Regulation of Phospholipase D2 Activity by Protein Kinase Cα

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 PKC␣ stimulated PLD2 activity to an even higher level than wild type PKC␣. Co-expression of wild type PKC␣, but not PKC␦, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKC␦-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKC␣ under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKC␣ 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 PKC␣ in COS-7 cells. However, the phosphorylation is not required for PKC␣ 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.Phospholipase D (PLD) 1 catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA), and choline (1). PA is an important "second messenger" in several physiological processes (2). PA can be further metabolized to diacylglycerol by PA phosphohydrolase or to lysophosphatidic acid by phospholipase A2. Diacylglycerol is a well known activator of protein kinase C (PKC), whereas lysophosphatidic acid mediates many important physiological functions via its G protein-coupled receptors (3). Thus, PLD influences many important intracellular events via producing these downstream products. PLD activity is regulated by many stimuli such as growth factors, cytokines, hormones, neurotransmitters, and other molecules involved in extracellular communication (1). PLD is thought to play an important role in secretion, membrane trafficking, cytoskeleton reorganization, and apoptosis (1).PLD1 and PLD2 are two isoforms of mammalian PLD that share about 50% amino acid similarity. However, their regulatory properties are quite different, e.g. t...

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity

“…Further, PKC has been reported to phosphorylate PLD2 [65] leading to the inactivation of the enzyme suggesting a feedback loop for PLD/PKC mediated activation and inactivation.…”

Lysophosphatidylcholine suppresses apoptosis and induces neurite outgrowth in PC12 cells through activation of phospholipase D2

“…LPC has been shown to play a crucial role in the Ca 2+-dependent activation of PLD2 in L1210 and COS-7 cells (Kim et al, 1999b). PLD2 activity has been reported to be stimulated by PKC-dependent phosphorylation, interaction with ARF, and several unsaturated fatty acids (Lopez et al, 1998;Kim et al, 1999a;Sung et al, 1999;Han et al, 2002;Kim et al, 2003b;Koch et al, 2003;Chen and Exton, 2004). However, LPC could not activate directly the enzymatic activity of PLD2 in vitro (Kim et al, 1999b).…”

The uncovering of a novel regulatory mechanism for PLD2: Formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2

“…Although PLD2 can form a complex with the EGF receptor that leads to phosphorylation of Tyr 11 [21], sitedirected mutagenesis and pharmacological studies suggest that this phosphorylation is not essential for stimulating enzyme activity [21,22]. Serine and threonine phosphorylation by PKC is similarly dispensable [23]. More recently, others have show that PLD2 can be phosphorylated on tyrosine by Fyn and Fgr kinases in mast cells [24].…”