Apoptosis

Phil Dash
Basic Medical Sciences, St.Georges, University of London www.sgul.ac.uk/dept/immunology/~dash
Apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms. Cells die in response to a variety of stimuli and during apoptosis they do so in a controlled, regulated fashion. This makes apoptosis distinct from another form of cell death called necrosis in which uncontrolled cell death leads to lysis of cells, inflammatory responses and, potentially, to serious health problems. Apoptosis, by contrast, is a process in which cells play an active role in their own death (which is why apoptosis is often referred to as cell suicide). Apoptosis in health and disease Apoptosis occurs during the normal development of multicellular organisms and continues throughout adult life. The combination of apoptosis and cell proliferation is responsible for shaping tissues and organs in developing embryos. For example the apoptosis of cells located in-between the toes allows for their separation. Apoptosis is also an important part of the regulation of the immune system. T lymphocytes are cells of the immune system that are responsible for destroying infected or damaged cells in the body. They mature in the thymus, but before they can enter the bloodstream they are tested to ensure that they are effective against foreign antigens and are also not reactive against normal, healthy cells. Any ineffective or self-reactive T-cells are removed through the induction of apoptosis. Problems with the regulation of apoptosis have been implicated in a number of diseases. Cancer is a disease that is often characterized by too little apoptosis. Cancer cells typically possess a number of mutations that have allowed them to ignore normal cellular signals regulating their growth and become more proliferative than normal. Under normal circumstances damaged cells will undergo apoptosis, but in the case of cancer cells mutations may have occurred that prevent cells from undergoing apoptosis. In these cases there is no check on the cellular proliferation and consequently the disease can progress to the formation of tumors. In many cases these tumors can be difficult to kill as many cancer treatments rely on damaging the cells with radiation or chemicals and mutations in the apoptotic pathway often produce cells that are resistant to this type of attack. Understanding how apoptosis is regulated in cancer is therefore of major interest in the development of treatments for this disease. If cancer is a disease where too little apoptosis occurs there are other diseases where too much apoptosis is thought to be part of the problem. For example in neurodegenerative diseases such as Parkinsons or Alzheimers Diseases apoptosis is thought to account for much of the cell death and the progressive loss of neurons. Apoptosis is also important for normal placental development. During pregnancy trophoblast cells from the placenta invade the uterine environment in order to remodel the maternal blood vessels and help establish and maintain a successful pregnancy. Strict control over cell proliferation and apoptosis is required to achieve this. In some cases this process can be compromised and excessive apoptosis of the trophoblast cells is thought to be implicated in the failure to fully remodel the maternal environment that is observed in complications of pregnancy such as preeclampsia.

Apoptosis is also thought to play a role in the progression of many autoimmune diseases. For example, in the case of rheumatoid arthritis excessive proliferation of synovial cells is thought to be due in part to the resistance of these cells to apoptotic stimuli. In other cases poor regulation of apoptosis in Tlymphocytes can result in auto-reactive T-cells entering the circulation and contributing to the onset of autoimmune diseases. The apoptotic process Upon receiving specific signals instructing the cells to undergo apoptosis a number of distinctive changes occur in the cell. A family of proteins known as caspases are typically activated in the early stages of apoptosis. These proteins breakdown or cleave key cellular components that are required for normal cellular function including structural proteins in the cytoskeleton and nuclear proteins such as DNA repair enzymes. The caspases can also activate other degradative enzymes such as DNases, which begin to cleave the DNA in the nucleus. Apoptotic cells display distinctive morphology during the apoptotic process, and this can be seen in Figure 1. Typically, the cell begins to shrink following the cleavage of lamins and actin filaments in the cytoskeleton (A). The breakdown of chromatin in the nucleus often leads to nuclear condensation and in many cases the nuclei of apoptotic cells take on a horse-shoe like appearance (B). Cells continue to shrink (C), packaging themselves into a form that allows for their removal by macrophages. These phagocytic cells are responsible for clearing the apoptotic cells from tissues in a clean and tidy fashion that avoids many of the problems associated with necrotic cell death. In order to promote

Figure 1: Morphology of an apoptosic trophoblast cell as captured by timelapse microscopy (images taken over a 6 hour period).

APOPTOSIS
their phagocytosis by macrophages, apoptotic cells often ungergo plasma membrane changes that trigger the macrophage response. One such change is the translocation of phosphatidylserine from the inside of the cell to the outer surface. The end stages of apoptosis are often characterised by the appearance of membrane blebs (D) or blisters process. Small vesicles called apoptotic bodies are also sometimes observed (D, arrow). There are a number of mechanisms through which apoptosis can be induced in cells. The sensitivity of cells to any of these stimuli can vary depending on a number of factors such as the expression of pro- and anti-apoptotic proteins (eg. the Bcl-2 proteins or the Inhibitor of Apoptosis Proteins), the severity of the stimulus and the stage of the cell cycle. Some of the major stimuli that can induce apoptosis include virus infection, cell stress and DNA damage. In some cases the apoptotic stimuli comprise extrinsic signals such as the binding of death inducing ligands to cell surface receptors called death receptors. These ligands can either be soluble factors or can be expressed on the surface of cells such as cytotoxic T lymphocytes. The latter occurs when T-cells recognise damaged or virus infected cells and initiate apoptosis in order to prevent damaged cells from becoming neoplastic (cancerous) or virus-infected cells from spreading the infection. Apoptosis can also be induced by cytotoxic T-lymphocytes using the enzyme granzyme. In other cases apoptosis can be initiated following intrinsic signals that are produced following cellular stress. Cellular stress may occur from exposure to radiation or chemicals or to viral infection. It might also be a consequence of growth factor deprivation or oxidative stress caused by free radicals. In general intrinsic signals initiate apoptosis via the involvement of the mitochondria. The relative ratios of the various bcl-2 proteins can often determine how much cellular stress is necessary to induce apoptosis. Death Receptors Death receptors are cell surface receptors that transmit apoptotic signals initiated by specific ligands such as Fas ligand, TNF alpha and TRAIL. They play an important role in apoptosis and can activate a caspase cascade within seconds of ligand binding. Induction of apoptosis via this mechanism is therefore very rapid. Apoptotic signalling from the death receptors Although there are differences in the signalling pathways activated by the different death receptors it is possible to outline a general apoptotic signalling pathway. Binding of the death inducing ligand to its receptor can lead to a the generation of ceramide, typically produced by acid sphingomyelinase. This ceramide release is thought to promote lipid raft fusion which results in a large scale clustering of the death receptors. The large scale receptor clustering is important because it helps amplify the apoptotic signalling. In the absence of receptor clustering some cells, such as lymphocytes, are still able to trigger apoptosis but in most cases amplification of the signalling pathway is needed to activate the full apoptotic response. Following ligand binding a conformational change in the intracellular domains of the receptors reveals the presence of a death domain which allows the recruitment of various apoptotic proteins to the receptor. This protein complex is often called the DISC, or Death Inducing Signalling Complex. The final step in this process is the recruitment of one of the caspases, typically caspase 8, to the DISC. This results in activation of caspase 8 and the inititation of apoptosis. TNF receptor signalling TNF is produced by T-cells and activated macrophages in response to infection. By activating its receptor, TNFR1, TNF can have several effects (see Figure 2). In some cells it leads to activation of NFkB and AP-1 which leads to the induction of a wide range of genes. In some cells, however, TNF can also induce apoptosis, although receptor ligation is rarely enough on its own to initiate apoptosis as is the case with Fas ligand binding. Binding of TNF alpha to TNFR1 results in receptor trimerisation and clustering of intracellular death domains. This allows binding of an intracellular adapter molecule called TRADD (TNFR-

www.sgul.ac.uk/depts/immunology/~dash associated death domain) via interactions between death domains. TRADD has the ability to recruit a number of different proteins to the activated receptor. Recruitment of TRAF2 (TNF-associated factor 2) can lead to activation of NF-kB and the JNK pathway. TRADD can also associate with FADD, which leads to the induction of apoptosis via the recruitment and cleavage of pro-caspase 8.

Figure 2: Illustration of the main TNF receptor signalling pathways. Signaling by Fas (CD95) The ligand for Fas (FasL or CD95L) activated apoptosis in a similar way to the TNF receptor. Binding of the ligand promotes receptor clustering, DISC formation and the activation of the caspase cascade. However, signalling through the Fas receptor is slightly simpler than through the TNF receptor. The adapter protein FADD can be recruited directly to the death domain on the Fas receptor, without requiring the prior recruitment of TRADD. In addition the Fas receptor is generally though to only activate apoptosis and does not play an important role in other aspects of cell signalling like the TNF receptor.

APOPTOSIS
Induction of apoptosis by TRAIL In a number of ways TRAIL (TNF-related apoptosis inducing ligand) is similar in action to FasL. Binding of TRAIL to its receptors DR4 or DR5 triggers rapid apoptosis in many cells. Interestingly there are also decoy receptors that compete for binding of TRAIL with the DR4 and DR5 receptors. The decoy receptors are called DcR1 and DcR2. Both of these receptors are capable of competing with DR4 or DR5 receptors for binding to the ligand (TRAIL), however ligation of these receptors does not initiate apoptosis since DcR1 does not possess a cytoplasmic domain, while DcR2 has a truncated death domain lacking 4 out of 6 amino acids essential for recruiting adapter proteins. Role of mitochondria in apoptosis Mitochondria play an important role in the regulation of cell death. They contain many pro-apoptotic proteins such as Apoptosis Inducing Factor (AIF), Smac/ DIABLO and cytochrome C. These factors are released from the mitochondria following the formation of a pore in the mitochondrial membrane called the Permeability Transition pore, or PT pore. These pores are thought to form through the action of the pro-apoptotic members of the bcl-2 family of proteins, which in turn are activated by apoptotic signals such as cell stress, free radical damage or growth factor deprivation. Mitochondria also play an important role in amplifying the apoptotic signalling from the death receptors, with receptor recruited caspase 8 activating the pro-apoptotic bcl-2 protein, Bid. Role of Bcl-2 proteins The bcl-2 proteins are a family of proteins involved in the response to apoptosis. Some of these proteins (such as bcl-2 and bcl-XL) are anti-apoptotic, while others (such as Bad, Bax or Bid) are pro-apoptotic. The sensitivity of cells to apoptotic stimuli can depend on the balance of pro- and anti-apoptotic bcl2 proteins. When there is an excess of pro-apoptotic proteins the cells are more sensitive to apoptosis, when there is an excess of anti-apoptotic proteins the cells will tend to be more resistant. An excess of pro-apoptotic bcl-2 proteins at the surface of the mitochondria is thought to be important in the formation of the PT pore. The pro-apoptotic bcl-2 proteins are often found in the cytosol where they act as sensors of cellular damage or stress. Following cellular stress they relocate to the surface of the mitochondria where the anti-apoptotic proteins are located. This interaction between pro- and antiapoptotic proteins disrupts the normal function of the anti-apoptotic bcl-2 proteins and can lead to the formation of pores in the mitochondria and the release of cytochrome C and other pro-apoptotic molecules from the intermembrane space. This in turn leads to the formation of the apoptosome and the activation of the caspase cascade. The release of cytochrome C from the mitochondria is a particularly important event in the induction of apoptosis. Once cytochrome C has been released into the cytosol it is able to interact with a protein called Apaf-1. This leads to the recruitment of pro-caspase 9 into a multiprotein complex with cytochrome C and Apaf-1 called the apoptosome. Formation of the apoptosome leads to activation of caspase 9 and the induction of apoptosis.

www.sgul.ac.uk/depts/immunology/~dash The role of mitochondria in the induction of apoptosis is summarised in Figure 3. Caspases and apoptosis The caspases are a family of proteins that are one of the main executors of the apoptotic process. They belong to a group of enzymes known as cysteine proteases and exist within the cell as inactive proforms or zymogens. These zymogens can be cleaved to form active enzymes following the induction of apoptosis. Induction of apoptosis via death receptors typically results in the activation of an initiator caspase such as caspase 8 or caspase 10. These caspases can then activate other caspases in a cascade. This cascade eventually leads to the activation of the effector caspases, such as caspase 3 and caspase 6. These caspases are responsible for the cleavage of the key cellular proteins, such as cytoskeletal proteins, that leads to the typical morphological changes observed in cells undergoing apoptosis.

Figure 3: Illustration of the main apoptotic signalling pathways involving mitochondria

APOPTOSIS
The apoptosome There are a number of other mechanisms, aside from activation of the death receptors, through which the caspase cascade can be activated. Granzyme B can be delivered into cells by cytotoxic T lymphocytes and is able to directly activate caspases 3, 7, 8 and 10. The mitochondria are also key regulators of the caspase cascade and apoptosis. Release of cytochrome C from mitochondria can lead to the activation of caspase 9, and then of caspase 3. This effect is mediated through the formation of an apoptosome, a multi-protein complex consisting of cytochrome C, Apaf-1, procaspase 9 and ATP. The formation of the apoptosome is illustrated in Figure 4. 1) Inactivation of enzymes involved in DNA repair. The enzyme poly (ADP-ribose) polymerase, or PARP, is an important DNA repair enzyme and was one of the first proteins identified as a substrate for caspases. The ability of PARP to repair DNA damage is prevented following cleavage of PARP by caspase-3. 2) Breakdown of structural nuclear proteins. Lamins are intra-nuclear proteins that maintain the shape of the nucleus and mediate interactions between chromatin and the nuclear membrane. Degradation of lamins by caspase 6 results in the chromatin condensation and nuclear fragmentation. 3) Fragmentation of DNA. The fragmentation of DNA into nucleosomal units is caused by an enzyme known as CAD, or caspase activated DNase. Normally CAD exists as an inactive complex with ICAD (inhibitor of CAD). During apoptosis, ICAD is cleaved by caspases, such as caspase 3, to release CAD. Rapid fragmentation of the nuclear DNA follows. Role of Nitric Oxide in Apoptosis Nitric oxide (NO) is an important signaling molecule that acts in many tissues to regulate a diverse range of physiological processes including vasodilation, neuronal function, inflammation and

www.sgul.ac.uk/depts/immunology/~dash immune function. Nitric oxide has also been demonstrated to be involved in the regulation of apoptosis. The effects of apoptosis vary depending upon the dose of NO and the type of cell used and has been shown to be able to both induce apoptosis and to protect from apoptosis in different cell types. Nitric oxide has been demonstrated to inhibit apoptosis in a number of cell types including leukocytes, hepatocytes, trophoblasts and endothelial cells. Generally the anti-apoptotic effects of NO can be mediated through a number of mechanisms such as the nitrosylation and inactivation of many of the caspases including caspase 3, caspase 1 and caspase 8. Other mechanisms include activating p53, upregulating heat shock protein 70 (and consequently blocking recruitment of pro-caspase 9 to the Apaf-1 apoptosome), upregulating Bcl-2 and Bcl-XL (with subsequent inhibition of cytochrome C release from the mitochondria) and activating cGMP signaling leading to activation of cGMP-dependent protein kinases and suppression of caspase activity. The effects of NO on apoptosis are generally classified as cGMP dependent or independent. Nitric oxide is able to activate cGMP signaling through the interaction of NO with the haem group of guanylate cyclase. The production of cGMP leads to the activation of cGMPdependent protein kinases and possibly to increased expression of anti-apoptotic proteins.

Figure 4: Formation of the apoptosome Caspases and chromatin breakdown One of the hallmarks of apoptosis is the cleavage of chromosomal DNA into nucleosomal units. The caspases play an important role in this process by activating DNases, inhibiting DNA repair enzymes and breaking down structural proteins in the nucleus. The role of the caspases in the breakdown of chromatin is illustrated in Figure 5.

Figure 5: Breakdown of chromatin during apoptosis

APOPTOSIS
Further Reading 1. Adams, J. M. & Cory, S. Life-or-death decisions by the Bcl-2 protein family. Trends in Biochemical Sciences 26, 61-6 (2001). 2. Ahern, G. P., Klyachko, V. A. & Jackson, M. B. cGMP and S-nitrosylation: two routes for modulation of neuronal excitability by NO. Trends Neurosci 25, 510-7 (2002). 3. Algeciras-Schimnich, A. et al. Molecular ordering of the initial signaling events of CD95. Mol Cell Biol 22, 207-20 (2002). 4. Allaire, A. D., Ballenger, K. A., Wells, S. R., McMahon, M. J. & Lessey, B. A. Placental apoptosis in preeclampsia. Obstetrics and Gynecology 96, 271-276 (2000). 5. Ashkenazi, A. & Dixit, V. M. Apoptosis control by death and decoy receptors. Current Opinion in Cell Biology 11, 255-60 (1999). 6. Barnhart, B. C., Alappat, E. C. & Peter, M. E. The CD95 type I/type II model. Semin Immunol 15, 185-93 (2003). 7. Bedi, A. On the TRAIL from death receptors to prostate cancer therapy. Cancer Biol Ther 1, 638-9 (2002). 8. Beere, H. et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf1 apoptosome. Nature Cell Biology 2, 469-475 (2000). 9. Belka, C. et al. Differential role of caspase-8 and BID activation during radiation- and CD95induced apoptosis. Oncogene 19, 1181-1190 (2000). 10. Benhar, M. & Stamler, J. S. A central role for S-nitrosylation in apoptosis. Nat Cell Biol 7, 645-6 (2005). 11. Blanchard, H. et al. The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis. Structure with Folding & Design 7, 1125-1133 (1999). 12. Brune, B. Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ 10, 864-9 (2003). 13. Brune, B., von Knethen, A. & Sandau, K. B. Nitric oxide and its role in apoptosis. European Journal of Pharmacology 351, 261-272 (1998). 14. Brune, B., von Knethen, A. & Sandau, K. B. Nitric oxide (NO): an effector of apoptosis. Cell Death and Differentiation 6, 969-975 (1999). 15. Budihardjo, I., Oliver, H., Lutter, M., Luo, X. & Wang, X. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15, 269-90 (1999). 16. Cardone, M. H. et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318-1321 (1998). 17. Carey, G. B. et al. B-cell receptor and Fas-mediated signals for life and death. Immunol Rev 176, 105-15 (2000). 18. Chang, D. W. et al. c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95mediated apoptosis. Embo J 21, 3704-14 (2002). 19. Chen, M. & Wang, J. Initiator caspases in apoptosis signaling pathways. Apoptosis 7, 313-9 (2002). 20. Chou, J. J., Matsuo, H., Duan, H. & Wagner, G. Solution structure of the RAIDD CARD and model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment. Cell 94, 171-180 (1998). 21. Curtin, J. F. & Cotter, T. G. Live and let die: regulatory mechanisms in Fas-mediated apoptosis. Cell Signal 15, 98392 (2003). 22. Dash, P. R., Cartwright, J. E., Baker, P. N., Johnstone, A. P. & Whitley, G. S. Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation. Exp Cell Res 287, 314-24 (2003). 23. Dash, P. R., Cartwright, J. E. & Whitely, G. S. Nitric oxide inhibits polyamineinduced apoptosis in the human extravillous trophoblast

www.sgul.ac.uk/depts/immunology/~dash cell line SGHPL-4. Human Reproduction 18, 959-968 (2003). Dash, P. R., Whitley, G. S., Ayling, L. J., Johnstone, A. P. & Cartwright, J. E. Trophoblast apoptosis is inhibited by hepatocyte growth factor through the Akt and betacatenin mediated up-regulation of inducible nitric oxide synthase. Cellular Signalling 17, 571-80 (2005). Datta, S. R. et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231-241 (1997). Downward, J. How BAD phosphorylation is good for survival. Nature Cell Biology 1, E33-E35 (1999). Engelmann, I. & Bauer, G. How can tumor cells escape intercellular induction of apoptosis? Anticancer Research 20, 2297-2306 (2000). Engels, I. H., Totzke, G., Fischer, U., Schulze-Osthoff, K. & Janicke, R. U. Caspase10 sensitizes breast carcinoma cells to TRAIL-induced but not tumor necrosis factor-induced apoptosis in a caspase-3dependent manner. Mol Cell Biol 25, 2808-18 (2005). Eramo, A. et al. CD95 deathinducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. Eur J Immunol 34, 1930-40 (2004). Garofalo, T. et al. Association of the death-inducing signaling complex with microdomains after triggering through CD95/ Fas. Evidence for caspase-8ganglioside interaction in T cells. J Biol Chem 278, 8309-15 (2003). Guo, F. & Bhalla, K. The FLIP variation on the TRAIL DISC: doxorubicin conducts the swan song. Cancer Biol Ther 1, 528-9 (2002).

24.

25.

26.

27.

28.

29.

30.

31.

APOPTOSIS
32. Grassme, H., Cremesti, A., Kolesnick, R. & Gulbins, E. Ceramide-mediated clustering is required for CD95-DISC formation. Oncogene 22, 545770 (2003). 33. Hennino, A., Berard, M., Casamayor-Palleja, M., Krammer, P. H. & Defrance, T. Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells. J Immunol 165, 3023-30 (2000). 34. Krueger, A., Schmitz, I., Baumann, S., Krammer, P. H. & Kirchhoff, S. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex. J Biol Chem 276, 20633-40 (2001). 35. Kruidering, M. & Evan, G. I. Caspase-8 in apoptosis: The beginning of The End? IUBMB LIFE USA 50, 85-90 (2000). 36. Lloyd, A. Caspase-8: an initiator of Fas-mediated apoptosis. Drug Discovery Today 3, 525-525 (1998). 37. Medema, J. P. et al. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). Embo J 16, 2794-804 (1997). 38. Ozoren, N. & El-Deiry, W. S. Defining characteristics of Types I and II apoptotic cells in response to TRAIL. Neoplasia 4, 551-7 (2002). 39. Scaffidi, C. et al. Two CD95 (APO-1/Fas) signaling pathways. Embo J 17, 1675-87 (1998). 40. Sprick, M. R. et al. FADD/ MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity 12, 599609 (2000). 41. Srivastava, R. K. TRAIL/ Apo-2L: mechanisms and clinical applications in cancer. Neoplasia 3, 535-46 (2001). 42. Walsh, C. M., Luhrs, K. A. & Arechiga, A. F. The fuzzy logic of the deathinducing signaling complexin lymphocytes. J Clin Immunol 23, 333-53 (2003). Weber, C. H. & Vincenz, C. A docking model of key components of the DISC complex: death domain superfamily interactions edefined. FEBS Lett 492, 171-6 (2001). LaCasse, E. C., Baird, S., Korneluk,R. G. & MacKenzie, A. E. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17, 324759 (1998). Mahajan, N. P. et al. Bcl2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nature Biotechnology 16, 547-552 (1998). Medema, J. P., Scaffidi, C., Krammer, P. H. & Peter, M. E. Bcl-xL acts downstream of caspase-8 activation by the CD95 death-inducing signaling complex. J Biol Chem 273, 3388-93 (1998). Nuessler, V. et al. Bcl-2, bax and bcl-xL expression in human sensitive and resistant leukemia cell lines. Leukemia 13, 18641872 (1999). OConnor, L., Huang, D. C., OReilly, L. A. & Strasser, A. Apoptosis and cell division. Current Opinion in Cell Biology 12, 257-63 (2000). Sawada, N. et al. Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposideinduced apoptosis in C6 glioma cells. Cell Death and Differentiation 7, 761-772 (2000). Stennicke, H. R. et al. Procaspase-3 is a major physiologic target of caspase-8. J.Biol. Chem. 273, 27084-27090 (1998). Tan, Y., Demeter, M. R., Ruan, H. & Comb, M. J. BAD Ser-155 phosphorylation regulates BAD/ Bcl-XL interaction and cell survival. Journal of Biological Chemistry 275, 25865-25869 (2000).

www.sgul.ac.uk/depts/immunology/~dash

43.

44.

45.

46.

47.

48.

49.

50.

51.