Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-bisphosphate-specific PH domain

The signalling pathway leading, for example, to actin cytoskeletal reorgani sation, secretion or superoxide generation involves phospholipase D (PLD)-c atalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, w hich appears to mediate the messenger functions of this pathway [1-3]. Two PLD genes (PLD1 and PLD2) with similar domain structures have been cloned a nd progress has been made in identifying the protein regulators of PLD1 act ivation, for example Arf and Rho family members [4,5], The activities of bo th PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate Pl(4 ,5)P-2 and our sequence analysis suggested the presence of a pleckstrin hom ology (PH) domain in PLD1, although its absence has also been claimed [4], Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Fur thermore, PLD1 bound specifically and with high affinity to lipid surfaces containing Pl(4,5)P-2 independently of the substrate phosphatidylcholine, s uggesting a key role for the PH domain in PLD function. Importantly, a glut athione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayer s containing Pl(4,5)P-2. Point mutations within the PLD1 PH domain inhibite d enzyme activity, whereas deletion of the domain both inhibited enzyme act ivity and disrupted normal PLD1 localisation, Thus, the functional PH domai n regulates PLD by mediating its interaction with polyphosphoinositide-cont aining membranes; this might also induce a conformational change, thereby r egulating catalytic activity.