title: Phosphatidylinositol 4,5-bisphosphate modifies tubulin participation in phospholipase Cbeta1 signaling.
abstract: Tubulin forms the microtubule and regulates certain G-protein-mediated signaling pathways. Both functions rely on the GTP-binding properties of tubulin. Signal transduction through Galpha(q)-regulated phospholipase Cbeta1 (PLCbeta1) is activated by tubulin through a direct transfer of GTP from tubulin to Galpha(q). However, at high tubulin concentrations, inhibition of PLCbeta1 is observed. This report demonstrates that tubulin inhibits PLCbeta1 by binding the PLCbeta1 substrate phosphatidylinositol 4,5-bisphosphate (PIP2). Tubulin binding of PIP2 was specific, because PIP2 but not phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, or inositol 1,4,5-trisphosphate inhibited microtubule assembly. PIP2 did not affect GTP binding or GTP hydrolysis by tubulin. Muscarinic agonists promoted microtubule depolymerization and translocation of tubulin to the plasma membrane. PIP2 augmented this process in both Sf9 cells, containing a recombinant PLCbeta1 pathway, and SK-N-SH neuroblastoma cells. Colocalization of tubulin and PIP2 at the plasma membrane was demonstrated with confocal laser immunofluorescence microscopy. Although tubulin bound to both Galpha(q) and PLCbeta1, PIP2 facilitated the interaction between tubulin and PLCbeta1 but not that between tubulin and Galpha(q). However, PIP2 did augment formation of tubulin--Galpha(q)-PLCbeta1 complexes. Subsequent to potentiating PLCbeta1 activation, sustained agonist-independent membrane binding of tubulin at PIP2- and PLCbeta1-rich sites appeared to inhibit Galpha(q) coupling to PLCbeta1. Furthermore, colchicine increased membrane-associated tubulin and also inhibited PLCbeta1 activity in SK-N-SH cells. Thus, tubulin, depending on local membrane concentration, may serve as a positive or negative regulator of phosphoinositide hydrolysis. Rapid changes in membrane lipid composition or in the cytoskeleton might modify neuronal signaling through such a mechanism.
authors.affiliation: Department of Physiology and Biophysics, University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60612-7342, USA.
authors.forename: Juliana S
authors.initials: JS
authors.lastname: Popova
authors.forename: Arin K
authors.initials: AK
authors.lastname: Greene
authors.forename: Jia
authors.initials: J
authors.lastname: Wang
authors.forename: Mark M
authors.initials: MM
authors.lastname: Rasenick
mesh_terms: D000818:Animals; D001386:Azides / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018679:Cholinergic Agonists; D004305:Dose-Response Relationship, Drug; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D054799:Phospholipase C beta; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D018411:Spodoptera; D014162:Transfection; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D001386:Azides; D018679:Cholinergic Agonists; D007527:Isoenzymes; D008164:Luminescent Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins; C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D002217:Carbachol; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins