Nuclear phospholipase D in Madin-Darby canine kidney cells. Guanosine 5'-O-(thiotriphosphate)-stimulated activation is mediated by RhoA and is downstream of protein kinase C.
We have recently demonstrated the existence of an ATP-activated phospholipase D (PLD) in the nuclei of MDCK-D1 cells (Balboa, M. A., Balsinde, J., Dennis, E. A., and Insel, P. A. (1995) J. Biol. Chem. 270, 11738-11740). We have now found that nuclear PLD is synergistically activated by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and ATP in a time- and concentration-dependent manner, but these compounds do not alter the sensitivity of the enzyme to activation by Ca2+. The synergistic stimulation of PLD activity could be blocked by addition of the protein kinase C inhibitors chelerythrine and calphostin C. Stimulation by GTP gamma S was abolished by guanosine 5'-O-(2-thiodiphosphate). Incubation of isolated nuclei with Clostridium botulinum C3 exoenzyme inhibited the potentiating effect of GTP gamma S on ATP-dependent nuclear PLD activity. Moreover, use of the Rho GDP dissociation inhibitor to extract Rho family G proteins from cell nuclei also inhibits PLD activity. Western blot analyses of isolated nuclei revealed the presence of the small G protein RhoA, but not of RhoB or the ADP-ribosylation factor. GTP gamma S-stimulated ATP-dependent PLD activity could be reconstituted in Rho GDP dissociation inhibitor-washed nuclei by addition of recombinant prenylated RhoA, but not by addition of non-prenylated RhoA. Taken together, these results indicate that nuclear PLD activity is modulated via a RhoA-dependent activation that occurs downstream of protein kinase C. Nuclear PLD, which appears to be a previously unrecognized effector regulated by protein kinase C and G proteins, may be involved in the regulation of nuclear function or structure.
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
M A
MA
Balboa
P A
PA
Insel
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D001905:Botulinum Toxins; D002118:Calcium / Q000494:pharmacology; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D003014:Clostridium botulinum / Q000201:enzymology; D004285:Dogs; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D007668:Kidney; D007700:Kinetics; D009281:Naphthalenes / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D010617:Phenanthridines; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; C016299:chelerythrine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011493:Protein Kinase C; D010739:Phospholipase D; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; C058819:calphostin C; D002118:Calcium