Protein Phosphorylation Associated with Epipodophyllotoxin- Induced Apoptosis of Lymphoid Cells: Role of a Serine/Threonine Protein Kinase

Autor: Ye, Xiaodan, Mody, Neeta S., Hingley, Susan T., Coffman, Frederick D., Cohen, Stanley, Fresa, Kerin L.
Zdroj: Clinical Immunology and Immunopathology (Now Called Clinical Immunology); November 1998, Vol. 89 Issue: 2 p117-125, 9p
Abstrakt: 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 byN-(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 μM sangivamycin to thymocyte cultures up to 2 h after addition of either inducer resulted in virtually complete inhibition of apoptosis. Addition of 10 μM 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 ofde novophosphorylation 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 thatde novoprotein 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.
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