Caspase cleavage of phospholipase D1 in vitro alters its regulation and reveals a novel property of the "loop" region.
Phospholipase D (PLD) has been implicated in mediating vesicular transport, mitosis, differentiation and apoptosis. The product of PLD activity, phosphatidic acid (PA) has mitogenic potential and elevated PLD expression has been detected in many tumor cell lines. Several reports have demonstrated that distinct PLD domains regulate its activity and that truncated forms of PLD retain enzymatic activity. We hypothesized that during apoptosis caspase cleavage of PLDs could result in modification of their activities. To test this idea, we have used in vitro translation of PLD1 and PLD2 which generated active enzymes exhibiting properties mimicking those of the endogenous proteins. Here we demonstrate that PLD1 was rapidly cleaved in vitro by caspases-8, -3 and -7. In contrast, PLD2 cleavage was delayed and its activity was unaffected by incubation with caspase-3. Significantly, following caspase cleavage the response of PLD1 to regulatory stimuli was altered; it was no longer activated by PKC and instead exhibited an increased activity in response to small GTPases. Notably, this enhanced activity was due to cleavage of PLD1 in the "loop" domain, a region previously associated with negative regulatory function. Thus our data have identified a novel regulatory domain in PLD1.
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Christian
C
Riebeling
Sylvain
S
Bourgoin
Dennis
D
Shields
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D016244:Guanosine 5'-O-(3-Thiotriphosphate); C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D053148:Caspase 3; D020169:Caspases; D013755:Tetradecanoylphorbol Acetate