Activation of Phospholipase D1 by Cdc42 Requires the Rho Insert Region

Abstract

Members of the Rho subfamily of GTP-binding proteins are implicated in the regulation of phospholipase D (PLD). In the present study, we demonstrate a physical association between a Rho family member, Cdc42, and PLD1. Binding of Cdc42 to PLD1 and subsequent activation are GTP-dependent. Although binding of Cdc42 to PLD1 does not require geranylgeranylation, activation of PLD1 is dependent on this lipid modification of Cdc42. Specific point mutations in the switch I region of Cdc42 abolish binding to and, therefore, activation of PLD1 by Cdc42. Deletion of the Rho insert region, which consists of residues 120 -139, from Cdc42 does not interfere with binding to PLD1 but inhibits Cdc42 stimulated PLD1 activity. Interestingly, deletion of the insert region from Cdc42 also inhibits activation of PLD1 by Arf and protein kinase C. With the lack of specific inhibitors of PLD activity, the insert deletion mutant of Cdc42 (designated (⌬L8)Cdc42) is a novel reagent for in vitro studies of PLD1 regulation, as well as for in vivo studies of Cdc42-mediated signaling pathways leading to PLD1 activation. Because the insert region is required for the transforming activity of Cdc42, regulation of PLD1 by this region on Cdc42 is of major interest.

Specificity of Rho Insert-mediated Activation of Phospholipase D1

“…The data in Fig. 6 was generated with an SP-Sepharose-purified fraction of Sf21 cytosol expressing hPLD1; this fraction and its purification were described previously (9). Reactions were incubated for 30 min at 37°C with 10 M GTP␥S in the presence of lipid vesicles containing radiolabeled substrate ([ 3 H]dipalmitoyl phosphatidylcholine).…”

“…Protein Expression-Expression and purification of recombinant hPLD1, Cdc42, PKC␤II, and Arf1 were carried out as described previously (9). Recombinant human RhoA and Rac1 were expressed and purified using the same protocol as that for Cdc42 expression and purification.…”

“…PLD Activity Assay-Activity in the exogenous substrate assay was measured as described previously (9,12). The hPLD1 source for the exogenous assays was cytosol from Sf21 cells infected with hPLD1 baculovirus, and all assays contained 3 g of this cytosol unless otherwise noted on the graph label.…”

“…Mutations were confirmed by restriction digest analysis, when applicable, and sequence analysis in all cases. The generation of hexahistidine-tagged Cdc42, Rac, and Rho constructs in the pVL1392 vector (PharMingen) and subsequent virus production were completed as previously described (9). The D4 PLD1 fragment was generated using PCR with full-length hPLD1 in the pCMV3 vector as the template.…”

“…To study the specificity and contribution of each Rho family protein to PLD signaling necessitates an understanding of the protein-protein interactions required for the Rho proteins to bind PLD, as well as an understanding of the mechanism by which these interactions stimulate PLD activity. In previous work, we investigated the activation of hPLD1 by Cdc42 and demonstrated that there are three key features to the activation mechanism (9). First, activation of hPLD1 by Cdc42 requires prenylation of the GTPase.…”