Association of N- and C-terminal domains of phospholipase D is required for catalytic activity.
Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)4D, denoted HKD. RPLD1 contains two HKD domains, located in the N- and C-terminal regions. Deletion mutants of rPLD1 that contained only an N- or C-terminal HKD domain exhibited no catalytic activity when expressed in COS 7 cells. However, when N-terminal fragments containing one of the HKD domains were cotransfected with a C-terminal fragment containing the other HKD domain, PLD activity was restored. Furthermore, immunoprecipitation assays showed that the N- and C-terminal halves of rPLD1 were physically associated when expressed in COS 7 cells. In addition, deletion of 168 amino acids from the N terminus of rPLD1 significantly enhanced basal PLD activity while inhibiting the response to phorbol ester. Likewise, the coexpression of this truncated N-terminal half with the C-terminal half resulted in increased PLD activity. In summary, this study provides direct evidence that the enzymatic activity of rPLD1 requires the presence of the HKD domains in both the N- and C-terminal regions of the molecule. More importantly, the two halves of rPLD1 can associate, and this may be essential to bring the two HKD domains together to form an active catalytic center. These findings provide new insights into the catalytic mechanism of enzymes of the PLD superfamily.
Xie
Z
Z
Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
Ho
W T
WT
Exton
J H
JH
D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D005816:Genetic Complementation Test; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D010446:Peptide Fragments; D011994:Recombinant Proteins; D010739:Phospholipase D