University of Texas Health Science Center at Houston, Houston, TX, United States. Electronic address: guangwei.du@uth.tmc.edu.
authors.forename: G
authors.initials: G
authors.lastname: Du
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism* / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D008081:Liposomes; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D010739:Phospholipase D
keywords: Actin cytoskeletal reorganization; Liposomes; PI(4,5)P2; PIPKI; PLD; Phosphatidic acid; Phosphatidylinositol 4,5-bisphosphate; Phosphatidylinositol kinase; Phosphoinositides; Phospholipase D