title: Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells.
abstract: Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.
authors.affiliation: Department of Life Science and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, South Korea.
authors.forename: Jung Min
authors.initials: JM
authors.lastname: Han
authors.forename: Jae Ho
authors.initials: JH
authors.lastname: Kim
authors.forename: Byoung Dae
authors.initials: BD
authors.lastname: Lee
authors.forename: Sang Do
authors.initials: SD
authors.lastname: Lee
authors.forename: Yong
authors.initials: Y
authors.lastname: Kim
authors.forename: Yon Woo
authors.initials: YW
authors.lastname: Jung
authors.forename: Sukmook
authors.initials: S
authors.lastname: Lee
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
authors.forename: Motoi
authors.initials: M
authors.lastname: Ohba
authors.forename: Toshio
authors.initials: T
authors.lastname: Kuroki
authors.forename: Pann-Ghill
authors.initials: PG
authors.lastname: Suh
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Ryu
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009154:Mutation; D016716:PC12 Cells; D010449:Peptide Mapping; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051745:Protein Kinase C-delta; D021381:Protein Transport; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D000098:Acetophenones; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D020033:Protein Isoforms; C085746:rottlerin; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; C105606:phospholipase D2; D010739:Phospholipase D; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; C064758:Ro 31-8220