Phorbol ester-induced actin cytoskeletal reorganization requires a heavy metal ion, probably Zn2+.

14 pagesThe cell-permeant heavy metal chelator N,N,N',Ntetrakis( 2-pyridylmethyl)ethylenediamine (TPEN) was found to counteract phorbol ester-induced actin reorganization in PTK2 and Swiss 3T3 cells. By us ing fluorescence and the higher resolution tech nique of photoelectron microscopy to monitor actin patterns, 15-min pretreatment with 25-50 pM TPEN was found to dramatically reduce actin alterations resulting from subsequent phorbol ester treatment in PTK2 cells. Similar results were obtained with Swiss 3T3 cells using 50 mM TPEN for 1.5 h. Phorbol ester-induced actin alterations are thought to de pend on activation of protein kinase C (PKC). In contrast to the phorbol ester effect, the PKC-independent actin cytoskeletal disruption caused by staurosporine and cytochalasin B was unaffected by TPEN pretreatment. TPEN did not block phorbol ester-induced activation of PKC in Swiss 3T3 cells, as observed by the phosphorylation of the 80K PKC substrate protein (MARCKS protein). TPEN also did not inhibit partially purified PKC from Swiss 3T3 cells in an in vitro PKC-specific commercial assay. To establish that the effect of TPEN is the removal of metal ions and not some other nonspecific effect of TPEN, a series of transition metal ions was added at the end of the TPEN pretreatment. The results indicate that the transient but dramatic phorbol ester-induced reorganization of the actin cytoskeleton in cultured cells depends on an inter action of PKC with a heavy metal, probably zinc