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.
Highlights
Phospholipase D (PLD)1 catalyzes phosphatidylcholine hydrolysis to generate phosphatidic acid
We described the phosphorylation-dependent activation of PLD1 by protein kinase C (PKC)␣ in 3Y1 fibroblast cells and in PLD1-transfected COS-7 cells [29] and PKC␣-mediated PLD1 phosphorylation with the activation occurring in caveolin-enriched microdomains within the plasma membrane [30]
phorbol 12myristate 13-acetate (PMA)-induced PLD2 Activation in PC12 Cells—It has been reported that PMA, an activator of PKC, stimulated PLD activity in PC12 cells [37] and that PLD2 was prominently expressed in PC12 cells [33]
Summary
Phospholipase D (PLD) catalyzes phosphatidylcholine hydrolysis to generate phosphatidic acid. Phosphatidic acid can be further metabolized to lysophosphatidic acid and diacylglycerol by phospholipase A2 (PLA2) and phosphatidic acid phosphohydrolase, respectively [1] These reactions are involved in receptor-mediated signal transductions and several cellular processes, such as membrane trafficking, cell growth and differentiation, cytoskeletal reorganization, respiratory burst, and apoptosis [1,2,3,4]. The details of the regulation of PLD2 by PKC are not well understood, recently, it was reported that PLD2 could be activated in different cell types in response to PMA [31,32,33]. The aim of this study was to examine the involvement of PKC in PMA-induced PLD2 stimulation in PC12 cells and to investigate whether PKC-mediated PLD2 activation is phosphorylation-dependent. Phosphorylated in response to PMA treatment and that PKC␦ mediates the phosphorylation-dependent activation of PLD2 in PC12 cells
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