Hee-Sae Park

DJ-1 modulates UV-induced oxidative stress signaling through the suppression of MEKK1 and cell death

DJ-1 is a multifunctional protein that performs functions in transcriptional regulation and oxidative stress, and the loss of its function is believed to result in the onset of Parkinsons disease (PD). In this study, we report that DJ-1 protects against UV-induced cell death through the suppression of the JNK1 signaling pathway. The results of both binding and kinase studies have revealed that MEKK1 is the direct target of DJ-1. The C-terminus of DJ-1 was crucial to the inhibition of the MEKK1 kinase activity. Wild-type DJ-1 sequesters MEKK1 within the cytoplasm and the L166P mutant facilitates the translocation of MEKK1 toward the nucleus without physical association. Both DJ-1 knockdown and pathogenic L166P mutant were determined to be highly susceptible to the UV-induced activation of the MEKK1-SEK1-JNK1 signaling cascade and cell death. Taken together, our findings show that missense mutation in DJ-1 sensitizes cells to stress-induced cell death through the MEKK1-SEK1-JNK1 signaling pathway, a process, which may trigger the early onset of PD.

Beta-catenin modulates the level and transcriptional activity of Notch1/NICD through its direct interaction

Wnt and Notch1 signaling pathways play an important role in a variety of biological processes including embryonic induction, the polarity of cell division, cell fate, and cell growth. Although there is evidence that the two main signaling pathways can modulate each other, the precise mechanism is not completely understood. This report shows that beta-catenin can regulate the level and transcriptional activity of the Notch1 and Notch1 intracellular domain (NICD). The in vivo and in vitro results demonstrate that beta-catenin binds with Notch1 and NICD, for which its Armadillo repeat domain is essential. It was further demonstrated that beta-catenin could upregulate the level of Notch1 and NICD, possibly by competing the common ubiquitin-dependent degradation machinery. In addition, beta-catenin enhanced the transcriptional activity of NICD on the hairy and enhancer of split 1 (HES1) and CSL through its C-terminal transactivation domain. This effect of cooperative regulation by beta-catenin could also be observed in bone morphogenetic protein 2 (BMP2) induced osteogenic differentiation of C2C12 cells. beta-catenin coexpression with NICD enhanced the alkaline phosphatase (ALP) activity in C2C12 cells compared with either beta-catenin or NICD expression alone. Culturing C2C12 cells on Delta-1 coated dishes together with Wnt3-conditioned media induced noticeable increases in ALP staining, verifying that employed physiological levels of NICD and beta-catenin are sufficient to induce ALP activation. Furthermore, effects of beta-catenin on Notch1 were dramatically diminished by overexpressed LEF1. Overall, our data suggest that beta-catenin can act as a switching molecule between the classical TCF/LEF1 mediated pathway and NICD mediated pathway.

Integrin-Linked Kinase Controls Notch1 Signaling by Down-Regulation of Protein Stability through Fbw7 Ubiquitin Ligase

ABSTRACT Integrin-linked kinase (ILK) is a scaffold and protein kinase that acts as a pivotal effector in integrin signaling for various cellular functions. In this study, we found that ILK remarkably reduced the protein stability of Notch1 through Fbw7. The kinase activity of ILK was essential for the inhibition of Notch1 signaling. Notably, the protein level and transcriptional activity of the endogenous Notch1 intracellular domain (Notch1-IC) were higher in ILK-null cells than in ILK wild-type cells, and the level of endogenous Notch1-IC was increased by the blocking of the proteasome, suggesting that ILK enhances the proteasomal degradation of Notch1-IC. ILK directly bound and phosphorylated Notch1-IC, thereby facilitating proteasomal protein degradation through Fbw7. Furthermore, we found down-regulation of Notch1-IC and up-regulation of ILK in basal cell carcinoma and melanoma patients but not in squamous cell carcinoma patients. These results suggest that ILK down-regulated the protein stability of Notch1-IC through the ubiquitin-proteasome pathway by means of Fbw7.

Tip60 Histone Acetyltransferase Acts as a Negative Regulator of Notch1 Signaling by Means of Acetylation

ABSTRACT The Notch signaling pathway appears to perform an important function in a wide variety of organisms and cell types. In our present study, we provide evidence that UV irradiation-induced Tip60 proteins reduced Notch1 activity to a marked degree. Accumulated UV irradiation-induced Tip60 suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-CSL complex. The binding between endogenous Tip60 and Notch1-IC in UV radiation-exposed cells was verified in this study by coimmunoprecipitation. Interestingly, the physical interaction of Tip60 with Notch1-IC occurs to a more profound degree in the presence of CSL but does not exist in a trimeric complex. Using Notch1-IC and Tip60 deletion mutants, we also determined that the N terminus, which harbors the RAM domain and seven ankyrin repeats of Notch1-IC, interacts with the zinc finger and acetyl coenzyme A domains of Tip60. Furthermore, here we report that Notch1-IC is a direct target of the acetyltransferase activity of Tip60. Collectively, our data suggest that Tip60 is an inhibitor of the Notch1 signaling pathway and that Tip60-dependent acetylation of Notch1-IC may be relevant to the mechanism by which Tip60 suppresses Notch1 signaling.

DJ-1 modulates the p38 mitogen-activated protein kinase pathway through physical interaction with apoptosis signal-regulating kinase 1.

DJ‐1 has been reported as a gene linked to early onset familial Parkinsons disease, and is functionally involved in transcriptional regulation and oxidative stress‐induced cell death. To understand the role of DJ‐1 in cellular stress, this study investigated DJ‐1s effect on stress‐activated protein kinase signaling and H2O2‐induced activation of apoptosis signal‐regulating kinase 1 (ASK1). According to the results, the overexpression of DJ‐1 inhibited H2O2‐induced activation of ASK1 as well as the activation of downstream kinases in the p38 mitogen‐activated protein kinase (MAPK) signaling cascade. The results of both in vivo binding and kinase studies have revealed that ASK1 is the direct target of DJ‐1, whereas it has shown no effect on either MKK3 or p38. DJ‐1 blocked both the homo‐oligomerization of ASK1 and inhibited ASK1 activity. Taken together, our data strongly suggest that DJ‐1, by directly inhibiting ASK1, may act as a negative regulator in ASK1 signaling cascades. J. Cell. Biochem. 110: 229–237, 2010.

Activation of nuclear PTEN by inhibition of Notch signaling induces G2/M cell cycle arrest in gastric cancer

Mutation in PTEN has not yet been detected, but its function as a tumor suppressor is inactivated in many cancers. In this study we determined that, activated Notch signaling disables PTEN by phosphorylation and thereby contributes to gastric tumorigenesis. Notch inhibition by small interfering RNA or γ-secretase inhibitor (GSI) induced mitotic arrest and apoptosis in gastric cancer cells. Notch inhibition induced dephosphorylation in the C-terminal domain of PTEN, which led to PTEN nuclear localization. Overexpression of activated Notch1-induced phosphorylation of PTEN and reversed GSI-induced mitotic arrest. Dephosphorylated nuclear PTEN caused prometaphase arrest by interaction with the cyclin B1-CDK1 complex, resulting in their accumulation in the nucleus and subsequent apoptosis. We found a correlation between high expression levels of Notch1 and low survival rates and, similarly, between reduced nuclear PTEN expression and increasing the TNM classification of malignant tumours stages in malignant tissues from gastric cancer patients. The growth of Notch1-depleted gastric tumors was significantly retarded in xenografted mice, and in addition, PTEN deletion restored growth similar to control tumors. We also demonstrated that combination treatment with GSI and chemotherapeutic agents significantly reduced the orthotopically transplanted gastric tumors in mice without noticeable toxicity. Overall, our findings suggest that inhibition of Notch signaling can be employed as a PTEN activator, making it a potential target for gastric cancer therapy.

The intracellular domain of Jagged-1 interacts with Notch1 intracellular domain and promotes its degradation through Fbw7 E3 ligase.

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. Jagged-1 also undergoes proteolytic cleavage by gamma-secretase and releases an intracellular fragment. In this study, we have demonstrated that the Jagged-1 intracellular domain (JICD) inhibits Notch1 signaling via a reduction in the protein stability of the Notch1 intracellular domain (Notch1-IC). The formation of the Notch1-IC-RBP-Jk-Mastermind complex is prevented in the presence of JICD, via a physical interaction. Furthermore, JICD accelerates the protein degradation of Notch1-IC via Fbw7-dependent proteasomal pathway. These results indicate that JICD functions as a negative regulator in Notch1 signaling via the promotion of Notch1-IC degradation.

SAG protects human neuroblastoma SH-SY5Y cells against 1-methyl-4-phenylpyridinium ion (MPP+)-induced cytotoxicity via the downregulation of ROS generation and JNK signaling

Sensitive to apoptosis gene (SAG), a novel zinc RING finger protein, exhibits anti-apoptotic and antioxidant activity against a variety of redox reagents. In the present study, we have determined that SAG suppresses 1-methyl-4-phenylpyridinium ion (MPP(+))-induced neurotoxicity via the downregulation of ROS generation and c-Jun N-terminal kinase 1 (JNK1) activity. Both transient and constitutively overexpressed SAG were found to inhibit the MPP(+)-induced neurotoxicity of SH-SY5Y neuroblastoma cells. In the SAG-expressing cells, MPP(+) induced ROS generation was suppressed to a significant degree as compared to the cells treated only with MPP(+). MPP(+)-induced JNK1 activation was also determined to be suppressed markedly by SAG. Furthermore, SAG inhibits MEKK1 dependent c-Jun transcription activity in SH-SY5Y cells. Thus, we concluded that SAG is a cellular protective molecule, which appears to function as an antioxidant, suppressing MPP(+)-induced neurotoxicity.

FE65 as a link between VLDLR and APP to regulate their trafficking and processing

BackgroundSeveral studies found that FE65, a cytoplasmic adaptor protein, interacts with APP and LRP1, altering the trafficking and processing of APP. We have previously shown that FE65 interacts with the ApoE receptor, ApoER2, altering its trafficking and processing. Interestingly, it has been shown that FE65 can act as a linker between APP and LRP1 or ApoER2. In the present study, we tested whether FE65 can interact with another ApoE receptor, VLDLR, thereby altering its trafficking and processing, and whether FE65 can serve as a linker between APP and VLDLR.ResultsWe found that FE65 interacted with VLDLR using GST pull-down and co-immunoprecipitation assays in COS7 cells and in brain lysates. This interaction occurs via the PTB1 domain of FE65. Co-transfection with FE65 and full length VLDLR increased secreted VLDLR (sVLDLR); however, the levels of VLDLR C-terminal fragment (CTF) were undetectable as a result of proteasomal degradation. Additionally, FE65 increased cell surface levels of VLDLR. Moreover, we identified a novel complex between VLDLR and APP, which altered trafficking and processing of both proteins. Furthermore, immunoprecipitation results demonstrated that the presence of FE65 increased the interaction between APP and VLDLR in vitro and in vivo.ConclusionsThese data suggest that FE65 can regulate VLDLR trafficking and processing. Additionally, the interaction between VLDLR and APP altered both proteins trafficking and processing. Finally, our data suggest that FE65 serves as a link between VLDLR and APP. This novel interaction adds to a growing body of literature indicating trimeric complexes with various ApoE Receptors and APP.

Wnt5a controls Notch1 signaling through CaMKII mediated degradation of the SMRT corepressor protein

Background: Wnt and Notch signaling pathways belong to a highly conserved network that controls gene expression. Results: Wnt5a enhances Notch1 signaling through down-regulation of SMRT by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Conclusion: CaMKII is crucial for the regulation between Notch and Wnt5a signaling. Significance: The findings of this study may begin to shed some light onto what may be a signal cross-talk mechanism of Notch1 signaling and the CaMKII. Serine-threonine Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the key component in noncanonical Wnt5a signaling and has been shown to regulate its signaling. In this study, we found that CaMKII induced by Wnt5a remarkably reduced the protein stability of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), a co-repressor of Notch signaling, through proteasomal degradation. Wnt5a was found to enhance Notch1 intracellular domain (Notch1-IC) transcription activity, which could be inhibited by treatment with KN93, a CaMKII inhibitor. The kinase activity of CaMKII was essential for the activation of Notch signaling. We also determined that CaMKII could enhance the association between Notch1-IC and RBP-Jk. Furthermore, the physical association between RBP-Jk and SMRT was substantially suppressed by CaMKII. We demonstrated that CaMKII directly bound and phosphorylated SMRT at Ser-1407, thereby facilitating SMRT translocation from the nucleus to the cytoplasm and proteasome-dependent degradation. These results suggest that CaMKII down-regulated the protein stability of SMRT through proteasomal degradation.