Eunhee Kim

Human Fas-Associated Factor 1, Interacting with Ubiquitinated Proteins and Valosin-Containing Protein, Is Involved in the Ubiquitin-Proteasome Pathway

ABSTRACT Human Fas-associated factor 1 (hFAF1) is a novel protein having multiubiquitin-related domains. We investigated the cellular functions of hFAF1 and found that valosin-containing protein (VCP), the multiubiquitin chain-targeting factor in the degradation of the ubiquitin-proteasome pathway, is a binding partner of hFAF1. hFAF1 is associated with the ubiquitinated proteins via the newly identified N-terminal UBA domain and with VCP via the C-terminal UBX domain. The overexpression of hFAF1 and a truncated UBA domain inhibited the degradation of ubiquitinated proteins and increased cell death. These results suggest that hFAF1 binding to ubiquitinated protein and VCP is involved in the ubiquitin-proteasome pathway. We hypothesize that hFAF1 may serve as a scaffolding protein that regulates protein degradation in the ubiquitin-proteasome pathway.

Modification of Daxx by small ubiquitin-related modifier-1

Small ubiquitin-related modifier-1 (SUMO-1) is a protein that is covalently modified to various cellular proteins and protects cells against both anti-Fas and TNF-induced cell death. Previously, we reported that the C-terminus of Daxx interacted with Ubc9, an E2 type SUMO-1 conjugating enzyme, as well as with SUMO-1. In BOSC23 cells expressing FLAG-Daxx together with HA-SUMO-1, 110 and 130kDa Daxx appeared and the 130kDa band bound to both anti-HA and anti-FLAG antibodies. This means that Daxx can be covalently modified by SUMO-1. Substitution of K630 and K631 abrogated the modification of Daxx by SUMO-1, implying that K630 and K631 were essential for sumoylation. Daxx (K630, 631A) and Daxx (K634, 636, 637A) in which the putative C-terminal nuclear localization signals (NLSs) were disrupted appeared in the nucleus, suggesting that the C-terminal NLS was not functional. Daxx (K630, 631A), the sumoylation defective mutant, was able to interact with PML and co-localized with PML in the PML oncogenic domains (PODs). Thus, our data show that sumoylation status of Daxx does not affect its presence in PODs.

Interaction of ALG-2 with ASK1 influences ASK1 localization and subsequent JNK activation.

ALG‐2 (apoptosis linked gene‐2) is an essential protein for the execution of apoptosis whose function is largely unknown. Here, we demonstrate that ALG‐2 could interact with the C‐terminus (amino acids 941–1375) of the apoptosis signal‐regulating kinase 1 (ASK1) in BOSC23 cells as well as in vitro. ASK1 failed to bind to an isotype of ALG‐2 found in the liver, ALG‐2,1, in which two amino acids (Gly‐121 and Phe‐122) are deleted. This implies that the interaction is very specific. Cotransfection with ALG‐2 resulted in the nuclear presence of ASK1 and inhibited the activation of c‐Jun N‐terminal kinase (JNK) by ASK1 in BOSC23 cells. This study reports that ALG‐2 could regulate the subcellular localization and the JNK activity modulation of ASK1 by direct interaction.

FAF1 Suppresses IκB Kinase (IKK) Activation by Disrupting the IKK Complex Assembly

This study presents a molecular inhibitory mechanism by Fas-associated factor 1 (FAF1) on IκB kinase (IKK) activation, where divergent NF-κB-activating stimuli converge. FAF1 interacts with IKKβ in response to proinflammatory stimuli (such as tumor necrosis factor-α, interleukin-1β, and lipopolysaccharide) and suppresses IKK activation. Interaction of the leucine-zipper domain of IKKβ with FAF1 affected the IKK heterocomplex (IKKα/β) and homocomplex (IKKα/α, IKKβ/β) formations and attenuated IKKγ recruitment to IKKβ. Overexpression of FAF1 reduced the level of IKKβ activity, whereas FAF1 depletion increased the activity. These results indicate that FAF1 inhibits IKK activation and its downstream signaling by interrupting the IKK complex assembly through physical interaction with IKKβ. Taken together, FAF1 robustly suppresses NF-κB activation through the inhibition of IKK activation in combination with previously reported cytoplasmic retention of NF-κB p65 (Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol. Chem. 279, 2544–2549). Such redundant suppression would prevent inadvertent activation of the NF-κB pathway.

Role of FLASH in caspase-8-mediated activation of NF-κB: Dominant-negative function of FLASH mutant in NF-κB signaling pathway

Caspase-8 is the most receptor-proximal, upstream caspase in the caspase cascade and plays a key role in cell death triggered by various death receptors. Here, we addressed the role of endogenous caspase-8 in tumor necrosis factor (TNF)-α-induced activation of NF-κB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-α-induced activation of NF-κB in NIH3T3, HeLa, and HEK293 cells as determined with luciferase reporter gene and cell fractionation assays. Reconstitution of caspase-8-deficient C33A cells with processing-defective (P/D) mutant of caspase-8 sensitized the cells to TNF-α for NF-κB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-κB and to bind FLICE-associated huge protein (FLASH) in vitro and in vivo. Instead, caspase-8 (D73A) mutant bound to caspase-8 and blocked NF-κB activation triggered by TNF-α and caspase-8. In addition, expression of an NF-κB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-α and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-κB. Further, immunoprecipitation assays showed that caspase-8 formed triple complex with TRAF2 and FLASH. Taken together, these results suggest that endogenous caspase-8 mediates TNF-α-induced activation of NF-κB via FLASH.

Cooperative Phosphorylation of FADD by Aur-A and Plk1 in Response to Taxol Triggers Both Apoptotic and Necrotic Cell Death

Administration of the antimitotic chemotherapeutic taxol is known to cause accumulation of the mitotic kinase Aurora-A (Aur-A). Here, we report that Aur-A phosphorylates S203 of the Fas associated with death domain protein (FADD) in response to taxol treatment. In addition, polo-like kinase 1 (Plk1) failed to phosphorylate the Aur-A-unphosphorylatable FADD substitution mutant S203A, indicating that phosphorylation of S203 by Aur-A serves to prime FADD for Plk1-mediated phosphorylation at S194. The double-phosphorylation-mimicking mutant form of FADD, FADD-S194D/S203D (FADD-DD), recruited caspase-8, activating the caspase-dependent cell death pathway. FADD-DD also dissociated the cell death protein RIP1 from FADD, resulting in activation of RIP1 and triggering of caspase-independent cell death. Consistent with its death-promoting potential, FADD-DD showed robust tumor suppressor activity. However, single-phosphorylation-mimicking mutant forms of FADD, FADD-S194D/S203A (FADD-DA) and FADD-S194A/S203D (FADD-AD), were incapable of carrying out such functions, indicating that double phosphorylation of FADD is critical for the execution of cell death and tumor suppression. Collectively, our data show the existence of cooperative actions between Aur-A and Plk1 mitotic kinases in response to taxol, providing a molecular explanation for the action mechanism of taxol.

Physical Interactions and Functional Coupling between Daxx and Sodium Hydrogen Exchanger 1 in Ischemic Cell Death

Daxx, a death domain-associated protein, is implicated in ischemic cell death. To clarify the mechanism of cell death mediated by Daxx, a yeast two-hybrid assay was performed. Sodium hydrogen exchanger isoform 1 (NHE1) was identified as a Daxx-interacting protein. During ischemic stress, Daxx translocates from the nucleus to the cytoplasm, where it colocalizes with NHE1. Daxx binds to the ezrin/radixin/moesin-interacting domain of NHE1, in competition with ezrin. Consistent with this finding, transfection of the constitutively cytoplasmic mutant, Daxx(W621A), inhibited ezrin-mediated Akt-1 activation. Moreover, transfection of Daxx(W621A), but not the Daxx(S667A) mutant that is confined to the nucleus, accelerated pHi recovery from an acid load, indicating that the cytoplasmic protein activates NHE1. Based on the results, we propose that ischemic insult triggers the nucleocytoplasmic translocation of Daxx, following which cytoplasmic Daxx stimulates the NHE1 transporter activity and suppresses activation of the NHE1-ezrin-Akt-1 pathway. Our data support a novel molecular function of Daxx as an upstream regulator of NHE1 in ischemic cell death.

Fas-associated factor-1 mediates chemotherapeutic-induced apoptosis via death effector filament formation

Fas‐associated factor‐1 (FAF1) is a newly introduced member of the Fas death‐inducing signaling complex and potentiates Fas‐mediated apoptosis. Clinical study has revealed that FAF1 is significantly reduced in gastric carcinomas. The present study demonstrates that FAF1 mediates chemotherapeutic‐induced apoptosis via participation in the formation of death effector filament (DEF), a cytoskeleton‐like structure found in receptor‐independent apoptosis. Overexpression of FAF1 enhanced DEF assembly and cell death induced by chemotherapeutics such as staurosporine (STS), cisplatin (CDDP) and etoposide (VP16). FAF1 sensitized cells to STS, CDDP and VP16 in dose‐ and time‐dependent manner. Introduction of antisense FAF1 construct inhibited DEF assembly and chemotherapeutic‐induced apoptosis. Analysis using FAF1 truncates showed that the FAF1 domain interacting with DEDs of FADD and caspase‐8 was sufficient to enhance DEF assembly. Confocal microscopy revealed that FAF1 was present in DEFs together with FADD and caspase‐8. Collectively, our data provide a molecular mechanism for the chemosensitization by FAF1 (i.e., mediating DEF assembly).

Negative Feedback Regulation of Aurora-A via Phosphorylation of Fas-associated Factor-1

This study reports that Aurora-A (Aur-A) phosphorylates Fas-associated factor-1 (FAF1) at Ser-289 and Ser-291. Forced expression of a FAF1 mutant mimicking phosphorylation at Ser-289 and Ser-291 (FAF1 DD), but not phosphorylation-deficient FAF1 (FAF1 AA), reduced Aur-A expression. However, transfection of FAF1 DD failed to reduce Aur-A expression in the presence of MG132 and MG115, indicating that this decrease is proteasome-mediated. Additionally, transfection of FAF1 DD suppressed the expression of Aur-A in ts20-BALB cells lacking E1 ubiquitin (Ub) activating enzyme activity at restrictive temperatures and also reduced the expression of Aur-A S51D, a mutant resistant to Ub-dependent degradation. Our data indicate that phosphorylated FAF1 mediates the ubiquitin-independent, proteasome-dependent degradation of Aur-A. Overexpression of FAF1 DD blocked Aur-A-induced centrosome amplification and accumulated cells in G2/M phase, representing cellular phenotypes consistent with the anticipated loss of Aur-A. Collectively, our findings support the negative feedback regulation of Aur-A via phosphorylation of the death-promoting protein, FAF1, and disclose the presence of molecular cross-talk between constituents of the cell cycle and cell death machinery.

Transactivation potential of the C-terminus of human Nm23-H1

The transactivation potential of Nm23‐H1, a homolog of c‐myc transcription factor Nm23‐H2/PuF was assessed in yeast as a fusion protein with the DNA binding domains (DBDs) of GAL4 and LexA. The C‐terminal half of Nm23‐H1 exhibited strong transactivation of the reporter genes, LacZ and Leu2 carrying GAL4 and LexA upstream activating sequences (UASs), whereas the full‐length Nm23‐H1 and its N‐terminal did not. Similar results were also obtained with Nm23‐H2/PuF transactivating the reporter genes only by the C‐terminus fused to GAL4 and LexA DBDs. Hence, our results suggested a possible regulatory role of the N‐termini of Nm23 isotypes upon transactivation.