REVIEW Phosphorylation of Bcl2 and regulation of apoptosis - Nature

Leukemia (2001) 15, 515–522  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu

REVIEW Phosphorylation of Bcl2 and regulation of apoptosis PP Ruvolo, X Deng and WS May University of Florida Shands Cancer Center and Department of Medicine, Box 100232, Gainesville, Florida 32610–0232, USA

Members of the Bcl2 family of proteins are important regulators of programmed cell death pathways with individual members that can suppress (eg Bcl2, Bcl-XL) or promote (eg Bax, Bad) apoptosis. While the mechanism(s) of Bcl2’s anti-apoptotic function is not yet clear, introduction of Bcl2 into most eukaryotic cell types will protect the recipient cell from a wide variety of stress applications that lead to cell death. There are, however, physiologic situations in which Bcl2 expression apparently fails to protect cells from apoptosis (eg negative selection of thymocytes). Further, Bcl2 expression in patient tumor samples does not consistently correlate with a worse outcome or resistance to anticancer therapies. For example, patient response and survival following chemotherapy is independent of Bcl2 expression at least for pediatric patients with ALL. These findings indicate that simple expression of Bcl2 may not be enough to functionally protect cells from apoptosis. The finding that Bcl2 is post-translationally modified by phosphorylation suggests another level of regulation of function. Recent studies have shown that agonist-activated phosphorylation of Bcl2 at serine 70 (single site phosphorylation), a site within the flexible loop domain (FLD), is required for Bcl2’s full and potent anti-apoptotic function, at least in murine IL-3dependent myeloid cell lines. Several protein kinases have now been demonstrated to be physiologic Bcl2 kinases indicating the importance of this post-translational modification. Since Bcl2 phosphorylation has been found to be a dynamic process involving both a Bcl2 kinase(s) and phosphatase(s), a mechanism exists to rapidly and reversibly regulate Bcl2’s activity and affect cell viability. In addition, multisite Bcl2 phosphorylation induced by anti-mitotic drugs like paclitaxel may inhibit Bcl2 indicating the potential wide range of functional consequences that this post-translational modification may have on function. While post-translational mechanisms other than phosphorylation may also regulate Bcl2’s function (eg ubiquitination), this review will focus on the regulatory role for phosphorylation and discuss its potential clinical ramifications. Leukemia (2001) 15, 515–522. Keywords: Bcl2; apoptosis; signal transduction; IL-3; bryostatin; ceramide

ily is evolutionarily conserved.10–12 For instance, the nematode C. elegans expresses an anti-apoptotic Bcl2 homologue, CED-9.10 The functional and structural similarity between CED-9 and Bcl2 is apparent as human Bcl2 can substitute for CED-9 in C. elegans,11,12 indicating the ancient importance of this activity in regulating cell survival. Structurally, Bcl2 family members are related since they contain one or more of four functional homology domains (BH for Bcl2 homology) found in Bcl2 (Figure 1; Refs 3, 4). Presently there are 16 family members that have been described.5 Interestingly, some of these related proteins exhibit an anti-apoptotic function similar to Bcl2 (eg Bcl-XL, Mcl1), while others are potent pro-apoptotic agents (eg Bax, Bad, Bid). At present, however, the mechanism(s) by which these proteins regulate apoptosis remains unclear, but is the subject of intense investigation and effort. The exact mechanism(s) by which Bcl2 and related family members regulate apoptosis is not yet clear. Interestingly, apoptosis can be induced by diverse stimuli13,14 and Bcl2 is capable of suppressing apoptosis following most stress applications.9,15–22 Examples include apoptosis induced by cytokine treatment or growth factor withdrawal,9,15–17 ␥ irradiation,18,19 glucocorticoids,18 dexamethasome,9,15,18 ceramide20 or chemotherapeutic drugs.21,22 Bcl2 has emerged as a negative prognostic factor when expressed at relatively high levels in acute myeloid leukemia23 and prostate cancer.24 Moreover, ectopic introduction of recombinant Bcl2 into many tissue types will result in increased protection from a variety of apoptotic stimuli.2,18 However, there are circumstances where Bcl2 is apparently ineffective at preventing apoptosis.18,25–27 Theses include the negative selection of thymocytes18 and cytotoxic T cell killing, as examples.25 In addition, expression levels of Bcl2 do not always correlate with prolonged cell survival in patients with leukemia. For example, even though Bcl2 is expressed at high levels in acute

Introduction Bcl2 is the founding member of a family of proteins that regulate programmed cell death pathways (reviews Refs 1–5). Bcl2 was identified as the cellular oncogene product associated with the t(14,18) translocation commonly seen in B cell lymphomas.6–8 While other oncogenes known at the time of Bcl2’s discovery were found to affect cell proliferation, Bcl2’s function to prolong cell survival was shown experimentally to be novel.1,2,9 Bcl2 was demonstrated to be a potent suppressor of the process of programmed cell death because expression of exogenous Bcl2 could protect (IL-3-dependent) cells from apoptosis in response to growth factor withdrawal.9 Subsequently, other Bcl2 family members were identified based on structural homology.3–5 Interestingly, the Bcl2 protein famCorrespondence: WS May; Fax: 352 846 1193 Received 15 November 2000; accepted 12 January 2001

Figure 1 Bcl2 contains a flexible loop region that may regulate its function. Bcl2 contains four Bcl2 homology domains (BH1–4) and seven alpha helix regions (␣ 1–7) that contribute to its regulation and function. Listed are regions associated with dimerization (eg for homodimerization or hetero-dimerization with BAX), putative channel/pore structures, and possible protein docking (eg Apaf 1, PP2B, RAF-1). The regulatory loop lies between a1 and a2 and contains the functional serine 70 (S70) phosphorylation site and a caspase cleavage site at aspartate 34 (D34).

Phosphorylation of Bcl2 and regulation of apoptosis PP Ruvolo et al

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lymphoblastic leukemia (ALL) cells from previously untreated pediatric patients, these cells apparently remain highly chemosensitive and do not display de novo resistance to initial induction-remission chemotherapy.26,27 Interestingly, a comparison of the human pre-B acute leukemia cell line REH with the myeloid HL60 cell line, derived from a patient with acute promyelocytic leukemia, reveals that the REH cells are at least 10-fold more sensitive to the clinically useful chemotherapeutic drugs adriamycin, ara-C, and etoposide.28 The relative chemosensitivity of the REH cells (compared to the HL60 cells) initially appears inconsistent with expectations based on Bcl2 expression since REH cells express over threefold more Bcl2. In addition to such experimental data, recent clinical data also indicate that increased expression of Bcl2 can apparently not suppress drug-induced apoptosis and Bcl2 expression levels also cannot explain the chemoresistance observed in patients with small cell lung carcinoma29 or breast cancer.30 Collectively then, such examples indicate that Bcl2 expression in and of itself is not sufficient to explain its full and potent anti-apoptotic activity. Therefore, other factors or regulatory mechanism(s) must be involved. What type of regulatory mechanism may be involved? Since Bcl2 can directly interact with several proteins (eg hetero-dimerization with Bax, Bad, Bcl-XL), appropriate ‘docking’ interactions may represent another mechanism to regulate Bcl2’s function in apoptosis. Such regulation may occur independently of Bcl2 expression levels.31 Indeed, a popular model holds that the hetero-dimerization of Bcl2 and Bax abrogates Bax’s proapoptotic effect.31 For example, in the comparison between REH and HL60 cells, while the expression levels of Bax were essentially equivalent, the ratio of Bcl2 to Bax was significantly higher (⬎ three-fold) in the more chemosensitive REH cells.28 Although current clinical data indicate that the Bcl2:Bax ratio may be important for cell survival in patients with AML,32 there is no clear correlation between chemoresistance and the ratio of Bcl2:Bax expressed in other (eg pediatric ALL) acute leukemias.33 These observations then suggest that there are also other mechanisms in addition to expression or interaction with other protein partners, such as Bax, that contribute to Bcl2’s function and role in chemoresistance. It has been discovered that phosphorylation of Bcl2 may regulate its function.34 Furthermore, recent studies have revealed that this post-translational modification may be necessary for Bcl2’s full and potent anti-apoptotic function.34,35 This review will focus on the mechanism(s) by which Bcl2 is phosphorylated and the role for phosphorylation in regulating the anti-apoptotic function of Bcl2 and other Bcl2 family members. Functional phosphorylation of Bcl2 occurs at serine 70 Bcl2 was initially identified as a phosphoprotein when it was found that exogenous Bcl2 introduced into SF9 insect cells by Baculovirus infection could prolong SF9 cell survival.36 Later, it was discovered that addition of IL-3 to growth factordeprived murine myeloid cells promoted serine phosphorylation of Bcl2.34 Importantly, Bcl2 phosphorylation was also correlated with increased cell survival since addition of IL-3 protected cells from apoptosis induced by the chemotherapeutic drug etoposide.34 In later studies, an intact phosphorylation site (ie ser 70) was shown to be required for Bcl2’s full and potent survival phenotype.35 Interestingly, the potent PKC agonist, bryostatin-1 (bryo), which is able to induce robust Bcl2 phosphorylation and promote cell survival, was found to

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be able to substitute for IL-3 in cell survival.34 This finding initially suggested that Bcl2 phosphorylation might be involved in the anti-apoptotic function of Bcl2.17 However, the mechanism by which post-receptor signals are generated following IL-3 binding to its surface receptor to induce Bcl2 phosphorylation was not clear. Since the addition of IL-3 or bryo did not significantly affect the expression levels of endogenous Bcl2, at least in the short term, and phosphorylation apparently did not alter the protein half-life, phosphorylation was concluded to directly influence Bcl2’s function.34 Since bryo is a potent PKC agonist and IL-3 also activates PKC,17 PKC was initially tested as a Bcl2 kinase.34,35 Certain protein kinase inhibitors relatively specific for PKC, including staurosporine37,38 and the methylpiperazine, H7,38,39 were found to partially inhibit Bcl2 phosphorylation in association with blocking the anti-apoptotic effect of Bcl2.28,34,35 PKC was found to be a direct Bcl2 kinase when it was shown that a mixture of pan-PKC was able to phosphorylate Bcl2 in vitro exclusively on serine residues, the same site(s) phosphorylated in vivo.34 Mutating each of the seven common and conserved PKC-consensus serine phosphorylation sites found in murine and human Bcl2 (ie residues 24, 70, 102, 158, 164, 202, and 213),35,40 revealed that only the mutation at serine 70 (S70A) resulted in a nonphosphorylatable Bcl2. Importantly, this mutation also rendered Bcl2 unable to support prolonged cell survival following either IL-3 growth factor withdrawal or treatment with chemotherapeutic agents (even in the presence of IL-335). These results indicate the physiologic relevance of Bcl2 phosphorylation at serine 70. A functional role for the potential charge conferred by phosphorylation at serine 70 was established when it was discovered that mutation of serine 70 to glutamate (S70E) resulted in a gain of survival function phenotype.35 Presumably this mutation was able to mimic phosphorylation by conferring a charge at this site.35 Thus, cells from clones expressing the S70E Bcl2 were found to be more hearty to survival than cells from clones expressing similar levels of either WT Bcl2 or S70A Bcl2 after growth factor withdrawal or chemotherapy treatment. These data indicated that serine 70 is the functional phosphorylation site of Bcl2. Taxane-induced Bcl2 hyper-phosphorylation involves both serine and threonine residues at multiple sites In addition to IL-3 and bryo, phosphorylation of Bcl2 was also shown to occur following treatment of cells with taxanes and other antimitotic agents including vincristine, dolistatin, and vinblastine,41–47 retinoic acid48 and aurinotricarboxylic acid (ATA).49 However, the effect of the taxane, paclitaxel, appears to be opposite to that of IL-3, bryo, ATA, or retinoic acid in that paclitaxelinduced Bcl2 phosphorylation was initially reported to be associated with cell death.41–47 The morphological pattern of paclitaxel-induced Bcl2 phosphorylation41–47 is dramatically different from that observed following growth factor,34,35 bryo,28,34,35 retinoic acid48 or ATA-induced phosphorylation.49 Only paclitaxel (or the other antimititoic agents) stimulated formation of a slower migrating, band-shifted form of Bcl2 detected by denaturing gel electrophoresis with a Mr 28 000 daltons, as compared to unmodified Bcl2 at Mr 26 000 daltons (Figure 2). Moreover, the timing of the Bcl2 phosphorylation observed following paclitaxel treatment differed significantly from that following IL-3 or bryo. Paclitaxelinduced phosphorylation was detected only after 2 h of drug treatment,41 compared to the rapid (ie minutes) phosphorylation observed following growth agonist treatment.35 In


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Figure 2 Bcl2 phosphorylation by growth factor agonist differs from phosphorylation induced by taxol. Bcl2 contains at least three sites that have been identified as phosphorylation sites (T69, S70 and S87). While growth factor agonist promotes survival, taxol promotes increased susceptibility to death. NSF/N1.H7 murine myeloid cells were grown as previously described.34,35 Western analysis of Bcl2 protein in the presence and absence of IL-3 (above) and the presence and absence of taxol (below) demonstrates that only taxol treatment results in the formation of p28 Bcl2.

addition, phosphorylation of Bcl2 occurred at multiple sites including threonine 69, serine 70, and serine 87 following treatment of cells with paclitaxel,44 indicating that phosphorylation was not exclusively at serine 70 as occurs following growth agonist-induced phosphorylation.28,34,35,47,49 These results indicate that paclitaxel induces a multisite, hyperphosphorylated form of Bcl2 vs the mono-site phosphorylation that is associated with cell survival (Figure 2). Further, the paclitaxel-induced shifted band Bcl2 is stable while IL-3 or bryoinduced phosphorylation is dynamic.34,35,49 Indeed, the mechanism of Bcl2 phosphorylation by taxanes is markedly different from that of survival agonists. For example, Blagosklonny and colleagues46,47 demonstrated that paclitaxel-induced Bcl2 phosphorylation does not involve PKC or mitogen-activated protein kinases (MAPKs), ERK1/2 as does survival agonistinduced Bcl2 phosphorylation.28,34,35,49 Still, the precise effect of paclitaxel on Bcl2 function is not clear. It has been suggested that such multi-site phosphorylation results in inhibition of Bcl2’s anti-apoptotic function.41–47,50 While it appears that paclitaxel-induced hyperphosphorylation does correlate with cell death,43,47 it has also been shown in a number of studies that overexpression of exogenous, WT Bcl2 protects cells from the cytotoxic effects of the taxanes.51,52 In contrast, however, a recent study using human Jurkat or murine WEHI-231 cells engineered to overexpress exogenous WT or phosphorylation site mutant Bcl2 found that S87A mutant Bcl2 which also contains an intact, phosphorylatable S70 site, was apparently able to significantly protect these cells from paclitaxel or TNF-␣-induced death.44 Thus phosphorylation of the ser87 site may somehow abrogate or reduce survival function even if ser70 is intact. By contrast, in murine IL-3-dependent cells, only the S70A Bcl2 mutant fails to protect cells from the stress of IL-3 withdrawal or chemotherapeutic drug treatment even in the presence of growth factor.35 Further-

more, induction of Bcl2 phosphorylation either by treatment with PKC agonist (ie bryo) or by exogenous expression of a direct Bcl2 kinase (ie PKC ␣) in the human pre-B REH cells correlated with increased cell survival following chemotherapeutic drug treatment.28 Thus from the collective data, it is not possible to conclude that phosphorylation of Bcl2 at ser70 is inactivating; rather phosphorylation of Bcl2 at ser70 is required for Bcl2’s full and anti-apoptotic function. Serine 70 is an evolutionarily conserved site in human, mouse and chicken Bcl2 that lies within the flexible loop domain region.53,54 The flexible loop domain (FLD) is an approximately 60 amino acid region (ie aa 30–90) that resides between the putative ␣1 and ␣2 helices that separate the amino-terminal BH4 domain from the BH3 domain in Bcl2 (Figure 1).53 An analogous FLD occurs in Bcl-XL as well.53,54 Interestingly, deletion of the FLD from either Bcl-XL or Bcl2 results in enhanced cell survival when expressed in WEHI231 cells induced to undergo apoptosis by IgM.54,55 Thus, it was proposed that the ‘loop domain’ region negatively regulates Bcl2’s anti-apoptotic function.54 While the mechanism is not clear, these large loop deletion mutants have been demonstrated to protect cells from paclitaxel more efficiently than WT Bcl2.50,51 How phosphorylation within the flexible loop domain of Bcl2 may actually contribute to the anti-apoptotic function of this death regulator is a critical question and will be discussed later. Bcl2 can be phosphorylated by diverse protein kinases involved in various intracellular signal transduction pathways PKC was initially implicated as a potential physiologic Bcl2 kinase. A member(s) of the classical PKC family (ie ␣, ␤I, ␤II Leukemia


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and ␥) was the most likely candidate since in vitro phosphorylation of Bcl2 with pan PKC required Ca+2.34,35 Recent studies have indicated that the functional cellular site of Bcl2 is the mitochondrial outer membrane56–58 and a physiologic Bcl2 kinase might be expected to co-localize in the mitochondrial membranes. Analysis of the Bcl2 phosphorylation status in REH vs HL60 leukemic cells indicated that the relatively chemosensitive REH cells expressed little, if any, phosphorylated Bcl2 while the relatively chemoresistant HL60 cells displayed robust phosphorylation of Bcl2.28 PKC ␣ was identified as the candidate physiologic Bcl2 kinase when it was demonstrated that: (1) HL60, but not REH, cells expressed mitochondrial PKC ␣; (2) bryo treatment caused translocation of PKC ␣ to mitochondrial membranes in REH cells and promoted Bcl2 phosphorylation; (3) forced mitochondrial localization of PKC ␣ by exogenous overexpression in REH cells resulted in basal phosphorylation of Bcl2 and promoted greater chemoresistance in transfectants; and (4) PKC ␣ was able to phosphorylate Bcl2 at the functional serine 70 site in vitro. The existence of a second Bcl2 kinase(s) was predicted on the basis that overexpression of exogenous Bcl2 could protect cells from apoptosis induced by high concentrations of the potent PKC inhibitor, staurosporine.59 As a clue, the stress kinase JNK/SAPK kinase p54-SAPK␤ was recently found to phosphorylate Bcl2 in vitro and following co-transfection in COS7 cells,60 suggesting that stress kinases may also play a role. The possibility that a MAP kinase (ie ERK, JNK, or p38 kinase) may be a Bcl2 kinase was considered when it was reported that activation of the MAP kinase phosphatase-1 by angiotensin led to the dephosphorylation of Bcl2 in association with apoptosis following nerve growth factor withdrawal from PC12 neuroblastoma cells.61 Furthermore, recent studies on the effect of various protein kinase inhibitors on IL-3-mediated Bcl2 phosphorylation in myeloid cells revealed that the MEK1 inhibitor, PD98059, could also partially inhibit agonistinduced Bcl2 phosphorylation in vivo.49 Importantly, treatment of cells with PD98059 potentiated cell killing when combined with staurosporine suggesting that the MEK1/MAPK (ERK) pathway may be involved.49 Both ERK1 (p44) and ERK2 (p42) were identified to be direct Bcl2 kinases since both: (1) co-localized with Bcl2 in the mitochondria; (2) were able to phosphorylate Bcl2 at serine 70 in vitro; and (3) were able to phosphorylate Bcl2 in co-transfection experiments using COS-7 cells.49 In these latter studies, cells were transiently transfected with three plasmids simultaneously including Bcl2, ERK1/2, and constitutively active MEK-1 (ie to activate WT ERK). The cells were treated with kinase inhibitors and Bcl2 phosphorylation monitored.49 Only cells transfected with all three plasmids demonstrated Bcl2 phosphorylation that could be blocked by treatment with PD98059, but not staurosporine.49 Thus, the ERKs 1/2 are staurosporine-resistant Bcl2 kinases (SRKs).49 These findings provide a potential answer to how Bcl2 is able protect cells from staurosporine treatment which potentially inhibits the activity of classical PKCs. In addition to PKC and the ERKs1/2, some stress-activated Bcl2 kinases have been reported. These include PKA,50 ASK,51 and JNK1.44 Bcl2 phosphorylation is a dynamic process involving the Bcl2 phosphatase, PP2A The kinetics of IL-3 or bryo-induced Bcl2 phosphorylation reveal that maximal phosphorylation occurs within 30 min after agonist addition and that phosphorylation levels decline

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to a lower ambient level of phosphorylation with extended time.35 Okadaic acid, a potent phosphatase inhibitor of PP1 and PP2A, can enhance Bcl2 phosphorylation, suggesting that a potential PP1 or PP2A Bcl2 phosphatase is involved.43 Other studies revealed that PP2A was a physiologic Bcl2 phosphatase since okadaic acid-sensitive PP2A, but not PP1, colocalized with Bcl2 at the mitochondrial membranes.62 Furthermore, PP2A was found to be superior in dephosphorylation of Bcl2 in vitro when compared to PP1 or PP2B.62 Thus, the co-localization or ability to translocate from cytosol to membrane of the Bcl2 kinases PKC ␣ and ERK1/2 and PP2A at the mitochondrial membrane could explain the rapid and reversible regulation of Bcl2 phosphorylation following agonist stimulation. The dynamic nature of agonist-activated Bcl2 phosphorylation differs from that induced by paclitaxel since the slower mobility bands of Bcl2 that result are maintained.41–47 Ceramide regulates Bcl2 phosphorylation The sphingolipid ceramide is a well known apoptogenic agent and important effector molecule of both cellular stress- and cytokine-activated signal transduction pathways.63–66 Ceramide is also a potent activator of a ceramide-activated protein phosphatase (CAPP), which appears to be a member of the PP2A family.67,68 Recently, we discovered that ceramide can also activate a mitochondrial PP2A responsible for Bcl2 dephosphorylation in association with cell death.69 Since expression of S70E Bcl2, a non-phosphorylatable, activated form of Bcl2, can protect cells from ceramide-induced apoptosis, one likely mechanism by which ceramide induces cell death is by functionally inactivating Bcl2.69 Thus, dynamic phosphorylation–dephosphorylation of Bcl2 may serve as a ‘survival sensor’ during stress stimuli.69 If favorable growth and survival conditions are achieved, Bcl2 is phosphorylated. As death-inducing conditions are encountered, however, ceramide (and possibly other negative regulators) is likely produced and Bcl2 dephosphorylated in association with cell death. Bcl2 may act as a ‘survival sensor’ Diacylglycerol, a potent PKC agonist, induces Bcl2 phosphorylation and promotes cell growth and proliferation.70 Conversely, ceramide induces Bcl2 dephosphorylation and promotes growth arrest and cell death.63,64 DAG and ceramide are simultaneously regulated by at least one enzyme, phosphatidylcholine-specific phospholipase C.71 Thus, one possible role for Bcl2 phosphorylation may be to reflect the overall ‘survival health’ of the cell as defined by the contrasting levels of these effector molecules. Figure 3 represents a model that illustrates how DAG and ceramide levels may regulate the cell’s ‘survival potential’ by affecting, at least in part, Bcl2 phosphorylation status. The presence or absence of an exogenous growth/survival stimulus like IL-3 can determine not only which signal pathways are activated but also (ie ultimately) whether the cell survives.17 Under conditions favorable for growth such as in the presence of ample growth factor in the medium, rapid, agonist-induced intra-cellular phosphorylation of PLC ␥ by tyrosine kinases occurs which can activate this phospholipase, resulting in the generation of DAG and inositol 1,4,5 triphosphate (IP-3).72 Generation of IP-3 is known to mobilize intracellular Ca+2 stores.73 DAG


Figure 3 DAG and ceramide levels have opposing effects on Bcl2 phosphorylation status and survival. A model is presented where Bcl2 function is regulated by effector molecules in response to IL-3. In the presence of IL-3, sphingomyelin synthetase promotes DAG production at the expense of ceramide. DAG stimulates Bcl2 kinases both directly (PKC ␣) and indirectly (ERK 1/2). Conversely, IL-3 withdrawal induces stress conditions that promote ceramide production by activating sphingomyelinase and/or ceramide synthetase. Ceramide promotes Bcl2 dephosphorylation by activation of mitochondrial PP2A and by indirectly inhibiting PKC ␣.

production and release of Ca+2 can then activate classical PKC isozymes resulting in substrate (including Bcl2) phosphorylation.74 Conditions that favor stimulation of PKC ␣ have been demonstrated to support cell growth and survival through the activation of Raf38,75 and the phosphorylation of Bcl2.28 Conversely, stress conditions that can stimulate sphingomyelinase and/or ceramide synthetase activation would favor the production of ceramide, at the expense of DAG, and potentially lead to dephosphorylation of Bcl2.76,77 Interestingly, addition of DAG blocks the effects of ceramide or sphingomyelinase thus suggesting that both effector molecules are functionally linked.76 While loss of DAG itself may be detrimental to cells, since the classical and novel PKC isoform activity would not be stimulated,74 ceramide production can also lead to the rapid inactivation of PKC ␣, apparently by activating the phosphatase PP2A.78 Furthermore, C2-ceramide has been shown to inhibit ERK2 activity when added to HL60 cells, although the mechanism of inhibition is not yet clear.66 Importantly, ceramide may also promote a decrease in Bcl2’s anti-apoptotic function by activating mitochondrial PP2A and dephosphorylating Bcl2.69 Further, the opposite effects of DAG and ceramide on cell signaling and survival appear to be cumulative and where Bcl2 regulation is involved, may result in diverse outcomes for post-translational modification of Bcl2 that can act as a cell’s ‘survival sensor’. Agonist-induced phosphorylation of other Bcl2 family members may also regulate apoptosis Other Bcl2 family members (ie Bad, Bcl-XL, Mcl-1) are also phosphorylated.79–82 Preliminary reports indicate that Mcl-1 is also phosphorylated by a TPA-activated kinase (ie likely a PKC isoform).79 Similar to Bcl2, IL-3 and erythropoietin also can induce Mcl-1 phosphorylation under survival conditions while paclitaxel treatment leads to apparent multisite hyperphosphorylation by a different mechanism(s).79 Bcl-XL apparently is phosphorylated following paclitaxel treatment

although the significance for Bcl-XL function is not yet clear.80 Serine phosphorylation of Bad may occur following growth agonist activation of Akt,81,82 PKA,83 and direct or indirect activation of MAPK.84 Bad displays pro-apoptotic activity when heterodimerizing with Bcl2 or Bcl-XL.85 Following addition of a survival stimulus such as GM-CSF or IL-3 to cells, Bad, like Bcl2,34,86 is rapidly phosphorylated.87 Bad phosphorylation occurs on serine residues S112 and S136 and results in an increased binding affinity for 14–3-3 protein.88 When phosphorylated and bound to the 14–3-3 protein, Bad is sequestered in the cytosol where it cannot access mitochondrial Bcl-XL or Bcl2 and thus the cells survive.81–88 However, at least in IL-3 hematopoietic cells, when IL-3 is withdrawn, Bad becomes dephosphorylated and displays an increased affinity to bind Bcl-XL which is closely associated with cell death.85,87 Bad binding to Bcl-XL causes the release of Bax from heterodimerizing with Bcl-XL, presumably resulting from a competitive mechanism and now Bax displays its death effector phenotype.81–85 Thus, like Bcl2, Bad is also dynamically regulated by phosphorylation. In addition to Bcl2 family members, other proteins involved in the apoptotic machinery may be regulated by phosphorylation. For example, phosphorylation of the xymogen form of caspase 9 by Akt may block pro-caspase proteolytic cleavage and thus may block apoptosis.89

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How might Bcl2 phosphorylation regulate function While the mechanism by which Bcl2 functions is not yet clear, it is evident that phosphorylation of the FLD region of Bcl2 can regulate its function (Figure 1). If indeed the role of Bcl2 is to ultimately maintain mitochondrial integrity during apoptotic stress, there are several possibilities how this molecular modification might work. Studies have demonstrated that cleavage of Bcl2 and Bcl-XL by caspases, the molecular scissors effecting intracellular apoptosis, may affect their antiapoptotic function.90–92 Caspases are cysteine proteases that cleave target proteins specifically at aspartic acid residues.93 Caspases have emerged as critical players in apoptotic pathways.93–95 Caspase 3 cleaves Bcl2 at aspartic acid residue 34 (which resides within the FLD) to apparently create a proapoptotic molecule.92 The deletion of the BH4 domain and resulting possible ‘exposure’ of the BH3, pore/channel forming region apparently can turn Bcl2 from an anti-apoptotic to pro-apoptotic molecule, much like a Jekyll and Hyde plot.91 The putative caspase cleavage site for Bcl-XL occurs within the flexible loop region (at D61).91 Other mechanisms involving the proteolytic degradation of Bcl2 may also be involved in its regulation. One recent study has found that phosphorylation of Bcl2 blocks TNF␣-induced apoptosis in both HeLa and HUVECs by a mechanism that may involve ubiquitination of Bcl2.95 Cells expressing Bcl2 mutants that mimic phosphorylation were more resistant to TNF ␣-induced apoptosis and Bcl2 expression was prolonged because Bcl2 was not degraded by the proteasome pathway.96 Another mechanism by which Bcl2 phosphorylation may regulate function involves Bcl2’s ability to dimerize with other partners such as Bax and Bad.31,85 The ratio of Bcl2 to Bax affects survival of cells in response to stress apparently as a result of the association of the two molecules.31 Murine factor dependent cells expressing a high ratio of Bcl2 to Bax (ie 2:1) were more viable after IL-3 withdrawal than cells with a lower Bcl2:Bax ratio (ie 1:2).31 We have recently found that the phosphorylation status of Bcl2 can affect its ability to stably Leukemia


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associate with Bax.46 In co-immunoprecipitation experiments, WT Bcl2 but not the loss of function, nonphosphorylatable S70A Bcl2 mutant was found to stably associate with Bax.49 Bcl-XL and Bcl2 are also thought to potentially regulate apoptosis by their intrinsic pore/channel forming properties.58,97 Thus, regulation of such channel/pore formation in mitochondrial membranes may affect the membrane potential and ability to sequester activators of pro-caspases such as cytochrome C within the mitochondria.97 However, it remains to be determined whether phosphorylation may affect Bcl2’s ability to form pores/channels.

has been reported in a number of hematologic malignancies including mantle cell lymphoma (approximately 100%), multiple myeloma and plasmacytoma (60%), and acute myeloid leukemia (40%).101 Since the role of Bcl2 and proteins that affect Bcl2 function will likely vary from patient to patient, it will be important to identify patients who one would predict would exhibit poor outcome due to chemoresistance, failure to achieve induction-remission success, and therefore are at risk for survival. With such knowledge, the future direction of leukemia treatment will likely move toward strategies ‘tailored’ for the individual.

Potential clinical implications for post-translational regulation of Bcl2 in patients with leukemia or lymphoma

References

Phosphorylation of Bcl2 may contribute to the cellular chemoresistance of human and murine leukemic cell lines.28,34,35 Thus, a recently identified physiologic Bcl2 kinase, PKC ␣, has been found in retrospective studies to be a prognostic factor for outcome in patients in with AML.32 In this study, PKC ␣ and Bax each was found not to be prognostic alone, but were shown to modulate the ability of Bcl2 to predict outcome in AML patients with favorable or intermediate cytogenetics (FIPC).32 Consistent with a model where phosphorylated Bcl2 promotes Bcl2 association with Bax (resulting in loss of Bax’s pro-apoptotic function), AML patients with molecular determinants favoring cell survival (a high ratio of Bcl2* PKC␣/Bax) fared much worse than patients with molecular determinants favoring apoptosis (a low ratio of Bcl2* PKC␣/Bax). FIPC AML patients with leukemic cells exhibiting a high Bcl2*PKC␣/Bax ratio had a median remission rate of 65% and median survival of 55 weeks, while FIPC AML patients with leukemic cells exhibiting a low Bcl2*PKC␣/Bax ratio had a median remission rate of 92% and median survival of 141.5 weeks.32 Importantly, PKC has also been implicated as a prognostic factor in pediatric ALL.98 If leukemic cells from patients expressing phosphorylated Bcl2 (at ser70) exhibit greater resistance to chemotherapy, it might be a novel therapeutic strategy to attempt to use inhibitors of PKC signal transduction pathways to block Bcl2 phosphorylation and thereby disable or defeat Bcl2’s anti-apoptotic function. This strategy may help sensitize otherwise resistant cells to standard antileukemic treatment regimens. Considering the effects of the serine 70 Bcl2 mutations in murine leukemic cells,34,35 it will be important to identify any genetic mutations of Bcl2 in cells of leukemic patients and determine if such mutations can contribute to chemoresistance. Previous studies have identified Bcl2 mutations exist in cells from patients with non-Hodgkin’s lymphoma (NHL).99,100 Interestingly, the frequency of such point mutations is associated with the presence of the t(14;18) translocation.99,100 It appears that the juxtaposition of the Bcl2 gene with the immunoglobulin heavy chain locus may also result in hypermutation of Bcl2.99,100 While only five NHL patient samples were studied, Tanaka et al99 demonstrated that cells from three of these patients had point mutations that resulted in one or more missense mutations. Furthermore, all three NHL patient cell sets had mutations that occurred within the FLD of Bcl2.99 Thus, mutations in the FLD of Bcl2 may be clinically relevant. While it is clear that there are many factors influencing chemoresistance in leukemia, it is important to understand the contribution that modification of Bcl2 and other Bcl2 family members play in this process. High level expression of Bcl2 Leukemia

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