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: Jung Min Han, Jae Ho Kim, Byoung Dae Lee, Sang Do Lee, Yong Kim, Yon Woo Jung, Sukmook Lee, Wonhwa Cho, Motoi Ohba, Toshio Kuroki, Pann-Ghill Suh, Sung Ho Ryu
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 277 Issue 10 Pg. 8290-7 (Mar 08 2002) ISSN: 0021-9258 [Print] United States
• PMID: 11744693 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Acetophenones
• Benzopyrans
• Enzyme Inhibitors
• Indoles
• Isoenzymes
• Maleimides
• Protein Isoforms
• rottlerin
• Prkcd protein, rat
• Protein Kinase C
• Protein Kinase C-delta
• phospholipase D2
• Phospholipase D
• bisindolylmaleimide I
• Tetradecanoylphorbol Acetate
• Bradykinin
• Ro 31-8220

Topics

• Acetophenones (pharmacology)
• Adenoviridae (genetics)
• Animals
• Benzopyrans (pharmacology)
• Bradykinin (pharmacology)
• Cell Membrane (metabolism)
• Cytosol (metabolism)
• Dose-Response Relationship, Drug
• Enzyme Inhibitors (pharmacology)
• Immunoblotting
• Immunohistochemistry
• Indoles (pharmacology)
• Isoenzymes (chemistry, metabolism)
• Maleimides (pharmacology)
• Microscopy, Fluorescence
• Mutation
• PC12 Cells
• Peptide Mapping
• Phospholipase D (chemistry, metabolism)
• Phosphorylation
• Precipitin Tests
• Protein Binding
• Protein Isoforms
• Protein Kinase C (chemistry, metabolism)
• Protein Kinase C-delta
• Protein Transport
• Rats
• Tetradecanoylphorbol Acetate (pharmacology)
• Time Factors
• Up-Regulation