Induction of apoptosis is driven by nuclear retention of protein kinase C delta

Protein kinase C delta (PKC delta) mediates apoptosis downstream of many apoptotic stimuli. Because of its ubiquitous expression, tight regulation of the proapoptotic function of PKC delta is critical for cell survival. Full-length PKC delta is found in all cells, whereas the catalytic fragment of PKC delta, generated by caspase cleavage, is only present in cells undergoing apoptosis. Here we show that full-length PKC delta transiently accumulates in the nucleus in response to etoposide and that nuclear translocation precedes caspase cleavage of PKC delta. Nuclear PKC delta is either cleaved by caspase 3, resulting in accumulation of the catalytic fragment in the nucleus, or rapidly exported by a Crm1-sensitive pathway, thereby assuring that sustained nuclear accumulation of PKC delta is coupled to caspase activation. Nuclear accumulation of PKC delta is necessary for caspase cleavage, as mutants of PKC delta that do not translocate to the nucleus are not cleaved. However, caspase cleavage of PKC delta per se is not required for apoptosis, as an uncleavable form of PKC delta induces apoptosis when retained in the nucleus by the addition of an SV-40 nuclear localization signal. Finally, we show that kinase negative full-length PKC delta does not translocate to the nucleus in apoptotic cells but instead inhibits apoptosis by blocking nuclear import of endogenous PKC delta. These studies demonstrate that generation of the PKC delta catalytic fragment is a critical step for commitment to apoptosis and that nuclear import and export of PKC delta plays a key role in regulating the survival/death pathway.     

Cytosolic delivery of granzyme B by bacterial toxins: evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% (51)Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal (51)Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.     

The nuclear pore complex, nuclear transport, and apoptosis

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eukaryotic cells, and it mediates all trafficking between these 2 cellular compartments. As such, the NPC and nuclear transport play central roles in translocating death signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes occurring during apoptosis. Recent findings suggest that the correlation between the NPC, nuclear transport, and apoptosis goes beyond the simple fact that NPCs mediate nuclear transport of key players involved in the cell death program. In this context, the accessibility of key regulators of apoptosis appears to be highly modulated by nuclear transport (e.g., impaired nuclear import might be an apoptotic trigger). In this review, recent findings concerning the unexpected tight link between NPCs, nuclear transport, and apoptosis will be presented and critically discussed.     

A presumed human nuclear autoantigen that translocates to plasma membrane blebs during apoptosis

The structure and subcellular localization of a number of molecules change during apoptosis. These molecules are recognized by the immune system, leading to the development of autoimmunity when apoptotic cells fail to be effectively cleared by phagocytosis. We searched for such molecules by analyzing sera from 12 individuals who suffered from autoimmune diseases and from 3 patients with amyotrophic lateral sclerosis. One serum sample, designated 681, detected an antigen that fulfilled the above criteria. In Western blotting of lysates of human Jurkat T cells, the 681 antigen appeared as a distinct signal with a molecular mass of 60 kDa in normal cells, and 2 additional signals with faster mobilities were detected in apoptotic cells. The results of subcellular fractionation and immunofluorescence experiments revealed this antigen to be strictly localized in the nucleus of normal cells, but to be translocated to a region near the plasma membrane, to membrane blebs in particular, after the induction of apoptosis. Under conditions in which membrane blebbing was inhibited in apoptotic cells, the antigen still moved away from the nucleus, but its accumulation at the periplasmic region was completely abolished. The apparent partial cleavage and intracellular redistribution of the 681 antigen in apoptotic cells mimics changes previously reported for the nuclear autoantigen La, but the 681 antigen was clearly distinct from La. These results suggest that cleavage-dependent exit from the nucleus during apoptosis is a phenomenon common to nuclear autoantigens.     

Perforin-dependent nuclear targeting of granzymes: A central role in the nuclear events of granule-exocytosis-mediated apoptosis?

Programmed cell death, apoptosis, involves very distinctive changes within the target cell nucleus, including margination of the chromatin, DNA fragmentation and breakdown of the nuclear envelope. Cytolytic granule-mediated target cell apoptosis is effected, in part, through synergistic action of the membrane-acting protein perforin and serine proteases, such as granzymes A or B. Recent work using confocal laser scanning microscopy as well as other techniques supports the idea that perforin-dependent translocation of granzymes to the nucleus of target cells plays a central role in effecting the nuclear changes associated with apoptosis. In vitro experiments indicate that granzyme nuclear import follows a novel pathway, being independent of ATP, not inhibitable by non-hydrolysable GTP analogues and involving binding within the nucleus, unlike conventional signal- dependent nuclear protein import. In intact cells, perforin-dependent nuclear entry of granzymes precedes the nuclear events of apoptosis such as DNA fragmentation and nuclear envelope breakdown; prevention of granzyme nuclear translocation through bcl2 overexpression or treatment of target cells with inhibitors of caspase activation blocks these events. Nuclear localization of granzymes thus appears to be central to induction of the nuclear changes associated with cytolytic granule-mediated apoptosis.     

Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine

The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores.     

A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli

Unlike other caspases, caspase-2 appears to be a nuclear protein although immunocytochemical studies have suggested that it may also be localized to the cytosol and golgi. Where and how caspase-2 is activated in response to apoptotic signals is not clear. Earlier immunocytochemistry studies suggest that caspase-2 is activated in the nucleus and through cleavage of BID leads to increased mitochondrial permeability. More recent studies using bimolecular fluorescence complementation found that caspase-2 oligomerization that leads to activation only occurs in the cytoplasm. Thus, apoptotic signals may lead to activation of caspase-2 which may already reside in the cytoplasm or lead to release of nuclear caspase-2 to the extra-nuclear cytoplasmic compartment. It has not been possible to study release of nuclear caspase-2 to the cytoplasm by cell fractionation studies since cell lysis is known to release nuclear caspase-2 to the extra-nuclear fraction. This is similar to what is known about unliganded nuclear estrogen receptor-α (ERα ) when cells are disrupted. In this study we found that pre-treatment of cells with N-ethylmaleimide (NEM), which alkylates cysteine thiol groups in proteins, completely prevents redistribution of caspase-2 and ERα from the nucleus to the extra-nuclear fraction when cells are lysed. Using this approach we provide evidence that apoptotic signals rapidly leads to a shift of caspase-2 from the nucleus to the extra-nuclear fraction, which precedes the detection of apoptosis. These findings are consistent with a model where apoptotic signals lead to a rapid shift of caspase-2 from the nucleus to the cytoplasm where activation occurs.     

Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) functions as a glycolytic enzyme within the cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, which trigger the translocation of GAPDH into the nucleus. As apoptosis can be induced by serum withdrawal, which otherwise causes cell cycle arrest, the linkage between serum deprivation, cell cycle and nuclear transport of GAPDH has been investigated. The intracellular distribution of GAPDH was monitored by confocal laser scanning microscopy of either immuno-stained NIH 3T3 fibroblasts or of cells overexpressing GFP-tagged GAPDH. Serum withdrawal led to an accumulation of GAPDH in the nucleus. In contrast to investigations published so far, this nuclear translocation was a reversible process: cytoplasmic location of endogenous GAPDH or of GFP-GAPDH could be recovered upon serum addition to arrested cells and was not inhibited by cycloheximide treatment. In addition, the nuclear import upon serum depletion had no influence neither on the catalytic activity nor on the expression level of GAPDH. The nuclear export of GFP-GAPDH in serum-deprived cells could be stimulated by serum or directly by the growth factors EGF or PDGE The transport process is not regulated via an initiation of cell cycle arrest, as olomoucine, which causes G1-arrest neither stimulated nuclear accumulation nor prevented nuclear export after serum addition to serum-depleted cultures. Moreover, SV40-transformed 3T3 cells transported GAPDH into the nucleus upon serum deprivation, though the expression of the viral large T-antigen enabled growth factor-independent cell proliferation in this cell line. The recruitment of GAPDH to the cytoplasm upon serum stimulation of arrested cells was not impaired by the inhibition of the MAPK signalling pathway with PD 098059. However, further analysis of the growth factor signalling pathway with specific inhibitors revealed that nuclear export was prevented by LY 294002, an inhibitor of the PI-3 kinase. PI3K links the growth factor signalling pathway with cell death via the repression of an apoptotic inducer. Thus, the nuclear accumulation of GAPDH upon growth factor depletion is a reversible process not related directly to cell cycle and likely triggered by survival signals.     

Functional histone antibody fragments traverse the nuclear envelope

Factors important in the translocation process of proteins across the nuclear membrane were studied by microinjecting either fluoresceinated nonimmune IgG and F(ab)2 or the corresponding molecules, prepared from antisera to histones, into the nucleus and cytoplasm of human fibroblasts. Intact IgG from both preparations remained at the site of injection regardless of whether it was injected into the nucleus or the cytoplasm. In contrast, nonimmune F(ab)2 distributed uniformly throughout the cell. The F(ab)2 derived from affinity-pure antihistone moves into the nucleus after cytoplasmic injection and remains in the nucleus after nuclear microinjection. The migration of the antihistone F(ab)2 into the nucleus results in inhibition of uridine incorporation in the nuclei of the microinjected cells. We conclude that non-nuclear proteins, devoid of specific signal sequences, traverse the nuclear membrane and accumulate in the nucleus provided their radius of gyration is less than 55A and the nucleus contains binding sites for these molecules. These findings support the model of "quasibifunctional binding sites" as a driving force for nuclear accumulation of proteins. The results also indicate that active F(ab)2 fragments, microinjected into somatic cells, can bind to their antigenic sites suggesting that microinjection of active antibody fragments can be used to study the location and function of nuclear components in living cells.     

Nuclear translocation of granzyme B in target cell apoptosis

Granzyme B is the prototypic member of a family of serine proteases localized to the cytolytic granules of cytotoxic lymphocytes. Together with another granule protein, perforin, granzyme B is capable of inducing all aspects of apoptotic death in target cells. A number of granzyme B substrates have been identified and it has been demonstrated that granzyme B is responsible, directly or indirectly, for the morphological nuclear changes observed in target cell apoptosis, including DNA fragmentation. In an earlier study, we showed that granzyme B binds to a nuclear protein in a manner dependent on its enzymatic activity. Here, we demonstrate that granzyme B is translocated rapidly to the nucleus in cells that have been induced to undergo apoptosis by a granzyme-dependent process, and that translocation is dependent on caspase activity. Appearance of granzyme B in the nucleus of target cells precedes the detection of DNA fragmentation. Although not directly responsible for DNA fragmentation, these data suggest a nuclear role for granzyme B in target cell apoptosis. c-Abl nuclear functions.     

Nucleocytoplasmic protein traffic in single mammalian cells studied by fluorescence microphotolysis

Fluorescence microphotolysis was employed to measure in single living cells the kinetics of nucleocytoplasmic transport and the coefficients of intracellular diffusional mobility for the nuclear non-chromosomal protein nucleoplasmin. Nucleoplasmin was isolated from Xenopus ovary and labeled fluorescently. By injection into Xenopus oocytes it was ascertained that fluorescent labeling did not interfere with normal nuclear accumulation. Upon injection into the cytoplasm of various mammalian cell types nucleoplasmin was rapidly taken up by the nucleus. In rat hepatoma cells the half-time of nuclear uptake was approx. 5 min at 37 degrees C; the nucleocytoplasmic equilibrium concentration ratio had a maximum of 6.5 +/- 1.4 and depended on the injected amount. Upon co-injection of ATPases or reduction of temperature to 10 degrees C a nucleocytoplasmic equilization but no nuclear accumulation was observed. Equilization was fast (time constant 65 s at 23 degrees C), similar to that of 10-kDa dextran permeating the nuclear envelope by simple diffusion through functional pores. Nucleoplasmin (160 kDa), however, is too large to permeate passively the nuclear envelope, which is apparent from the fact that its tryptic 'core' fragment (100 kDa) could not permeate the nuclear envelope. On the other hand, a large fluorescent protein, phycoerythrin (240 kDa), was targeted to the nucleus by conjugation with nucleoplasmin. In the nucleus-to-cytoplasm direction the nuclear envelope was completely impermeable to nucleoplasmin, independently of temperature or ATP depletion. Nucleoplasmin, its core fragment, phycoerythrin and the phycoerythrin-nucleoplasmin conjugate were mobile in both cytoplasm and nucleus.     

A nuclear export signal and phosphorylation regulate Dok1 subcellular localization and functions

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.     

Nuclear translocation of a leukocyte elastase Inhibitor/Elastase complex during staurosporine-induced apoptosis: role in the generation of nuclear L-DNase II activity

Using L1210 murine leukemia cells, we have previously shown that in response to treatment with drugs having different targets, apoptotic cell death occurs through at least two different signaling pathways. Here, we present evidence that nuclear extracts from staurosporine-treated cells elicit DNase II activity that is not detected in nuclear extracts from cisplatin-treated cells. This activity correlates with the accumulation of two nuclear proteins (70 and 30 kDa) which are detected by an anti-L-DNase II antibody. Partial purification of this DNase II activity suggests that the 30-kDa protein could be the nuclease responsible for staurosporine-induced DNA fragmentation. The 70-kDa protein is also recognized by an anti-elastase antibody, suggesting that it carries residues belonging to both L-DNase II and elastase. Since previous findings showed that L-DNase II was generated from the leukocyte inhibitor of elastase, we propose that the 70-kDa protein results from an SDS-stable association between these two proteins and is translocated from the cytoplasm to the nucleus during staurosporine-induced apoptosis.     

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background

We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner.

Methodology/Principal Findings

Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes.

Conclusions/Significance

These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.     

Nuclear translocation of PDCD5 (TFAR19): an early signal for apoptosis?

The programmed cell death 5 (PDCD5) protein is a novel protein related to regulation of cell apoptosis. In this report, we demonstrate that the level of PDCD5 protein expressed in cells undergoing apoptosis is significantly increased compared with normal cells, then the protein translocates rapidly from the cytoplasm to the nucleus of cells. The appearance of PDCD5 in the nuclei of apoptotic cells precedes the externalization of phosphatidylserine and fragmentation of chromosome DNA. This phenomenon is parallel to the loss of mitochondrial membrane potential, independent of the feature of apoptosis-inducing stimuli and also independent of the cell types and the apoptosis modality. In conclusion, the nuclear translocation of PDCD5 is a universal earlier event of the apoptotic process, and may be a novel early marker for apoptosis.     

From the nucleus to the plasma membrane: translocation of the nuclear proteins histone H3 and lamin B1 in apoptotic microglia

Nuclear autoantibodies have been found in patients with autoimmune diseases. One possible source for nuclear antigens are apoptotic cells. However, the mechanism of how apoptotic cells make nuclear factors accessible to the immune system is still elusive. In the present study, we investigated the redistribution of nuclear components after UV irradiation in the microglial cell line BV-2 and in primary mouse microglia at the ultrastructural level. We used transmission electron microscopy-coupled electron energy loss spectroscopy (EELS) to measure phosphorus as an indicator for nucleic acids and immunogold labeling to detect histone H3 and lamin B1 in apoptotic cells. EELS revealed elevated concentrations of phosphorus in nuclear and cytoplasmic condensed chromatin compared to the remaining cytoplasm. Furthermore, immunolabeling of lamin B1 and histone H3 was detected in apoptotic microglia not only in the nucleus, but also in the cytoplasm, and even at the plasma membrane. Confocal images of apoptotic microglia, which were not previously permeabilized, showed patches of histone H3 and lamin B1 labeling at the cell surface. The pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) prevented the occurrence of cytoplasmic condensed chromatin in apoptotic microglia. Our findings indicate that nuclear components leak from the nucleus into the cytoplasm in apoptotic microglia. At least histone H3 and lamin B1 reach the cell surface, this may promote autoreactive processes.     

Phosphatidylcholine-specific phospholipase C in mitogen-stimulated fibroblasts

To investigate expression, subcellular localization and mechanisms of translocation of phosphatidylcholine-specific phospholipase C (PC-PLC) during the cell proliferative response, biochemical, immunoblotting, and immunofluorescence analyses were performed on quiescent and mitogen-stimulated NIH-3T3 fibroblasts. Platelet-derived growth factor (PDGF), insulin and 12-O-tetradecanoylphorbol-13-acetate induced, in 10-60 min, PC-PLC translocation from a perinuclear cytoplasmic area to the plasma membrane. Following cell exposure to PDGF (60 min), the overall PC-PLC expression increased up to 2-3x, while the enzyme activity increased 5x in total cell lysates, 2x in the plasma membrane, and 4x in the nucleus; moreover, confocal laser scanning microscopy showed a progressive externalization of PC-PLC on the outer plasma membrane surface and its accumulation in the nuclear matrix. Pre-incubation of cells with the PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609), before PDGF-stimulation, not only reduced the enzyme activity in total cell lysates as well as in plasma membrane and nuclear fractions, but also blocked the mechanisms of PC-PLC subcellular redistribution. These effects were associated with a D609-induced long-lasting cell cycle block in Go.     

Mechanisms governing subcellular localization and function of human RGS2

RGS proteins negatively regulate heterotrimeric G proteins at the plasma membrane. RGS2-GFP localizes to the nucleus, plasma membrane, and cytoplasm of HEK293 cells. Expression of activated G(q) increased RGS2 association with the plasma membrane and decreased accumulation in the nucleus, suggesting that signal-induced redistribution may regulate RGS2 function. Thus, we identified and characterized a conserved N-terminal domain in RGS2 that is necessary and sufficient for plasma membrane localization. Mutational and biophysical analyses indicated that this domain is an amphipathic alpha-helix that binds vesicles containing acidic phospholipids. However, the plasma membrane targeting function of the amphipathic helical domain did not appear to be essential for RGS2 to attenuate signaling by activated G(q). Nevertheless, truncation mutants indicated that the N terminus is essential, potentially serving as a scaffold that binds receptors, signaling proteins, or nuclear components. Indeed, the RGS2 N terminus directs nuclear accumulation of GFP. Although RGS2 possesses a nuclear targeting motif, it lacks a nuclear import signal and enters the nucleus by passive diffusion. Nuclear accumulation of RGS2 does not limit its ability to attenuate G(q) signaling, because excluding RGS2 from the nucleus was without effect. RGS2 may nonetheless regulate signaling or other processes in the nucleus.     

Defective anchoring of JNK1 in the cytoplasm by MKK7 in Jurkat cells is associated with resistance to Fas-mediated apoptosis

The c-Jun N-terminal protein kinase (JNK) plays a context-dependent role in tumorigenesis. Stress-induced redistribution of JNK from the cytoplasm to the nucleus has been demonstrated as essential for stress-induced cell death. However, accumulation of basal JNK activity in the nucleus has frequently been seen in tumor cells. Our previous report revealed aberrant nuclear entry of JNK protein in Jurkat human leukemic T-cells even without JNK hyperactivation. Because inhibition of JNK activity, especially JNK1 activity, in Jurkat cells results in augmented Fas-mediated apoptosis, it is possible that aberrant subcellular localization of JNK, especially the JNK1 isoform, contributes to the resistance to Fas-mediated apoptosis. Here we report that MKK7 works as a cytoplasmic anchoring protein for JNK1 in various types of cells, including human peripheral blood mononuclear cell (PBMC) T-cells, but exhibits aberrant nuclear entry in Jurkat cells. Ectopic expression of a JNK1 mutant defective of nuclear entry or a nuclear JNK inhibitor leads to impaired UV-induced apoptosis in both PBMC T- and Jurkat cells. The same treatment shows no effect on Fas-mediated apoptosis of PBMC T-cells but sensitizes Jurkat cells to Fas-mediated apoptosis. Taken together, our work suggests that aberrant subcellular organization of the JNK pathway might render certain tumor cells resistant to Fas-mediated apoptosis.