Regulation of Phospholipase D2 Activity by Protein Kinase Cα

Jun-Song Chen,John H Exton

doi:10.1074/jbc.m311033200

Jun-Song Chen, John H Exton

Open Access

https://doi.org/10.1074/jbc.m311033200

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Abstract

It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, Gö 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.

Highlights

Phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA), and choline [1]
protein kinase C (PKC) Mediates phorbol 12myristate 13-acetate (PMA)-induced PLD2 Activation—Because it is well established that PMA activates conventional and novel PKC isozymes, we used PKC inhibitors to explore the involvement of PKC in the PLD2 activation
Our group showed that PLD2, like PLD1, exhibited a large response to PMA when overexpressed in COS-7 cells [21]

Summary

EXPERIMENTAL PROCEDURES

Materials—4␤-Phorbol 12-myristate 13-acetate, phosphatidylinositol 4,5-bisphosphate, bovine serum albumin, and Triton X-100 were from Sigma. In Vivo PLD Assay—After a 6-h transfection with FuGENE 6, COS-7 cells in 6-well plates were serum-starved (0.5% fetal bovine serum in Dulbecco’s modified Eagle’s medium in the presence of 1 ␮Ci/ml [3H]myristic acid). Cells were centrifuged and resuspended in 500 ␮l of ice-cold lysis buffer (25 mM Hepes, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture). The cells were washed twice with ice-cold phosphate-buffered saline and harvested in 600 ␮l of immunoprecipitation buffer containing 25 mM Hepes, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 50 mM KC1, 1%. The mixture was spun, and the supernatant was incubated with 2 ␮l of anti-Xpress mouse antibody (1 mg/ml) and 20 ␮l of protein G-agarose beads overnight. The mixture was incubated at 30 °C for 30 min and electrophoresed through an 8% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane, and the membrane was exposed to a photographic film for autoradiography

RESULTS

Regulation of Phospholipase D by Protein Kinase C

DISCUSSION