Definition of the protein kinase C interaction site of phospholipase D.
Serial deletions of the N-terminal 319 amino acids of rPLD1 expressed in COS-7 cells resulted in increased basal PLD activity. Incubation of the cells with phorbol myristate acetate increased the activity of endogenous and wild-type rPLD1. The mutant rPLD1 with deletion of the first 50 amino acids responded to the phorbol ester, however, rPLD1 with deletions of 115 amino acids or more did not. In cells in which constitutively active V14RhoA was co-expressed with the mutant PLDs, stimulation of PLD activity was observed with all deletion mutants. In membranes from COS-7 cells in which the mutant PLDs were expressed, only the mutant with deletion of 50 N-terminal amino acids responded to added protein kinase C-alpha and phorbol ester, in agreement with the in vivo studies. When myristoylated ADP-ribosylation factor 3 (mARF3) was added together with guanosine 5'-3-O-(thio)triphosphate, all mutants showed stimulation of PLD activity. It is concluded that the site of interaction of protein kinase C with rPLD1 is located in the N-terminal region and that Rho and ARF interact at other sites.
Park
S K
SK
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Min
D S
DS
Exton
J H
JH
D020727:ADP-Ribosylation Factors; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein
D017931:DNA Primers; D011994:Recombinant Proteins; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate