A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis.
X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. Here we show that XIAP associates with the active caspase-9-Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevented caspase-9 association with XIAP and its concomitant inhibition. We note that the N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. Our studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis.
Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
S M
SM
Srinivasula
R
R
Hegde
A
A
Saleh
P
P
Datta
E
E
Shiozaki
J
J
Chai
R A
RA
Lee
P D
PD
Robbins
T
T
Fernandes-Alnemri
Y
Y
Shi
E S
ES
Alnemri
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; D002352:Carrier Proteins / Q000378:metabolism*; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004330:Drosophila; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein
C506592:APAF1 protein, human; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C412403:DIABLO protein, human; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases