TPCK-induced apoptosis and labelling of the largest subunit of RNA polymerase II in Jurkat cells.
N-alpha-Tosyl-L: -phenylalanyl chloromethyl ketone (TPCK) is an affinity label for chymotrypsin-like proteases and has been extensively used as an experimental tool in apoptosis research to probe the role of proteases in cell death. While TPCK blocks some apoptotic changes and induces others, the cellular target or targets for TPCK have not been identified. Here we investigated for the first time the cellular targets for TPCK using a polyclonal anti-tosyl antibody. We have found that TPCK rapidly and irreversibly labels numerous intracellular proteins and have identified one as RPB1, the largest subunit of RNA polymerase II. We show that TPCK inhibits DNA binding by RNA polymerase and that TPCK inhibits transcription. Inhibition of transcription is known to induce apoptosis and while TPCK may trigger death through interaction with multiple targets, our data suggests that the pro-apoptotic effects of TPCK may be explained in part by the inhibition of RNA polymerase II activity.
Fabian
Z
Z
National Centre for Biomedical Engineering Science, School of Science, NUI Galway, Galway, Ireland.
O'Brien
P
P
Pajecka
K
K
Fearnhead
H O
HO
D000478:Alkylation / Q000187:drug effects; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3 / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D003851:Deoxyribonucleases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D019169:Jurkat Cells; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D013194:Staining and Labeling; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000737:chemistry / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects
D000906:Antibodies; D051017:Apoptosis Regulatory Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; C109664:caspase-activated DNase inhibitor; D014108:Tosylphenylalanyl Chloromethyl Ketone; D004247:DNA; D012319:RNA Polymerase II; C410949:RNA polymerase II largest subunit; C113696:DFFB protein, human; D003851:Deoxyribonucleases; D053148:Caspase 3