Protein phosphorylation associated with epipodophyllotoxin-induced apoptosis of lymphoid cells: role of a serine/threonine protein kinase.
We have previously shown that apoptosis induced in thymocytes by dexamethasone or teniposide (VM-26) could be inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and sangivamycin, both relatively specific inhibitors for protein kinase C, but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor for cAMP-dependent protein kinases. Apoptosis in this model system was not blocked by EGTA and no increase in cytosolic Ca2+ was observed during apoptosis induced by either dexamethasone or VM-26, suggesting that this kinase was Ca2+-independent. In the present study, we demonstrate that addition of 10 microM sangivamycin to thymocyte cultures up to 2 h after addition of either inducer resulted in virtually complete inhibition of apoptosis. Addition of 10 microM sangivamycin at 3 or 4 h after addition of inducer resulted in partial inhibition of apoptosis. Computerized image analysis of two-dimensional PAGE analyses of whole-cell lysates demonstrated that treatment of mouse thymocytes with VM-26 resulted in a limited number of de novo phosphorylation events within 1 h of treatment. The most prominent phosphorylation events associated with VM-26-induced apoptosis were that two intracellular protein species (Protein 1: m.w. = 22.9 kDa, pI, 5.11; and Protein 2: m.w. = 22.9 kDa, pI, 4.98). Similar phosphorylation events were seen in cells treated with dexamethasone. Finally, Western blot analysis suggests that de novo protein phosphorylation induced by VM-26 is on serine/threonine residues. These results provide further evidence that the mechanism of VM-26-induced apoptosis of murine thymocytes involves the action of one or more serine/threonine kinases.
Ye
X
X
Department of Microbiology and Immunology, Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania, 19131, USA.
Mody
N S
NS
Hingley
S T
ST
Coffman
F D
FD
Cohen
S
S
Fresa
K L
KL
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D051379:Mice; D008809:Mice, Inbred C3H; D010766:Phosphorylation; D011034:Podophyllotoxin / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D013713:Teniposide / Q000494:pharmacology; D013950:Thymus Gland / Q000166:cytology*
D000972:Antineoplastic Agents, Phytogenic; D004791:Enzyme Inhibitors; D003907:Dexamethasone; D013713:Teniposide; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D011034:Podophyllotoxin