Hedberg K K, Birrell G B, Griffith O H
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Cell Regul. 1991 Dec;2(12):1067-79. doi: 10.1091/mbc.2.12.1067.
The cell-permeant heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine(TPEN) was found to counteract phorbol ester-induced actin reorganization in PTK2 and Swiss 3T3 cells. By using fluorescence and the higher resolution technique of photoelectron microscopy to monitor actin patterns, 15-min pretreatment with 25-50 microM 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 microM TPEN for 1.5 h. Phorbol ester-induced actin alterations are thought to depend 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 interaction of PKC with a heavy metal, probably zinc.
细胞渗透性重金属螯合剂N,N,N',N'-四(2-吡啶甲基)乙二胺(TPEN)被发现可对抗佛波酯诱导的PTK2和瑞士3T3细胞中的肌动蛋白重组。通过使用荧光以及更高分辨率的光电子显微镜技术来监测肌动蛋白模式，发现用25 - 50微摩尔TPEN预处理15分钟可显著减少PTK2细胞中后续佛波酯处理导致的肌动蛋白改变。在瑞士3T3细胞中使用50微摩尔TPEN处理1.5小时也获得了类似结果。佛波酯诱导的肌动蛋白改变被认为依赖于蛋白激酶C(PKC)的激活。与佛波酯的作用相反，由星形孢菌素和细胞松弛素B引起的不依赖PKC的肌动蛋白细胞骨架破坏不受TPEN预处理的影响。如通过80K PKC底物蛋白( MARCKS蛋白)的磷酸化所观察到的，TPEN不会阻断佛波酯诱导的瑞士3T3细胞中PKC的激活。在体外PKC特异性商业检测中，TPEN也不会抑制从瑞士3T3细胞中部分纯化的PKC。为了确定TPEN的作用是去除金属离子而不是TPEN的其他非特异性作用，在TPEN预处理结束时添加了一系列过渡金属离子。结果表明，培养细胞中佛波酯诱导的肌动蛋白细胞骨架的短暂但显著的重组依赖于PKC与一种重金属(可能是锌)的相互作用。
