Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer

The sensitivity to ABT-737, a prototype BH3 mimetic drug, varies in a broad range in small cell lung cancer (SCLC) cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents.     

MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

MCL-1ES Induces MCL-1L-Dependent BAX- and BAK-Independent Mitochondrial Apoptosis

MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.     

Coupling cellular localization and function of checkpoint kinase 1 (Chk1) in checkpoints and cell viability

Chk1 plays a key role in regulating the replication checkpoint and DNA damage response. Recent evidence suggests that mammalian Chk1 regulates both the nuclear and cytoplasmic checkpoint events. However, mechanisms regulating cellular mobilization of Chk1 were not well understood. Here, we report the identification of regions of human Chk1 that regulate its protein cellular localization and checkpoint function. We demonstrate that the two highly conserved motifs (CM1 and CM2) at the C terminus of Chk1 function as a nuclear export signal and nuclear localization signal, respectively. Mutating five highly conserved residues within these two motifs of Chk1 resulted in its accumulation mainly in the cytoplasm. These cytoplasmic Chk1 mutants were less stable and exhibited significantly reduced phosphorylation by DNA damage treatment, yet they retained, at least partially, checkpoint function. Using an adenovirus-mediated gene targeting technique, we attempted to create an HCT116 cell line in which endogenous Chk1 is mutated so that it is expressed exclusively in the cytoplasm. However, we failed to obtain homozygous mutant cell lines. We found that even the heterozygous mutant cell lines showed cell survival defects accompanied by spontaneous cell death. Together, these results reveal novel regulatory mechanisms that couple protein cellular localization with the checkpoint response and cell viability of Chk1.     

Synergistic apoptotic effect of Mcl-1 inhibition and doxorubicin on B-cell precursor acute lymphoblastic leukemia cells

Molecular Biology Reports - Myeloid cell leukemia-1 (MCL-1) is a component of the Bcl-2 anti-apoptotic family that plays a key role in cell proliferation and differentiation. Despite tremendous...     

人乳头瘤病毒次要衣壳蛋白L2核定位

在人乳头瘤病毒(human papillomavirus,HPV)次要衣壳蛋白L2的N端和C端,有大量带正电荷的氨基酸残基组成核定位信号(nuclear localization signal,NLS)。细胞的核结构域10(nuclear domain 10,ND10)是细胞周期和病毒生活周期的重要调节者。L2定位到ND10的过程不仅会受到早幼粒细胞白血病蛋白(promyleocytic leukaemia protein,PML)、死亡结构域相关蛋白(deathdomain-associated protein,Daxx)、Sp100核抗原(Sp100 nuclear antigen)等细胞蛋白的影响,也会与L1在ND10发生相互作用。在HPV感染和组装过程中,L2的核定位信号有着重要作用。     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondria protein with an N-terminal mitochondrial targeting sequence and induces apoptosis

Apoptosis is a genetically determined cell suicide program. Mitochondria play a central role in this process and various molecules have been shown to regulate apoptosis in this organelle. In the present study, we firstly identified that protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondrial protein, which may induce apoptosis in HEK293T and HeLa cell lines. PTPIP51 transfection resulted in the externalization of phosphatidylserine (PS), activation of caspase-3, cleavage of PARP, and condensation of nuclear DNA. Further investigation revealed that PTPIP51 over-expression caused a decrease in mitochondrial membrane potential and release of cytochrome c, suggesting that it may be involved in a mitochondria/cytochrome c mediated apoptosis pathway. We also found that a putative TM domain near the N terminus of PTPIP51 is required for its targeting to mitochondria, as evidenced by the finding that deletion of the PTPIP51 TM domain prevented the protein's mitochondiral localization. Furthermore, this deletion significantly influenced the ability of PTPIP51 to induce apoptosis. Taken together, the results of the present study suggest that PTPIP51 is a mitochondrial protein with apoptosis-inducing function and that the N-terminal TM domain is required for both the correct targeting of the protein to mitochondria and its apoptotic functions.     

Distinct domains for anti- and pro-apoptotic activities of IEX-1

IEX-1 (immediate early response gene X-1) is a stress-inducible gene. Its overexpression can suppress or enhance apoptosis dependent on the nature of stress, yet the polypeptide does not possess any of the functional domains that are homologous to those present in well characterized effectors or inhibitors of apoptosis. This study using sequence-targeting mutagenesis reveals a transmembrane-like integrated region of the protein to be critical for both pro-apoptotic and anti-apoptotic functions. Substitution of the key hydrophobic residues with hydrophilic ones within this region impairs the capacity IEX-1 to positively and negatively regulate apoptosis. Mutations at N-linked glycosylation and phosphorylation sites or truncation of the C terminus of IEX-1 also abrogated its potential to promote cell survival. However, distinguished from the transmembrane-like domain, these mutants preserved pro-apoptotic activity of IEX-1 fully. On the contrary, mutation of nuclear localization sequence, despite its importance in apoptosis, did not impede IEX-1-mediated cell survival. Strikingly, all the mutants that lose their anti-apoptotic ability are unable to prevent acute increases in production of intracellular reactive oxygen species (ROS) at the initial onset of apoptosis, whereas those mutants that can sustain anti-death function also control acute ROS production as sufficiently as wild-type IEX-1. These findings suggest a critical role of IEX-1 in regulation of intracellular ROS homeostasis, providing new insight into the mechanism underlying IEX-1-mediated cell survival.     

Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration

MCL-1, an anti-apoptotic BCL-2 family member that is essential for the survival of multiple cell lineages, is also among the most highly amplified genes in cancer. Although MCL-1 is known to oppose cell death, precisely how it functions to promote survival of normal and malignant cells is poorly understood. Here, we report that different forms of MCL-1 reside in distinct mitochondrial locations and exhibit separable functions. On the outer mitochondrial membrane, an MCL-1 isoform acts like other anti-apoptotic BCL-2 molecules to antagonize apoptosis, whereas an amino-terminally truncated isoform of MCL-1 that is imported into the mitochondrial matrix is necessary to facilitate normal mitochondrial fusion, ATP production, membrane potential, respiration, cristae ultrastructure and maintenance of oligomeric ATP synthase. Our results provide insight into how the surprisingly diverse salutary functions of MCL-1 may control the survival of both normal and cancer cells.     

Focal adhesion kinase regulation of N-WASP subcellular localization and function

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr(256). We found that phosphorylation of Tyr(256) affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr(256) of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr(256) plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.     

cGMP-dependent protein kinase I gamma encodes a nuclear localization signal that regulates nuclear compartmentation and function

cGMP-dependent protein kinase I (PKGI) plays an important role in regulating how cGMP specifies vascular smooth muscle cell (SMC) phenotype. Although studies indicate that PKGI nuclear localization controls how cGMP regulates gene expression in SMC, information about the mechanisms that regulate PKGI nuclear compartmentation and its role in directly regulating cell phenotype is limited. Here we characterize a nuclear localization signal sequence (NLS) in PKGIγ, a proteolytically cleaved PKGI kinase fragment that translocates to the nucleus of SMC. Immuno-localization studies using cells expressing native and NLS-mutant PKGIγ, and treated with a small molecule nuclear transport inhibitor, indicated that PKGIγ encodes a constitutively active NLS that requires importin α and β for regulation of its compartmentation. Moreover, studies utilizing a genetically encoded nuclear phospho-CREB biosensor probe and fluorescence lifetime imaging microscopy demonstrated that this NLS controls PKGIγ nuclear function. In addition, although cytosolic PKGIγ-activity was observed to stimulate MAPK/ERK-mediated nuclear CREB signaling in SMC, NLS-mediated PKGIγ nuclear activity alone was determined to increase the expression of differentiation marker proteins in these cells. These results indicate that NLS-mediated nuclear PKGIγ localization plays an important role in how PKGI regulates vascular SMC phenotype.     

Contribution of membrane localization to the apoptotic activity of PUMA

The BH3-only protein PUMA plays an important role in the activation of apoptosis in response to p53. In different studies, PUMA has been described to function by either directly activating the pro-apoptotic proteins Bax and Bak, or by neutralizing anti-apoptotic members of the Bcl2 family. We have examined the contribution of regions of PUMA other than the BH3 domain to its localization and function. Although the hydrophobic domain in the C-terminus of PUMA is necessary for efficient mitochondrial localization of PUMA itself, PUMA proteins lacking this region can still induce apoptosis and localize to the mitochondria through binding to Bcl2. Even a nuclear localization signal (NLS)-tagged PUMA protein retains apoptotic activity and can be efficiently relocalized from the nucleus after interaction with ectopically expressed Bcl2, underscoring the efficiency of this interaction. Interestingly, unlike the Bcl2 interaction, the binding of PUMA to Bax is severely compromised by the loss of the C-terminal domain of PUMA. However, since the loss of the C-terminus does not compromise the ability of PUMA to induce cell death, our results indicate that the key apoptotic function of PUMA is through interaction with anti-apoptotic proteins such as Bcl2.     

MCL-1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL-2 homologue MCL-1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL-1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation conditions where MCL-1 levels regulate activation of autophagy. Furthermore, deletion of MCL-1 in cortical neurons of transgenic mice activates a robust autophagic response. This autophagic response can, however, be converted to apoptosis by either reducing the levels of the autophagy regulator Beclin-1, or by a concomitant activation of BAX. Our results define a pathway whereby MCL-1 has a key role in determining cell fate, by coordinately regulating apoptosis and autophagy.     

The Rho exchange factor Net1 is regulated by nuclear sequestration

Net1 is a guanine nucleotide exchange factor specific for the small GTPase Rho. Oncogenic activation of Net1 occurs by truncation of the N-terminal part of the protein, which functions as a negative regulatory domain. Here, we have investigated the mechanism of Net1 regulation via its N terminus. We find that Net1 localizes to the nucleus, whereas oncogenic Net1 is found in the cytoplasm. Nuclear import of Net1 is mediated by two nuclear localization signals present in the N terminus of the protein, and forced cytoplasmic localization of Net1 is sufficient to activate Rho. In addition, the pleckstrin homology (PH) domain of Net1 acts as a nuclear export signal. Because an amino acid substitution in the PH domain that inhibits guanine nucleotide exchange factor activity does not inhibit nuclear export, we conclude that this PH domain has at least two functions. Together, our results suggest that Net1 can shuttle in and out of the nucleus, and that activation of Rho by Net1 is controlled by changes in its subcellular localization.     

Polo box domain of Plk3 functions as a centrosome localization signal, overexpression of which causes mitotic arrest, cytokinesis defects, and apoptosis

Polo-like kinase 3 (Plk3), an immediate early response gene product, plays an important role in the regulation of mitosis, DNA damage checkpoint activation, and Golgi dynamics. Similar to other members of the Plk family, Plk3 has a conserved kinase domain at the N terminus and a Polo box domain consisting of two Polo boxes at the C terminus. In this study, we demonstrate that the Polo box domain of Plk3 is sufficient for subcellular localization of this kinase to the centrosomes, the spindle poles, and the midbody when ectopically expressed in HeLa and U2OS cells. Both Polo boxes are required for the subcellular localization. Overexpression of the Polo box domain, not the kinase domain, of Plk3 causes significant cell cycle arrest and cytokinesis defects, eventually leading to mitotic catastrophe/apoptosis. Interestingly, the Polo box domain of Plk3 is more potent in inhibiting cell proliferation and inducing apoptosis than that of Plk1, suggesting that this domain can provide an additional structural basis for discovery of new anticancer drugs given the current emphasis on Plk1 as a therapeutic target.     

Parathyroid hormone related protein (PTHrP) inhibits TNFalpha-induced apoptosis by blocking the extrinsic and intrinsic pathways

Parathyroid hormone related protein (PTHrP) is expressed at low levels in many fetal and adult tissues where it plays a central role in regulating cell proliferation, cell death, and tissue homeostasis. In vivo and in vitro, PTHrP has been shown to promote the survival of a variety of cells by regulating expression of the anti-apoptotic protein Bcl2. Additional work has shown that intra-nuclear accumulation of PTHrP in CFK2 (PTH1R positive) and 27m21 (PTH1R negative) condrogenic cells promotes their survival by closing down ribosome biogenesis and promoting quiescence. The current studies were undertaken to examine the role of wild-type PTHrP and a mutant form that cannot translocate to the nucleus in protecting cells from TNFalpha-induced apoptosis. Both forms of the protein were equally effective in blocking the extrinsic pathway by inhibiting expression of the TNF receptor death domain, activating Bid, and promoting cleavage of caspase 8. These observations suggest a direct mechanism of PTHrP action on components of the extrinsic pathway, involving a region of the protein outside of the NTS. PTHrP and M1PTHrP also inhibited the intrinsic pathway by preventing the exchange of anti-apoptotic for pro-apoptotic proteins at the mitochondrial membrane, thus maintaining its integrity and preventing the release of caspase-activating factors into the cytosol. In general, this mitochondrial-related activity was somewhat delayed and was mediated more effectively by PTHrP than by M1PTHrP, suggesting an indirect mechanism of action that might require the presence of an intact NTS.