Transition metal chelator TPEN counteracts phorbol ester-induced actin cytoskeletal disruption in C6 rat glioma cells without inhibiting activation or translocation of protein kinase C.
Phorbol ester-induced reorganization of the actin cytoskeleton was investigated in C6 rat glioma cells. Observations by fluorescence microscopy and photoelectron microscopy indicated that pretreatment with the transition metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) for 1-2 h at 50 microM reduced the sensitivity of the actin cytoskeleton to disruption by the subsequent addition of 200 nM phorbol myristate acetate (PMA). The protective effect of TPEN was eliminated by adding back Zn2+ prior to PMA addition, implicating chelation of metal ions as the mechanism of action of TPEN. C6 cells exposed to PMA experience potent activation of protein kinase C (PKC) and substantial redistribution of the kinase from a soluble to a particulate cellular fraction (translocation). TPEN pretreatment did not block PKC translocation in PMA-exposed cells. By two-dimensional gel analysis, TPEN also did not reduce, but rather slightly increased, the PMA-stimulated phosphorylation of the acidic 80 kDa endogenous PKC substrate, as well as two other proteins at 18 kDa and 50 kDa. In contrast, TPEN significantly suppressed phosphorylation of a 20 kDa protein, both in cells treated with TPEN only and in TPEN-pretreated PMA-exposed cells. The results indicate that the ability of TPEN to protect against PKC-mediated actin cytoskeletal disruption is not due to either a block of PKC translocation or to general inhibition of PKC activity. Rather, the action of TPEN is more selective and probably involves chelation of Zn2+ at a critical Zn(2+)-dependent phosphorylation step downstream from the initial tumor promoter-induced effects on PKC.
Hedberg
K K
KK
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Birrell
G B
GB
Mobley
P L
PL
Griffith
O H
OH
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005029:Ethylenediamines / Q000494:pharmacology*; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D008670:Metals; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000199:Actins; D002614:Chelating Agents; D005029:Ethylenediamines; D008670:Metals; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine