Inhibition of GTP gamma S-dependent phospholipase D and Rho membrane association by calphostin is independent of protein kinase C catalytic activity.
We studied the relationships between the activation of phospholipase D (PLD) by guanine nucleotides and phorbol esters in permeabilized U937 promonocytes and in solubilized extracts prepared from U937 cell membranes. Treatment of permeabilized cells with phorbol myristate acetate (PMA) strongly potentiated GTP gamma S-dependent PLD activity at free Ca2+ < 100 nM. In the absence of GTP gamma S, PMA stimulated only minor PLD activity. This suggested synergistic interaction between regulatory G-proteins and a protein kinase C (PKC) family kinase. The potential role of PKC was evaluated by testing two mechanistically distinct PKC inhibitors, bisindolylmaleimide (BIM) and calphostin. BIM inhibits PKC enzymes via competition with ATP for binding to the catalytic domain, while calphostin competes with PMA or diglyceride for binding to the regulatory domain. The ability of PMA to potentiate the GTP gamma S-dependent PLD was not inhibited by BIM. In contrast, calphostin strongly inhibited the GTP gamma S-dependent PLD activity, both in the presence and absence of PMA as a potentiating agent. Calphostin also produced complete inhibition of a GTP gamma S-dependent PLD activity, present in solubilized membrane extracts, which was assayed using phospholipid vesicles of defined composition. Treatment of reconstituted membrane/cytosol mixtures with calphostin also produced complete inhibition of the GTP gamma S-induced translocation of Rho A from cytosol to membrane. In contrast to its effects on the U937 cell PLD, calphostin did not inhibit the activity of purified PLD from cabbage. These results suggest that the assembly of active RhoA/PLD signaling complexes on membranes involves a phorbol ester/calphostin-binding protein, but is not dependent on PKC-type catalytic activity.
Dubyak
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GR
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA. gxd3@po.cwru.edu
Kertesy
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SB
D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D020690:GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007938:Leukemia; D008081:Liposomes / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology*; D010539:Permeability; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; D007211:Indoles; D008081:Liposomes; D008301:Maleimides; D009281:Naphthalenes; C061465:calphostin complex; C085379:rho GTPase-activating protein; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate