Filamin (280-kDa actin-binding protein) is a caspase substrate and is also cleaved directly by the cytotoxic T lymphocyte protease granzyme B during apoptosis.
We used yeast two-hybrid screening to identify the cytoskeletal protein filamin as a ligand for the proapoptotic protease granzyme B, produced by cytotoxic T lymphocytes. Filamin was directly cleaved by granzyme B when target cells were exposed to granzyme B and the lytic protein perforin, but it was also cleaved in a caspase-dependent manner following the ligation of Fas receptors. A similar pattern of filamin cleavage to polypeptides of approximately 110 and 95 kDa was observed in Jurkat cells killed by either mechanism. However, filamin cleavage in response to granzyme B was not inhibited by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone at concentrations that abolished DNA fragmentation. Filamin staining was redistributed from the cell membrane into the cytoplasm of Jurkat cells exposed to granzyme B and perforin and following ligation of Fas receptors, coincident with the morphological changes of apoptosis. Filamin-deficient human melanoma cells were significantly (although not completely) protected from granzyme B-mediated death compared with isogenic filamin-expressing cells, both in clonogenic survival and (51)Cr release assays, whereas death from multiple other stimuli was not affected by filamin deficiency. Thus, filamin is a functionally important substrate for granzyme B, as its cleavage may account at least partly for caspase-independent cell death mediated by the granzyme.
Browne
K A
KA
Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett Street, Melbourne 8006, Australia.
Johnstone
R W
RW
Jans
D A
DA
Trapani
J A
JA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D001665:Binding Sites; D020169:Caspases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002860:Chromium Radioisotopes / Q000378:metabolism; D003285:Contractile Proteins / Q000378:metabolism* / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D064448:Filamins; D053804:Granzymes; D006801:Humans; D020287:In Situ Nick-End Labeling; D019169:Jurkat Cells; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000494:pharmacology; D008840:Microfilament Proteins / Q000378:metabolism* / Q000502:physiology*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011233:Precipitin Tests; D012697:Serine Endopeptidases / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D019014:fas Receptor / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D002860:Chromium Radioisotopes; D003285:Contractile Proteins; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D064448:Filamins; D008024:Ligands; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D052899:Pore Forming Cytotoxic Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D020169:Caspases; D019311:Staurosporine