LRRK2 functions as a scaffolding kinase of ASK1-mediated neuronal cell death.

Leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein, is a key causative factor in Parkinson's disease (PD). Identification of novel substrates and the molecular mechanisms underlying the effects of LRRK2 are essential for understanding the pathogenesis of PD. In this study, we showed that LRRK2 played an important role in neuronal cell death by directly phosphorylating and activating apoptosis signal-regulating kinase 1 (ASK1). LRRK2 phosphorylated ASK1 at Thr832 that is adjacent to Thr845, which serves as an autophosphorylation site. Moreover, results of binding and kinase assays showed that LRRK2 acted as a scaffolding protein by interacting with each components of the ASK1-MKK3/6-p38 MAPK pathway through its specific domains and increasing the proximity to downstream targets. Furthermore, LRRK2-induced apoptosis was suppressed by ASK1 inhibition in neuronal stem cells derived from patients with PD. These results clearly indicate that LRRK2 acts as an upstream kinase in the ASK1 pathway and plays an important role in the pathogenesis of PD.

Parkin mediates neuroprotection through activation of Notch1 signaling.

Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson's disease. Inactivation of Parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins and ensuing neurodegeneration. In this study, we show that Parkin positively regulates the Notch1 signaling pathway. Overexpression of Parkin stabilized Notch1-IC protein levels, whereas knockdown of Parkin decreased Notch1-IC protein stability. Notably, overexpression of Parkin disrupted oxidative stress-induced apoptosis in neuronal cells. However, knockdown of Notch1 inhibited Parkin-induced neuronal cell survival. Together, these results indicate that Parkin is a novel regulator of the Notch1 signaling pathway, which promotes neuronal cell survival.

Autophagy negatively regulates tumor cell proliferation through phosphorylation dependent degradation of the Notch1 intracellular domain.

Autophagy is a highly conserved mechanism that degrades long-lived proteins and dysfunctional organelles, and contributes to cell fate. In this study, autophagy attenuates Notch1 signaling by degrading the Notch1 intracellular domain (Notch1-IC). Nutrient-deprivation promotes Notch1-IC phosphorylation by MEKK1 and phosphorylated Notch1-IC is recognized by Fbw7 E3 ligase. The ubiquitination of Notch1-IC by Fbw7 is essential for the interaction between Notch1-IC and p62 and for the formation of aggregates. Inhibition of Notch1 signaling prevents the transformation of breast cancer cells, tumor progression, and metastasis. The expression of Notch1 and p62 is inversely correlated with Beclin1 expression in human breast cancer patients. These results show that autophagy inhibits Notch1 signaling by promoting Notch1-IC degradation and therefore plays a role in tumor suppression.

Tumor Suppressor HIPK2 Regulates Malignant Growth via Phosphorylation of Notch1.

The receptor Notch1 plays an important role in malignant progression of many cancers, but its regulation is not fully understood. In this study, we report that the kinase HIPK2 is responsible for facilitating the Fbw7-dependent proteasomal degradation of Notch1 by phosphorylating its intracellular domain (Notch1-IC) within the Cdc4 phosphodegron motif. Notch1-IC expression was higher in cancer cells than normal cells. Under genotoxic stress, Notch1-IC was phosphorylated constitutively by HIPK2 and was maintained at a low level through proteasomal degradation. HIPK2 phosphorylated the residue T2512 in Notch1-IC. Somatic mutations near this residue rendered Notch1-IC resistant to degradation, as induced either by HIPK2 overexpression or adriamycin treatment. In revealing an important mechanism of Notch1 stability, the results of this study could offer a therapeutic strategy to block Notch1-dependent progression in many types of cancer. Cancer Res; 76(16); 4728-40. ©2016 AACR.

NOTCH1 intracellular domain negatively regulates PAK1 signaling pathway through direct interaction.

p21-Activated kinase 1 (PAK1) is a serine/threonine protein kinase implicated in cytoskeletal remodeling and cell motility. Recent studies have shown that it also promotes cell proliferation, regulates apoptosis, and increases cell transformation and invasion. In this study, we showed that NOTCH1 intracellular domain (NOTCH1-IC) negatively regulated PAK1 signaling pathway. We found a novel interaction between NOTCH1-IC and PAK1. Overexpression of NOTCH1-IC decreased PAK1-induced integrin-linked kinase 1 (ILK1) phosphorylation, whereas inhibition of NOTCH1 signaling increased PAK1-induced ILK1 phosphorylation. Notably, ILK1 phosphorylation was higher in PS1,2(-/-) cells than in PS1,2(+/+) cells. As expected, overexpression of NOTCH1-IC decreased ILK1-induced phosphorylation of glycogen synthase kinase 3 beta (GSK-3beta). Furthermore, NOTCH1-IC disrupted the interaction of PAK1 with ILK1 and altered PAK1 localization by directly interacting with it. This inhibitory effect of NOTCH1-IC on the PAK1 signaling pathway was mediated by the binding of NOTCH1-IC to PAK1 and by the alteration of PAK1 localization. Together, these results suggest that NOTCH1-IC is a new regulator of the PAK1 signaling pathway that directly interacts with PAK1 and regulates its shuttling between the nucleus and the cytoplasm.

Fe65 negatively regulates Jagged1 signaling by decreasing Jagged1 protein stability through the E3 ligase Neuralized-like 1.

Fe65 is a highly conserved adaptor protein that interacts with several binding partners. Fe65 binds proteins to mediate various cellular processes. But the interacting partner and the regulatory mechanisms controlled by Fe65 are largely unknown. In this study, we found that Fe65 interacts with the C-terminus of Jagged1. Furthermore, Fe65 negatively regulates AP1-mediated Jagged1 intercellular domain transactivation in a Tip60-independent manner. We found that Fe65 triggers the degradation of Jagged1, but not the Jagged1 intracellular domain (JICD), through both proteasome and lysosome pathways. We also showed that Fe65 promotes recruitment of the E3 ligase Neuralized-like 1 (Neurl1) to membrane-tethered Jagged1 and monoubiquitination of Jagged1. These three proteins form a stable trimeric complex, thereby decreasing Jagged1 targeting by ubiquitin-mediated degradation. Consequently, Jagged1 is a novel binding partner of Fe65, and Fe65 may act as a novel effector of Jagged1 signaling.

Alpha-synuclein negatively regulates Notch1 intracellular domain protein stability through promoting interaction with Fbw7.

Notch signaling pathway is well known that it is involved in regulating cell fate, proliferation and homeostasis. In this study, we show a novel function of alpha-synuclein (SNCA) to promote degradation of Notch1 intracellular domain (Notch1-IC) through Fbw7, ubiquitin E3 ligase. We identified that SNCA inhibits Notch1 transcription activity and diminishes the interaction between Notch1-IC and RBP-Jk. We also found decrease of Notch1-IC protein stability by exogenous and endogenous SNCA through proteasomal pathway, not through lysosomal pathway. And, we found that SNCA promotes interaction between Notch1-IC and Fbw7. Furthermore, SNCA directly interacts with Fbw7. SNCA increases ubiquitination of Notch-IC by Fbw7 through interaction with Fbw7. Together, these results suggest that SNCA is a novel regulator of Notch1-IC transcriptional activity with acting as an enhancer of the interaction of Notch1-IC and Fbw7 with increasing degradation of Notch1-IC.

Akt1 phosphorylates Nicastrin to regulate its protein stability and activity.

The gamma-secretase is a multiprotein complex that cleaves many type-I membrane proteins, such as the Notch receptor and the amyloid precursor protein. Nicastrin (NCT) is an essential component of the multimeric gamma-secretase complex and functions as a receptor for gamma-secretase substrates. In this study, we found that Akt1 markedly regulated the protein stability of NCT. Importantly, the kinase activity of Akt1 was essential for the inhibition of gamma-secretase activity through degradation of NCT. Notably, the protein level of endogenous NCT was higher in shAkt1-expressing cells than in shCon-expressing cells. Akt1 physically interacted with NCT and mediated its degradation through proteasomal and lysosomal pathways. We also found that Akt1 phosphorylates NCT at Ser437, resulting in a significant reduction in NCT protein stability. Importantly, a phospho-deficient mutation in NCT at Ser437 stabilized its protein levels. Collectively, our results reveal that Akt1 functions as a negative regulator of the gamma-secretase activity through phosphorylation and degradation of NCT. Generation of the amyloid peptide (A-beta) and the amyloid precursor protein (APP) intracellular domain (AICD) can happen by sequential proteolysis of APP by beta and gamma-secretase. The gamma-secretase complex consists of four essential proteins: presenilin (PS1 or PS2), presenilin enhancer 2 (PEN-2), anterior pharynx-defective 1 (APH-1), and the Nicastrin (NCT). NCT can interact and be phosphorylated by Akt1, and phosphorylated NCT promotes its proteasomal and lysosomal degradation. As a result, Akt1 plays role in reducing gamma-secretase activity through phosphorylation-dependent regulation of NCT protein degradation.

Notch1 modulates oxidative stress induced cell death through suppression of apoptosis signal-regulating kinase 1.

Notch1 genes encode receptors for a signaling pathway that regulates various aspects of cell growth and differentiation; however, the role of Notch1 signaling in p38 mitogen-activated protein kinase (MAPK) signaling pathway is still not well defined. In this study, we found that Notch1 intracellular domain (Notch1-IC) prevents oxidative stress-induced cell death through the suppression of the Apoptosis signal-regulating kinase (ASK) 1 signaling pathway. Notch1-IC inhibited H2O2-induced activation of ASK1 and the activation of downstream kinases in the p38 MAPK signaling cascade. The results of both in vivo binding and kinase studies have revealed that ASK1 is the direct target of Notch1-IC, whereas it produced no effect on either MAP kinase kinase (MKK) 3 or p38 MAPK. Notch1-IC blocked both the homooligomerization of ASK1 and inhibited ASK1 activity. Furthermore, Notch1-IC facilitated the translocation of activated ASK1 toward the nucleus. Notch1 knockdown was determined to be highly susceptible to oxidative stress-induced activation of ASK1-MKK3/MKK6-p38 MAPK signaling cascade and cell death. Taken together, our findings suggest that Notch1-IC may act as a negative regulator in ASK1 signaling cascades.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) enhances osteoclast differentiation via the up-regulation of Notch1 protein stability.

The Notch signaling pathway plays a crucial role in the regulation of cell fate decision, and is also a key regulator of cell differentiation, including bone homeostasis, in a variety of contexts. However, the role of Notch1 signaling in osteoclast differentiation is still controversial. In this study, we show that Receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation is promoted by the Notch1 intracellular domain (Notch1-IC) and Ca(2+)/Calmodulin dependent protein kinase IV (CaMKIV) signaling. Notch1-IC protein level was augmented by CaMKIV through escape from ubiquitin dependent protein degradation. In addition, CaMKIV remarkably increased Notch1-IC stability, and the kinase activity of CaMKIV was essential for facilitating Notch1 signaling. CaMKIV directly interacted with Notch1-IC and phosphorylates Notch1-IC, thereby decreasing proteasomal protein degradation through F-box and WD repeat domain-containing 7 (Fbw7). We also found that Notch1-IC prevented inhibition of osteoclast differentiation by KN-93 but not the phosphorylation deficient form of Notch1-IC. These results suggest that phosphorylated Notch1-IC by CaMKIV increases Notch1-IC stability, which enhances osteoclast differentiation.

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

Serine-threonine Ca(2+)/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.

Phosphorylation of nicastrin by SGK1 leads to its degradation through lysosomal and proteasomal pathways.

The gamma-secretase complex is involved in the intramembranous proteolysis of a variety of substrates, including the amyloid precursor protein and the Notch receptor. Nicastrin (NCT) is an essential component of the gamma-secretase complex and functions as a receptor for gamma-secretase substrates. In this study, we determined that serum- and glucocorticoid-induced protein kinase 1 (SGK1) markedly reduced the protein stability of NCT. The SGK1 kinase activity was decisive for NCT degradation and endogenous SGK1 inhibited gamma-secretase activity. SGK1 downregulates NCT protein levels via proteasomal and lysosomal pathways. Furthermore, SGK1 directly bound to and phosphorylated NCT on Ser437, thereby promoting protein degradation. Collectively, our findings indicate that SGK1 is a gamma-secretase regulator presumably effective through phosphorylation and degradation of NCT.

Presenilin-2 regulates the degradation of RBP-Jk protein through p38 mitogen-activated protein kinase.

Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk)--a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1-RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase ∂ (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.

Dual regulation of notch1 signaling pathway by adaptor protein fe65.

Notch1 receptor functions as a critical controller of cell fate decisions and also as a key regulator of cell growth, differentiation, and proliferation in invertebrates and vertebrates. In this study, we have demonstrated that the adaptor protein Fe65 attenuates Notch1 signaling via the accelerated degradation of the membrane-tethered Notch1 in the cytoplasm. Fe65 also suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-recombining binding protein suppressor of hairless (RBP)-Jk complex within the nucleus. Fe65 is capable of forming a trimeric complex with Itch and membrane-tethered Notch1, and Fe65 enhances the protein degradation of membrane-tethered Notch1 via an Itch-dependent proteasomal pathway. Collectively, our results demonstrate that Fe65 carries out different functions depending on its location in the regulation of Notch1 signaling.

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.

Regulation of Notch1 signaling by Delta-like ligand 1 intracellular domain through physical interaction.

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. The Delta-like ligand 1 also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. In this study, we have demonstrated that the Delta-like 1 intracellular domain (Dll1-IC) specifically binds to Notch1-IC in the nucleus, thereby disrupting the association of the Notch1-IC-RBP-Jk-MAM transcription activator complex. Additionally, the Notch1-mediated blockage of the induction of MyoD is abolished by the co-expression of Dll1-IC. Collectively, our results show that Dll1-IC functions as a negative regulator in Notch signaling via the disruption of the Notch1-IC-RBP-Jk complex.

Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk.

The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC-RBP-Jk complex after processing by γ-secretase. Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1-IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP-Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk protein degradation.

Inhibition of Notch1 signaling by Runx2 during osteoblast differentiation.

Notch1 genes encode receptors for a signaling pathway that regulates cell growth and differentiation in various contexts, but the role of Notch1 signaling in osteogenesis is not well defined. Notch1 controls osteoblast differentiation by affecting Runx2, but the question arises whether normal osteoblastic differentiation can occur regardless of the presence of Notch1. In this study, we observed the downregulation of Notch1 signaling during osteoblastic differentiation. BMPR-IB/Alk6-induced Runx2 proteins reduced Notch1 activity to a marked degree. Accumulated Runx2 suppressed Notch1 transcriptional activity by dissociating the Notch1-IC-RBP-Jk complex. Using deletion mutants, we also determined that the N-terminal domain of Runx2 was crucial to the binding and inhibition of the N-terminus of the Notch1 intracellular domain. Notably, upregulation of the Runx2 protein level paralleled reduced expression of Hes1, which is a downstream target of Notch1, during osteoblast differentiation. Collectively, our data suggest that Runx2 is an inhibitor of the Notch1 signaling pathway during normal osteoblast differentiation.

Serum- and glucocorticoid-inducible kinase 1 (SGK1) controls Notch1 signaling by downregulation of protein stability through Fbw7 ubiquitin ligase.

Notch is a transmembrane protein that acts as a transcriptional factor in the Notch signaling pathway for cell survival, cell death and cell differentiation. Notch1 and Fbw7 mutations both lead the activation of the Notch1 pathway and are found in the majority of patients with the leukemia T-ALL. However, little is known about the mechanisms and regulators that are responsible for attenuating the Notch signaling pathway through Fbw7. Here, we report that the serum- and glucocorticoid-inducible protein kinase SGK1 remarkably reduced the protein stability of the active form of Notch1 through Fbw7. The protein level and transcriptional activity of the Notch1 intracellular domain (Notch1-IC) were higher in SGK1-deficient cells than in SGK1 wild-type cells. Notch1-IC was able to form a trimeric complex with Fbw7 and SGK1, thereby SGK1 enhanced the protein degradation of Notch1-IC via a Fbw7-dependent proteasomal pathway. Furthermore, activated SGK1 phosphorylated Fbw7 at serine 227, an effect inducing Notch1-IC protein degradation and ubiquitylation. Moreover, accumulated dexamethasone-induced SGK1 facilitated the degradation of Notch1-IC through phosphorylation of Fbw7. Together our results suggest that SGK1 inhibits the Notch1 signaling pathway via phosphorylation of Fbw7.

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 Parkinson's 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-1's effect on stress-activated protein kinase signaling and H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). According to the results, the overexpression of DJ-1 inhibited H(2)O(2)-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.