PS? Engulfing phagocyte Apoptotic cell Phagocytosis ATP Phosphatidyl serine (PS) normally on the inner leaflet of the plasma membrane ‘flips’ to the outer leaflet TSP, macrophage secreted glycoprotein which provides a ‘bridge’ between the macrophage and an apoptotic cell Exposure of sugar residues on cell-surface carbohydrates Lectins recognize and interact with sugars CDI4 Plasma membrane-bound glycoprotein which binds lipid molecules Ion flux *α vB3 *CD36 Croquemort (drosophila) DOCK I80 (CED5 homolog) PS receptor (CDI4?) Contains SH3 domain which allows interaction with signaling pathways - regulation of cytoskeleton and cell motility ? ABC Transporter (CED7 homolog) role in phagocytosis unclear *Adhesion molecules which bind TSP and signal via tyrosine kinases Thrombospondin (TSP) moiety Mitochondria Pro-apoptotic Bcl-2 family members promote, and anti-apoptotic Bcl-2 family members block mitochondrial cytochrome c release Bax Cytochrome c Apaf-I Apoptosome Caspase-9 tetramer Procaspase-9 Bcl-x L blocks? Caspases- 3,6,7 BH2 BHI BH3 BH4 TM BH2 BHI BH3 BH4 TM BH2 BHI BH3 BH4 TM BH3 BH4 TM BH3 TM N C Bax, Bak, Mtd/Bok Bcl-x S Bik, Hrk, Bim, Blk Bad, Bid Pro-apoptotic Bcl-2, Bcl-x L , Mcl-I Bcl-w, A-I Anti-apoptotic BH3 The Bcl-2 Family TNF receptor (TNFR) (monomer, cysteines in yellow, contact residues in red) Ligand (TNF) (trimer, contact residues in red) Active caspase-8 tetramer Apoptosis TNF-family death receptors TNF FasL TRAIL TRAIL TNFR TRAIL-R2 FADD FADD c-IAP TRAF- I TRAF-2 ? TRAIL-RI TRADD Recruits and permits caspase-8 transactivation Recruits and permits caspase-8 transactivation Unknown adaptor recruits and permits caspase-8 transactivation Inhibits TNF - induced apoptosis p53 Nuclear signal IKKI/IKK2 Calcineurin JNK/SAPK EGR-3 AP-I IKKI/IKK2 Phosphorylation and dissociation Active Inactive complex Transcriptional regulation e.g.Bax Death NF-AT NF-AT Stress signals T-cell receptor activation DNA damage Fos/Jun heterodimers NF-κB NF-κB NF-κB NF-κB IκB IκB FasL transcription EGR-3 IL-3 IL-3R IGF-I IGF-IR PI 3-kinase AKT (PKB) SCF Ceramide wortmannin Bad Akt P Bcl-2 Bax Bcl-x L Bcl-x L Bad P Survival signals Kit (SCF-R ) Phosphorylation and inactivation of Bad *SerI36 P Bad *SerI36 SerII2 *SerI36 SerII2 Sequestration of Bad 14-3-3 Bad *Required for survival-promoting activity Dissociation of Bad/Bcl-2 and Bad/Bcl-x L heterodimers Neutralization of pro-apoptotic effects of Bax Perforin Granzyme B (GrB ) ? Perforin Death Granzyme B Cytotoxic T cell Gr B + rab5 + Gr B + rab4 + Gr B + Gr B + Non-classical receptor- mediated entry Mechanism of perforin entry unknown Executioner caspase Activated caspase tetramer Cytosolic granzyme B rab5 – rab4 – lgpI20 – Cathepsin D – Cytochr ome c – M-6-P-R – Target cell 3 6 7 8 10 9 I 4 5 2 Executioner caspases Initiator caspases Inflammatory caspases Executioner caspase Initiator caspase Adaptors Caspases Transactivation Active caspase tetramer (2 heterodimers) Active caspase-3 DNA fragmentation I80 bp oligo- nucleosomal fragments Internucleosomal DNA cleavage CAD/ DFF40/ CPAN Active nuclease iC AD/ DFF45 PARP Other target substrates: pro-IL-I β (caspase-I) lamins A/C (caspase-6) fodrin, Mdm2, UI -70 kDa, actin (caspase-3) FAK Focal adhesion kinase IC AD-L Inhibitor of caspase-activated DNAse PAK-2 p2I-associated kinase PARP Poly-ADP-ribose polymerase PKC Protein kinase C IGF-IR Insulin-like growth factor I receptor IL-3R Interleukin-3 receptor SCF-R Stem cell factor receptor PA-DEVD 213 G-RP DNA-PK DNA- DEVD 2712 N-PK Retinoblastoma Retino-DEAD 886 G-blastoma PKC- δ PKC- DMQD 330 N -δ PAK-2 PA-EVD 212 G -K-2 Gelsolin Gel- DQTD 352 G -solin MEKK- I ME-DTVD 874 G-KK- I Substrate cleavage Mediate membrane and morphological changes IC AD-L (-S) IC -DEPD 117 -AD-L IC -DAVD 224 -AD-S DNA fragmentation Disruption of cell-cycle control FAK F-VSWD 704 S-AK F-DQTD 772 S-AK Loss of cellular adhesion Disable DNA repair mechanisms Death effector domain Caspase recruitment domain Prodomain Protease subunits TNF TRAIL TRADD FADD TRAF IAP Death effector domain Death domain Tumor necrosis factor TNF-related apoptosis-inducing ligand TNF-receptor-associated death domain Fas-associated death domain TNFR-associated factor Inhibitor of apoptosis protein Death domain Fas SerII2 P Apoptosis Burial Execution Initiation LIT.APOP.2010.1 Copyright © 1998 Elsevier Science Ltd. All rights reserved. Based on an original design by Sheila Betts. Your first choice for antibodies! North & South America Tel: +1 800 265 7376 Fax: +1 919 878 3751 Email: Worldwide Tel: +44 1865 852 700 Fax: +44 1865 852 739 Email: Europe Tel: 00800 CALL 4ABD (00800 2255 4223) Fax: 00800 2FAX 2ABD (00800 2329 2223) Email: [email protected] [email protected] [email protected] Helen M. Beere and Douglas R. Green La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA With thanks to Joshua Goldstein, Carl Ware, Michael Pinkoski, Shailaja Kasibhatla and Nigel Waterhouse FREE PERSONALIZED Antibody Detective Service www.abdserotec.com/ADS