The C Terminus of Mammalian Phospholipase D Is Required for Catalytic Activity

Abstract

The activity of phospholipase D (PLD) is regulated by a variety of hormonal stimuli and provides a mechanistic pathway for response of cells to extracellular stimuli. The two identified mammalian PLD enzymes possess highly homologous C termini, which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the Cterminal threonine or the addition of a single alanine attenuates activity of the enzyme. Protein folding appears to be intact because mutant enzymes express to similar levels in Sf9 cells and addition of peptides representing the C-terminal amino acids, including the simple hexamer PMEVWT, restores partial activity to several of the mutants. Analysis of several mutants suggests a requirement for the hydrophobic reside at the ؊2-position but not an absolute requirement for the hydroxyl side chain of threonine at the C terminus. The inability of peptides amidated at their C termini to effect restoration of activity indicates the involvement of the C-terminal ␣ carboxyl group in functional activity of these enzymes. The ability of peptides to restore activity to PLD enzymes mutated at the C terminus suggests a flexible interaction of this portion of the molecule with a catalytic core constructed on conserved HKD motifs. Participation of these C termini residues in either stabilization of the catalytic site or the enzymatic reaction itself remains to be determined. This requirement for the C terminus provides an excellent potential site for interaction with regulatory proteins that may either enhance or down-regulate the activity of these enzymes in vitro.Phosphatidic acid plays a prominent role in phospholipid metabolism (1) and signal transduction. As a signaling molecule, phosphatidic acid is produced through the action of phospholipase D (PLD) 1 in response to a variety of cellular stimuli and acts directly as a second messenger or as a precursor for the formation of another second messenger, diacylglycerol (see Refs. 2-5 for reviews).Understanding of mechanisms by which mammalian PLD enzymes are regulated has been greatly accelerated by the development of a sensitive in vitro assay for the activity (6, 7) and identification of two mammalian genes (8 -10). The regulation of PLD activity in vitro allowed the identification of four distinct mechanisms for stimulation of the enzymes. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) was the first activator and a key element in establishing assays for the enzymes (6, 11-13). This was followed by the elucidation of activation by members of both the Arf (6, 14) and Rho (15-19) families of small monomeric GTPases and by classical isoforms of protein kinase C (PKC) via a phosphorylation independent pathway (20, 21). Subsequent studies with recombinant proteins in vitro have shown that PLD1 responds to all of these activators (22), while PLD2 is only stimulated by PIP 2 and Arf (9,23,24). Elucidation of these regulatory mechanisms and investigations to determine which of these pathways are utilized in the ce...

Mechanisms of Regulation of Phospholipase D1 and D2 by the Heterotrimeric G Proteins G13 and Gq

“…Our studies on rat PLD1 (rPLD1) showed that there is an interdomain association between the N-and C-terminal halves of the molecule and this is important for the catalytic activity (25,26). Another domain essential for the catalytic activity of PLD is located at the C terminus of the enzyme, and deletion of the last amino acid of rPLD1 abolishes its enzymatic activity (27,28,30). Besides the motifs essential for the catalysis, domains important for the PKC and Rho responses have been mapped to the N-terminal and C-terminal regions of the enzyme, respectively (29 -32).…”

Endosomal localization of phospholipase D 1a and 1b is defined by the C-termini of the proteins, and is independent of activity

“…It has also been demonstrated that deletion of the last four amino acids of rat PLD1 abolishes PLD activity of this protein, although substitution of these amino acids to other charged or less hydrophobic amino acids restored some PLD activity to these proteins and addition of protein with an intact C-terminus restored full PLD activity [40]. The importance of these C-terminal amino acids has been confirmed by a complementary analysis of human PLD1, which also established that PLD activity could be partially restored to deletion mutants by the addition of a peptide consisting of the six C-terminal amino acids [41]. These data would indicate that the C-terminus, absent in PLD1a2 and PLD1b2, should have a role in catalytic activity and therefore that PLD1a2 and PLD1b2 might be predicted to have no PLD activity at all.…”

“…Secondly, the C-terminus has a strong influence on the catalytic activity of PLD1. Deletion of the C-terminus destroys PLD1 activity ( Figure 6C, [16,[39][40][41]46]) ; however, it appears that the ten amino acids found in PLD1a2 and PLD1b2 are sufficient to restore partial activity to PLD1a or PLD1b that lack the C-terminal 113 amino acids. However, catalytic activity does not appear to influence endosomal localization.…”

Phospholipase D—Structure, regulation and function

“…One region is conserved sequence III and another is the extreme C-terminus, since limited mutations of these sequences abolish catalysis (Sung et al 1999b;Xie et al 2000b;Liu et al 2001). The C-terminal four amino acids are conserved in PLD1 and PLD2 and are similar to those in putative PLDs from C. elegans and D. melanogaster, but deviate from those in fungi, bacteria and plants (Fig.…”