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Partially Purified RhoA‐Stimulated Phospholipase D Activity Specifically Binds to Phosphatidylinositol 4,5‐Bisphosphate
Author(s) -
Yokozeki T.,
Kuribara H.,
Katada T.,
Touhara K.,
Kanaho Y.
Publication year - 1996
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1996.66031234.x
Subject(s) - rhoa , phospholipase d , vesicle , pleckstrin homology domain , phosphatidylinositol , adp ribosylation factor , chemistry , phosphatidic acid , phospholipase c , biochemistry , biophysics , biology , enzyme , phospholipid , kinase , signal transduction , golgi apparatus , membrane , cell
Phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) is absolutely required for the ADP‐ribosylation factor‐stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP 2 , but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP 2 for the RhoA‐stimulated PLD activation, too. It is interesting that the RhoA‐dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP 2 . The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP 2 that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA‐dependent PLD binds to PE/PC vesicles with PIP 2 . The degree of binding of the RhoA‐dependent PLD activity to the vesicles was totally dependent on the amount of PIP 2 in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β‐adrenergic receptor kinase fused to glutathione S ‐transferase, which specifically binds to PIP 2 , inhibited the PIP 2 ‐stimulated, RhoA‐dependent PLD activity in a concentration‐dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP 2 , owing to its specific interaction with PIP 2 , to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP 2 effectively extracted the RhoA‐dependent PLD activity from rat brain membranes. This indicates that PIP 2 serves as an anchor to localize PLD on plasma membranes in vivo.