Hydrogen peroxide induces association between glyceraldehyde 3-phosphate dehydrogenase and phospholipase D2 to facilitate phospholipase D2 activation in PC12 cells.
Oxidative stress or signaling is widely implicated in apoptosis, ischemia and mitogenesis. Previously, our group reported that the hydrogen peroxide (H2O2)-dependent activation of phospholipase D2 (PLD2) in PC12 cells is involved in anti-apoptotic effect. However, the precise mechanism of PLD2 activation by H2O2 was not revealed. To find H2O2-dependent PLD2-regulating proteins, we immunoprecipitated PLD2 from PC12 cells and found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) coimmunoprecipitated with PLD2 upon H2O2 treatment. This interaction was found to be direct by in vitro reconstitution of purified GAPDH and PLD2. In vitro studies also indicated that PLD2-associated GAPDH was modified on its reactive cysteine residues. Koningic acid, an alkylator of GAPDH on catalytic cysteine residue, also increased interaction between the two proteins in vitro and enhanced PLD2 activity in PC12 cells. Blocking H2O2-dependent modification of GAPDH with 3-aminobenzamide resulted in the inhibition of the GAPDH/PLD2 interaction and attenuated H2O2-induced PLD2 activation in PC12 cells. From the results, we suggest that H2O2 modifies GAPDH on its catalytic cysteine residue not only to inactivate the dehydrogenase activity of GAPDH but also to endow GAPDH with the ability to bind PLD2 and the resulting association is involved in the regulation of PLD2 activity by H2O2.
Kim
Jung Hwan
JH
Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea.
Lee
Sukmook
S
Park
Jong Bae
JB
Lee
Sang Do
SD
Kim
Jong Hyun
JH
Ha
Sang Hoon
SH
Hasumi
Keiji
K
Endo
Akira
A
Suh
Pann-Ghill
PG
Ryu
Sung Ho
SH
D000595:Amino Acid Sequence; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D020134:Catalytic Domain / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology*; D016716:PC12 Cells; D010673:Pheochromocytoma / Q000737:chemistry / Q000188:drug therapy / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D020539:Sequence Analysis, Protein; D012717:Sesquiterpenes / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
D001549:Benzamides; D004791:Enzyme Inhibitors; D016877:Oxidants; D012717:Sesquiterpenes; C027285:heptelidic acid; C025160:3-aminobenzamide; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C105606:phospholipase D2; D010739:Phospholipase D