Inhibition of GTPgammaS-Dependent Phospholipase D and Rho Membrane Association by Calphostin Is Independent of Protein Kinase C Catalytic Activity

Authors: Dubyak G.R.; Kertesy S.B.

Source: Archives of Biochemistry and Biophysics, Volume 341, Number 1, May 1997 , pp. 129-139(11)

Publisher: Academic Press

Abstract:

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 GTPgammaS-dependent PLD activity at free Ca 2+ < 100 n m . In the absence of GTPgammaS, 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 GTPgammaS-dependent PLD was not inhibited by BIM. In contrast, calphostin strongly inhibited the GTPgammaS-dependent PLD activity, both in the presence and absence of PMA as a potentiating agent. Calphostin also produced complete inhibition of a GTPgammaS-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 GTPgammaS-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.

Keywords: phospholipase D; protein kinase C; G-proteins; Rho; ARF; lipid second-messengers

Language: English

Document Type: Research article

Affiliations: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, 44106

Publication date: 1997-05-01

• In this: publication
• By this: publisher
• In this Subject: Anatomy & Physiology ,  Biology ,  Biochemistry
• By this author: Dubyak G.R. ;  Kertesy S.B.

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