D4-GDI, a substrate of CPP32, is proteolyzed during Fas-induced apoptosis.
Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis, ICE/CED-3 family cysteine proteases have been implicated directly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified. Here we report that D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of the apoptosis protease CPP32/Yama/Apopain. D4-GDI was rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine. Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk. D4-GDI was cleaved in vitro by recombinant CPP32 expressed in Escherichia coli to form a 23-kDa fragment. The CPP32-mediated cleavage of D4-GDI was completely inhibited by 1 microM DEVD-CHO, a reported selective inhibitor of CPP32. In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrations up to 50 microM. N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp19 and Ser20 of the poly(ADP-ribose) polymerase-like cleavage sequence DELD19S. These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI protein.
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Department of Molecular Sciences, Pfizer Inc., Groton, Connecticut 06340, USA.
Chuang
T H
TH
Cunningham
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Turi
T G
TG
Hanke
J H
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Bokoch
G M
GM
Danley
D E
DE
D000470:Alkaloids / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000378:metabolism; D019311:Staurosporine; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000276:immunology / Q000502:physiology*; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C569658:ARHGDIB protein, human; D000470:Alkaloids; D000906:Antibodies; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D020730:Guanine Nucleotide Dissociation Inhibitors; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D019014:fas Receptor; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D011493:Protein Kinase C; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D019204:GTP-Binding Proteins; D019311:Staurosporine