title: Protein kinase Calpha translocates to the perinuclear region to activate phospholipase D1.
abstract: The inhibition of phorbol ester activation of phospholipase D1 (PLD1) by protein kinase C (PKC) inhibitors has been considered proof of phosphorylation-dependent activation of PLD1 by PKCalpha. We studied the effect of the PKC inhibitors Ro-31-8220 and bisindolylmaleimide I on PLD1 activation and found that they inhibited the activation by interfering with PKCalpha binding to PLD1. Further studies showed that only unphosphorylated PKCalpha could bind to and activate PLD1 and that both inhibitors induced phosphorylation of PKCalpha. The phosphorylation status of either PLD1 or PKCalpha per se did not affect PLD1 activation in vitro. Immunofluorescence studies showed that PLD1 remained in the perinuclear region after phorbol ester treatment, whereas PKCalpha translocated from cytosol to both plasma membrane and perinuclear regions. Both Ro-31-8220 and bisindolylmaleimide I blocked the translocation of PKCalpha to the perinuclear region but not to the plasma membrane. Studies with okadaic acid suggested that phosphorylation regulated the relocation of PKCalpha from the plasma membrane to the perinuclear region. It is proposed that localization and interaction of PKCalpha with PLD1 in the perinuclear region is required for PLD1 activation and that PKC inhibitors inhibit this through phosphorylation of PKCalpha, which blocks its translocation.
authors.affiliation: Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37212, USA.
authors.forename: Tianhui
authors.initials: T
authors.lastname: Hu
authors.forename: John H
authors.initials: JH
authors.lastname: Exton
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D021381:Protein Transport; D051381:Rats
chemical_list: D004791:Enzyme Inhibitors; D007211:Indoles; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; C106801:phospholipase D1; C064758:Ro 31-8220