Galectin-3 phosphorylation is required for its anti-apoptotic function and cell cycle arrest.

Galectin-3, a beta-galactoside-binding protein, is implicated in cell growth, adhesion, differentiation, and tumor progression by interactions with its ligands. Recent studies have revealed that galectin-3 suppresses apoptosis and anoikis that contribute to cell survival during metastatic cascades. Previously, it has been shown that human galectin-3 undergoes post-translational signaling modification of Ser(6) phosphorylation that acts as an "on/off" switch for its sugar-binding capability. We questioned whether galectin-3 phosphorylation is required for its anti-apoptotic function. Serine to alanine (S6A) and serine to glutamic acid (S6E) mutations were produced at the casein kinase I phosphorylation site in galectin-3. The cDNAs were transfected into a breast carcinoma cell line BT-549 that innately expresses no galectin-3. Metabolic labeling revealed that only wild type galectin-3 undergoes phosphorylation in vivo. Expression of Ser(6) mutants of galectin-3 failed to protect cells from cisplatin-induced cell death and poly(ADP-ribose) polymerase from degradation when compared with wild type galectin-3. The non-phosphorylated galectin-3 mutants failed to protect cells from anoikis with G(1) arrest when cells were cultured in suspension. In response to a loss of cell-substrate interactions, only cells expressing wild type galectin-3 down-regulated cyclin A expression and up-regulated cyclin D(1) and cyclin-dependent kinase inhibitors, i.e. p21(WAF1/CIP1) and p27(KIP1) expression levels. These results demonstrate that galectin-3 phosphorylation regulates its anti-apoptotic signaling activity.     

Galectin-3 regulates RasGRP4-mediated activation of N-Ras and H-Ras

Galectin-3 (Gal-3) is a pleiotropic beta-galactoside-binding protein expressed at relatively high levels in human neoplasms. Its carbohydrate recognition domain (CRD) contains a hydrophobic pocket that can accommodate the farnesyl moiety of K-Ras. Binding of K-Ras to Gal-3 stabilizes K-Ras in its active (GTP-bound) state. Gal-3, which does not interact with N-Ras, was nevertheless shown to reduce N-Ras-GTP in BT-549 cells by an unknown mechanism that we explored here. First, comparative analysis of various cancer cell lines (glioblastomas, breast cancer cells and ovarian carcinomas) showed a positive correlation between low N-Ras-GTP/high K-Ras-GTP phenotype and Gal-3 expression levels. Next we found that epidermal growth factor-stimulated GTP loading of N-Ras, but not of K-Ras, is blocked in cells expressing high levels of Gal-3. Activation of Ras guanine nucleotide releasing proteins (RasGRPs) by phorbol 12-myristate 13-acetate (PMA) or downregulation of Gal-3 by Gal-3 shRNA increased the levels of N-Ras-GTP in Gal-3 expressing cells. We further show that the N-terminal domain of Gal-3 interacts with and inhibits RasGRP4-mediated GTP loading on N-Ras and H-Ras proteins. Growth of BT-549 cells stably expressing the Gal-3 N-terminal domain was strongly attenuated. Overall, these experiments demonstrate a new control mechanism of Ras activation in cancer cells whereby the Gal-3 N-terminal domain inhibits activation of N-Ras and H-Ras proteins.     

Nuclear localization signal and phosphorylation of Serine350 specify intracellular localization of DRAK2

DAP kinase-related apoptosis-inducing kinase 2 (DRAK2) is a serine/threonine kinase of the death-associated protein kinase family. DRAK2 mediates apoptosis induced by extracellular stimuli, including UV irradiation and interleukin-2, and also regulates T-cell receptor sensitivity in developing thymocytes. During these events, the subcellular localization of DRAK2 changes between the nucleus and cytoplasm. We found that DRAK2 has a putative nuclear-localization signal (NLS) sequence. Mutations in this sequence interfered with DRAK2 localization to the nucleus. Furthermore, green fluorescence protein fused to the putative NLS accumulated in the nucleus, indicating that the putative sequence functions as an NLS. We also found that the function of the NLS was regulated by phosphorylation. Phorbol myristate acetate (PMA) induced the accumulation of DRAK2 in the cytoplasm of NIH3T3 cells, whereas in the absence of PMA, DRAK2 was localized to the nucleus. Ectopic expression of PKC-gamma induced cytoplasmic localization of DRAK2 and PKC-gamma phosphorylated Ser350 flanking the NLS. DRAK2, but not the Ser350Asp mutant, accumulated in the nuclei of ACL-15 cells in response to UV-irradiation. These results suggest that phosphorylation of Ser350 plays an essential role in regulating translocation of DRAK2 to the nucleus from the cytoplasm, possibly by affecting the activity of the NLS.     

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with ¹⁵N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.     

Phosphorylation of Bax Ser184 by Akt regulates its activity and apoptosis in neutrophils

Although important for apoptosis, the mechanism of Bax regulation is poorly understood. This study demonstrates that phosphorylation of Ser(184) regulates Bax activity. The phosphorylation required phosphatidylinositol 3-kinase/Akt activation and appeared to be mediated by Akt itself. In the serine-phosphorylated form, Bax was detected in the cytoplasm, could not be immunoprecipitated with the activation-specific antibody 6A7, and promoted heterodimerization with Mcl-1, Bcl-x(L), and A1. Apoptotic neutrophils possessed reduced levels of serine-phosphorylated Bax correlating with an increase in activated Bax as well as an increase in the amount of Bax found translocated to the mitochondria. We suggest that Bax is regulated by phosphorylation of Ser(184) in an Akt-dependent manner and that phosphorylation inhibits Bax effects on the mitochondria by maintaining the protein in the cytoplasm, heterodimerized with antiapoptotic Bcl-2 family members.     

Protein kinase Cepsilon activates protein kinase B/Akt via DNA-PK to protect against tumor necrosis factor-alpha-induced cell death

We have previously shown that protein kinase Cepsilon (PKCepsilon) protects breast cancer cells from tumor necrosis factor-alpha (TNF)-induced cell death. In the present study, we have investigated if the antiapoptotic function of PKCepsilon is mediated via Akt and the mechanism by which PKCepsilon regulates Akt activity. TNF caused a transient increase in Akt phosphorylation at Ser473 in MCF-7 cells. Overexpression of PKCepsilon in MCF-7 cells increased TNF-induced Akt phosphorylation at Ser473 resulting in its activation. Knockdown of PKCepsilon by small interfering RNA (siRNA) decreased TNF-induced Akt phosphorylation/activation and increased cell death. Introduction of constitutively active Akt protected breast cancer MCF-7 cells from TNF-mediated cell death and partially restored cell survival in PKCepsilon-depleted cells. Depletion of Akt in MCF-7 cells abolished the antiapoptotic effect of PKCepsilon on TNF-mediated cell death. Akt was constitutively associated with PKCepsilon and DNA-dependent protein kinase (DNA-PK), and this association was increased by TNF treatment. Overexpression of PKCepsilon enhanced the interaction between Akt and DNA-PK. Knockdown of DNA-PK by siRNA inhibited TNF-induced Akt phosphorylation and the antiapoptotic effect of Akt and PKCepsilon. These results suggest that PKCepsilon activates Akt via DNA-PK to mediate its antiapoptotic function. Furthermore, we report for the first time that DNA-PK can regulate receptor-initiated apoptosis via Akt.     

Protein kinase B/Akt binds and phosphorylates PED/PEA-15, stabilizing its antiapoptotic action

The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser(116). In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser(116) PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser(116). In addition, a mutant of PED/PEA-15 featuring the substitution of Ser(116)-->Gly (PED(S116-->G)) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also induced phosphorylation of PED/PEA-15 at Ser(116). Based on pull-down and coprecipitation assays, PED/PEA-15 specifically bound Akt, independently of Akt activity. Serum activation of Akt as well as BAD phosphorylation by Akt showed no difference in 293 cells transfected with PED/PEA-15 and in untransfected cells (which express no endogenous PED/PEA-15). However, the antiapoptotic action of PED/PEA-15 was almost twofold reduced in PED(S116-->G) compared to that in PED/PEA-15(WT) cells. PED/PEA-15 stability closely paralleled Akt activation by serum in 293 cells. In these cells, the nonphosphorylatable PED(S116-->G) mutant exhibited a degradation rate threefold greater than that observed with wild-type PED/PEA-15. In the U373MG glioma cells, blocking Akt also reduced PED/PEA-15 levels and induced sensitivity to tumor necrosis factor-related apoptosis-inducing ligand apoptosis. Thus, phosphorylation by Akt regulates the antiapoptotic function of PED/PEA-15 at least in part by controlling the stability of PED/PEA-15. In part, Akt survival signaling may be mediated by PED/PEA-15.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

The localization of protein kinase Cdelta in different subcellular sites affects its proapoptotic and antiapoptotic functions and the activation of distinct downstream signaling pathways

Protein kinase Cdelta (PKCdelta) regulates cell apoptosis and survival in diverse cellular systems. PKCdelta translocates to different subcellular sites in response to apoptotic stimuli; however, the role of its subcellular localization in its proapoptotic and antiapoptotic functions is just beginning to be understood. Here, we used a PKCdelta constitutively active mutant targeted to the cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER) and examined whether the subcellular localization of PKCdelta affects its apoptotic and survival functions. PKCdelta-Cyto, PKCdelta-Mito, and PKCdelta-Nuc induced cell apoptosis, whereas no apoptosis was observed with the PKCdelta-ER. PKCdelta-Cyto and PKCdelta-Mito underwent cleavage, whereas no cleavage was observed in the PKCdelta-Nuc and PKCdelta-ER. Similarly, caspase-3 activity was increased in cells overexpressing PKCdelta-Cyto and PKCdelta-Mito. In contrast to the apoptotic effects of the PKCdelta-Cyto, PKCdelta-Mito, and PKCdelta-Nuc, the PKCdelta-ER protected the cells from tumor necrosis factor-related apoptosis-inducing ligand-induced and etoposide-induced apoptosis. Moreover, overexpression of a PKCdelta kinase-dead mutant targeted to the ER abrogated the protective effect of the endogenous PKCdelta and increased tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. The localization of PKCdelta differentially affected the activation of downstream signaling pathways. PKCdelta-Cyto increased the phosphorylation of p38 and decreased the phosphorylation of AKT and the expression of X-linked inhibitor of apoptosis protein, whereas PKCdelta-Nuc increased c-Jun NH(2)-terminal kinase phosphorylation. Moreover, p38 phosphorylation and the decrease in X-linked inhibitor of apoptosis protein expression played a role in the apoptotic effect of PKCdelta-Cyto, whereas c-Jun NH(2)-terminal kinase activation mediated the apoptotic effect of PKCdelta-Nuc. Our results indicate that the subcellular localization of PKCdelta plays important roles in its proapoptotic and antiapoptotic functions and in the activation of downstream signaling pathways.     

Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells

Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)-induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 --> Ala mutant of paxillin (paxS85A) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing paxS85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and paxS85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than paxS85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization.     

Phosphorylation of Serine 402 Regulates RacGAP Protein Activity of FilGAP Protein

FilGAP is a Rho GTPase-activating protein (GAP) that specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at Ser-402 is localized to the cytoplasm but not at the cytoskeleton. Although Ser-402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of Ser-402 is accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin, whereas cells transfected with FilGAP S402A mutant were not affected by calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP, and phosphorylation of Ser-402 may play a role in the regulation of cell spreading on fibronectin.     

Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL(Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit.     

Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1

Mcl-1 is an antiapoptotic Bcl-2 family member that is highly regulated and when dysregulated contributes to cancer. The Mcl-1 protein is phosphorylated at multiple sites in response to different signaling events. Phosphorylations at Thr163 (by ERK) and Ser159 (by glycogen-synthase kinase 3beta) have recently been shown to slow and enhance, respectively, Mcl-1 protein turnover. Phosphorylation is also known to be stimulated at other, as-yet uncharacterized sites in the G2/M phase of the cell cycle. Using an S peptide-tagged Mcl-1 T163A mutant, Ser64 was identified as a novel Mcl-1 phosphorylation site by mass spectrometry. Immunoblotting demonstrated that phosphorylation at this site was maximal in cells in G2/M phase, was enhanced by tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) treatment, was blocked by inhibitors of CDK (but not ERK or glycogen-synthase kinase 3beta), and was stimulated in vitro by CDK 1, CDK2, and JNK1. The half-life of a nonphosphorylatable S64A Mcl-1 mutant was indistinguishable from that of the wild type polypeptide. In contrast, this mutant failed to protect cells from TRAIL-mediated apoptosis, whereas reconstitution with the phosphomimetic S64E Mcl-1 mutant rendered cells TRAIL-resistant. This anti-apoptotic phenotype of the S64E Mcl-1 mutant was also associated with enhanced binding to the proapoptotic proteins Bim, Noxa, and Bak. A pharmacological CDK inhibitor that reduced Ser64 phosphorylation also sensitized cells to TRAIL cytotoxicity. Collectively, these observations not only identify G2/M-associated phosphorylation at Ser64 as a critical determinant of the antiapoptotic activity of Mcl-1 but also elucidate a novel mechanism by which CDK1/2 inhibitors can enhance the effectiveness of the cytotoxic cytokine TRAIL.     

Functional Interaction of the Ras Effector RASSF5 with the Tyrosine Kinase Lck: Critical Role in Nucleocytoplasmic Transport and Cell Cycle Regulation

RASSF5 is a member of the Ras association domain family, which is known to be involved in cell growth regulation. Expression of RASSF5 is extinguished selectively by epigenetic mechanism(s) in different cancers and cell lines, and reexpression usually suppresses cell proliferation and tumorigenicity. To date, the mechanism regulating RASSF5 nuclear transport and its role in cell growth regulation remains unclear. Using heterokaryon assay, we have demonstrated that RASSF5 shuttles between the nucleus and the cytoplasm, and its export from the nucleus is sensitive to leptomycin B, suggesting that RASSF5 is exported from the nucleus by a CRM-1-dependent export pathway. We further demonstrate that RASSF5 contains a hydrophobic-rich nuclear export signal (NES) towards the C-terminus and two nuclear localization signals—one each at the N-terminus and the C-terminus. Combination of mutational and immunofluorescence analyses suggests that the functional NES residing between amino acids 260 and 300 in the C-terminus is necessary for the efficient export of RASSF5 from the nucleus. In addition, substitution of conserved hydrophobic residues within the minimal NES impaired RASSF5 export from the nucleus. Furthermore, exchange of proline residues within the putative Src homology 3 binding motifs altered the export of RASSF5 from the nucleus despite the presence of functional NES, suggesting that multiple domains independently modulate the nucleocytoplasmic transport of RASSF5. Interestingly, the present investigation provided evidence that RASSF5 interacts with the tyrosine kinase Lck through its C-terminal Src homology 2 binding motif and showed that Lck-mediated phosphorylation is critical for the efficient translocation of RASSF5 into the nuclear compartment. Interestingly, our data demonstrate that wild type and nuclear export defective (ΔNES) mutant of RASSF5 but not the import defective mutant of accumulate the cells at G1/S phase and induce apoptosis. Furthermore, the Lck-interaction-defective mutant of RASSF5 induces apoptosis without altering cell cycle progression, suggesting that RASSF5 induces apoptosis independent of cell cycle arrest. Together, our data demonstrate that interaction with Lck is critical for RASSF5 phosphorylation, which in turn regulates the cell growth control activity of RASSF5. Finally, we have shown that RASSF5 encodes four splice variants and is translocated to the nucleus by the classical nuclear import pathway. One of the splice variants, RASSF5C, was found to be localized in the cytoplasm and translocated into the nucleus upon leptomycin B treatment despite the absence of N-terminal nuclear localization signal, suggesting that distribution of RASSF5 variants in different cellular compartments may be critical for Ras-dependent cell growth regulation. Collectively, the present investigation provided evidence that Lck-mediated phosphorylation regulates the nucleocytoplasmic shuttling and cell growth control activities of RASSF5.     

Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity

Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.     

Compartmentalized phosphorylation of IAP by protein kinase A regulates cytoprotection

Cell death pathways are likely regulated in specialized subcellular microdomains, but how this occurs is not understood. Here, we show that cyclic AMP-dependent protein kinase A (PKA) phosphorylates the inhibitor of apoptosis (IAP) protein survivin on Ser20 in the cytosol, but not in mitochondria. This phosphorylation event disrupts the binding interface between survivin and its antiapoptotic cofactor, XIAP. Conversely, mitochondrial survivin or a non-PKA phosphorylatable survivin mutant binds XIAP avidly, enhances XIAP stability, synergistically inhibits apoptosis, and accelerates tumor growth, in vivo. Therefore, differential phosphorylation of survivin by PKA in subcellular microdomains regulates tumor cell apoptosis via its interaction with XIAP.     

Death inducer-obliterator 1 triggers apoptosis after nuclear translocation and caspase upregulation

Death inducer-obliterator 1 (DIO-1) is a gene that is upregulated early in apoptosis. Here we report that in healthy cells, the DIO-1 gene product was located in the cytoplasm, where it formed oligomers. After interleukin-3 starvation or c-Myc-induced apoptosis in serum-free conditions, DIO-1 translocated to the nucleus, where it upregulated caspase levels and activity. A nuclear localization signal deletion mutant (DIO-1deltaNLS) was unable to translocate to the nuclear compartment in the absence of interleukin-3 and failed to upregulate procaspase levels or trigger cell death. In addition, cells stably expressing DIO-1deltaNLS were protected from apoptosis induced by interleukin-3 withdrawal. These results indicate that DIO-1 has a relevant role in regulating the early stages of cell death.