Inhibition of muscarinic receptor-linked phospholipase D activation by association with tubulin.
Mammalian phospholipase D (PLD) is considered a key enzyme in the transmission signals from various receptors including muscarinic receptors. PLD activation is a rapid and transient process, but a negative regulator has not been found that inhibits signal-dependent PLD activation. Here, for the first time, we report that tubulin binding to PLD2 is an inhibition mechanism for muscarinic receptor-linked PLD2 activation. Tubulin was identified in an immunoprecipitated PLD2 complex from COS-7 cells by peptide mass fingerprinting. The direct interaction between PLD2 and tubulin was found to be mediated by a specific region of PLD2 (amino acids 476-612). PLD2 was potently inhibited (IC50 <10 nM) by tubulin binding in vitro. In cells, the interaction between PLD2 and tubulin was increased by the microtubule disrupting agent nocodazole and reduced by the microtubule stabilizing agent Taxol. Moreover, PLD2 activity was found to be inversely correlated with the level of monomeric tubulin. In addition, we found that interaction with and the inhibition of PLD2 by monomeric tubulin is important for the muscarinic receptor-linked PLD signaling pathway. Interaction between PLD2 and tubulin was increased only after 1-2 min of carbachol stimulation when carbachol-stimulated PLD2 activity was decreased. The expression of the tubulin binding region of PLD2 blocked the later decrease in carbachol-induced PLD activity by masking tubulin binding. Taken together, these results indicate that an increase in local membrane monomeric tubulin concentration inhibits PLD2 activity, and provides a novel mechanism for the inhibition of muscarinic receptor-induced PLD2 activation by interaction with tubulin.
Chae
Young Chan
YC
Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
Lee
Sukmook
S
Lee
Hye Young
HY
Heo
Kyun
K
Kim
Jung Hwan
JH
Kim
Jong Hyun
JH
Suh
Pann-Ghill
PG
Ryu
Sung Ho
SH
D000818:Animals; D019556:COS Cells; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D018679:Cholinergic Agonists / Q000494:pharmacology; D006801:Humans; D008870:Microtubules / Q000378:metabolism; D016716:PC12 Cells; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D018679:Cholinergic Agonists; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D002217:Carbachol; C105606:phospholipase D2; D010739:Phospholipase D