Post-translational Modification of PLD

Several studies have shown that PLDl and yeast PLD (Spol4) are phosphorylated under basal conditions. The phosphorylation involves Ser and Thr residues and results in relocalization of the enzymes and the appearance of slower migrating enzyme species on SDS polyacrylamide-gel electrophoresis [18, 24, 43, 44] (Fig. 4.3). The phosphorylation of PLDl occurs predominantly in the N-terminal half of the enzyme and appears to have little effect on the intrinsic catalytic activity of the enzyme [18]. However, cell fractionation studies have shown that the phosphorylated form of overexpressed PLDl is not present in the cytosol, in contrast to the non-phosphorylated form, indicating that phosphorylation increases membrane association of the enzyme [18]. Treatment of cells with phorbol ester or overexpression of PKCa results in further phosphorylation of PLDl, but this is associated with a loss of activity [45]. Basal Ser/Thr phosphorylation of PLD2 is low compared with that of PLDl. However, addition of phorbol ester or expression of PKCa greatly increases this. 

Another post-translational modification of PLDl and PLD2 is palmitoylation [25, 46]. This occurs on two specific Cys residues (C240, C241 in PLDl and C223, C224 in PLD2) [24—26]. The requirements for palmitoylation have been studied in detail for PLDl. The modification requires the presence of the N-terminal 168 

The determinants of membrane association of PLDl and PLD2 are complex. As indicated above, phosphorylation and palmitoylation play a role. There is also evidence for involvement of the PH domain [9]. Surprisingly, mutation of a PIP2 binding site located between conserved sequences II and III did not alter membrane association of PLD2 [8]. 

Studies utilizing intact mammalian cells have shown that neomycin and C. difficile Toxin B, which reduce PIP2 levels, inhibit PLD activity [49-51]. Conversely, coexpression of PLDl or PLD2 and PI 4-P 5-kinase, which synthesizes PIP2, results in increased PLD activity [52]. When a kinase-dead mutant of PI 4-P 5-kinase is transfected in place of the wild-type enzyme, the increase in PLD activity is much less [52]. 

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