HDGF is dephosphorylated during the early steps of endothelial cell apoptosis in a caspase-dependent way.
We were looking by a proteomic approach for new phospho-proteins involved during the early steps of the TNF + cycloheximide (CHX)-induced apoptosis-preceding mitochondrial membrane permeabilization-of endothelial cells (BAEC). In the present study, we observed on the autoradiography from 2D gel of (32)P-labeled samples a string of proteins undergoing a complete dephosphorylation after 1 h of stimulation with TNF + CHX-while mitochondrial membrane permeabilization was observed after 3 h-identified the different spots by mass spectrometry as one and only protein, HDGF, and confirmed the identity by western blot. The intensity of the 2D phosphorylation pattern of HDGF was correlated with the amount of apoptosis induced by TNF + CHX and TNF or CHX alone and this event was inhibited by the Caspase specific inhibitor zVADfmk. Moreover the TNF + CHX-treatment did not affect the nuclear localization of GFP-HDGF. Taken together, our data suggest an involvement of HDGF during the initiation phase of the apoptotic process downstream from an initiator Caspase and a regulation of this protein by phosphorylation in the nucleus.
Clermont
Frederic
F
Institute of Interdisciplinary Research (IRIBHM), Institute of Molecular Biology and Medicine, Free University of Brussels (ULB), 12 rue des professeurs Jeener et Brachet, B-6041 Gosselies, Belgium.
Gonzalez
Nathalie Suarez
NS
Communi
David
D
Franken
Sebastian
S
Dumont
Jacques E
JE
Robaye
Bernard
B
D000818:Animals; D001011:Aorta / Q000166:cytology; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000502:physiology*; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D036341:Intercellular Signaling Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha; C051610:hepatoma-derived growth factor; D003513:Cycloheximide; D020169:Caspases