Increased activity of the protein kinase C-delta holoenzyme in the cytoplasmic particulate fraction precedes the activation of caspases in polyomavirus-transformed pyF111 rat fibroblasts exposed to calphostin C or topoisomerase-II inhibitors.
A caspase-mediated release of the 40-kDa catalytic fragment of the delta isoform (CF-delta) of protein kinase C (PKC-delta) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-delta, but cannot make the antiapoptotic Bcl-2 and Bcl-X(L) proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-delta activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101-108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737-743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-delta holoenzyme (holo-PKC-delta) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-delta fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-delta-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-delta. However, while calphostin C did not affect the delta-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the delta activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-delta activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of delta-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.
Dal Pra
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I
Histology & Embryology Unit, University of Verona, Verona, I-37134, Italy.
Whitfield
J F
JF
Chiarini
A
A
Armato
U
U
D000818:Animals; D020169:Caspases / Q000378:metabolism*; D002461:Cell Line, Transformed; D003593:Cytoplasm / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D007527:Isoenzymes / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051381:Rats; D059005:Topoisomerase II Inhibitors
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009281:Naphthalenes; D059005:Topoisomerase II Inhibitors; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D020169:Caspases; C058819:calphostin C