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Caspase cleavage of phospholipase D alters its regulatory response during cell death
Author(s) -
Riebeling Christian,
Shields Dennis
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb44-a
Subject(s) - phospholipase d , microbiology and biotechnology , caspase , gtp' , apoptosis , cleavage (geology) , pld2 , gtpase , enzyme activator , chemistry , caspase 3 , biology , biochemistry , phosphorylation , programmed cell death , signal transduction , enzyme , phosphatidic acid , phospholipid , membrane , paleontology , fracture (geology)
Phospholipase D (PLD) has been implicated in mediating vesicular transport, mitosis and apoptosis. Several reports have demonstrated that distinct domains regulate PLD activity and that truncated forms of PLD retain enzymatic activity. We hypothesized that PLD could be similarly truncated by caspases during apoptosis, retaining or modifying its activity. To test this idea, we used in vitro ‐translated PLDs treated with purified caspases to identify PLD cleavage products and to determine their enzymatic activity. We demonstrate that PLD1 is rapidly cleaved by caspase‐2, −3, −7 and −8, whereas PLD2 is relatively resistant. Consistent with its caspase‐resistance, the activity of PLD2 was unchanged after incubation with activated caspase‐3. Strikingly, the activity of PLD1 cleaved by caspase‐3 was no longer stimulated by TPA, an activator of protein kinase C; however its activity was increased by incubation with the non‐hydrolyzable analog of GTP, GTPγS, which activates small GTPases. Using site‐directed mutagenesis we identified three distinct caspase cleavage sites that are responsible for the decreased stimulation by TPA, the increased activation by GTPγS or had no effect on enzymatic properties, respectively. Our results suggest that although PLD1 is cleaved during apoptosis, it retains enzymatic activity which can be stimulated by small GTP‐binding proteins but not protein kinase C. We speculate that this activity enhances fragmentation of the Golgi apparatus, a hallmark of early apoptosis. This work is supported by the DFG (RI‐1222) and by the NIH (DK21860).