Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the interaction site of human PLD1 for RhoA is located in its C-terminus, but the exact locus has not been determined. The purpose of the present study was to determine the interaction site of rat PLD1 (rPLD1) with RhoA. Selection with phage display of different peptides of rPLD1 confirmed that GTP-bound RhoA interacted with a site in the amino acid sequence 873–1024 at the C-terminus of rPLD1. RhoA also associated with this peptide in a GTP-dependent manner in COS-7 cell lysates and the peptide inhibited RhoA stimulation of PLD activity in membranes from COS-7 cells expressing rPLD1. A series of alanine mutations of non-conserved residues were made in this sequence, and the enzymes were expressed in COS-7 cells and checked for responses to activation of PKC, which interacts with the N-terminus of PLD1, and also to the constitutively active V14RhoA. Mutations in the C-terminus of rPLD1 (K946A, V950A, R955A and K962A) caused partial loss of V14RhoA stimulation, and double mutations (K946A/K962A, K946A/V950A and K962A/V950A) caused an almost total loss. Co-immunoprecipitation studies also showed that the mutated forms of rPLD1 described above failed to bind V14RhoA compared with wild-type rPLD1, whereas rPLD1 with mutations outside the region K946–K962 bound V14RhoA normally. It is concluded that basic amino acids in a restricted C-terminal region of rPLD1 are important for binding of RhoA and its activation of PLD activity.
Research Article|April 24 2001
Determination of interaction sites of phospholipase D1 for RhoA
John H. EXTON
John H. EXTON
1Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0295, U.S.A.
1To whom correspondence should be addressed (e-mail email@example.com).
Search for other works by this author on:
- Views Icon Views
- PDF LinkPDF
- Share Icon Share
- Cite Icon Cite
Songmin CAI, John H. EXTON; Determination of interaction sites of phospholipase D1 for RhoA. Biochem J 1 May 2001; 355 (3): 779–785. doi: https://doi.org/10.1042/bj3550779
Download citation file: