The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity

Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-J kappa. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. In this report we explore the molecular basis for this functional difference between Notch 1 and Notch 3 IC. We find that Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-J kappa on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-J kappa in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-J kappa. We show that two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC. First, the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators. Second, we identify a novel important region in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors.     

High levels of Notch signaling down-regulate Numb and Numblike

Inhibition of Notch signaling by Numb is critical for many cell fate decisions. In this study, we demonstrate a more complex relationship between Notch and the two vertebrate Numb homologues Numb and Numblike. Although Numb and Numblike at low levels of Notch signaling negatively regulated Notch, high levels of Notch signaling conversely led to a reduction of Numb and Numblike protein levels in cultured cells and in the developing chick central nervous system. The Notch intracellular domain but not the canonical Notch downstream proteins Hes 1 and Hey 1 caused a reduction of Numb and Numblike. The Notch-mediated reduction of Numblike required the PEST domain in the Numblike protein and was blocked by the proteasome inhibitor MG132. Collectively, these observations reveal a reciprocal negative regulation between Notch and Numb/Numblike, which may be of relevance for stabilizing asymmetric cell fate switches and for tumor development.     

Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.     

Lysosome-dependent degradation of Notch3

Notch signaling plays an essential role in diverse biological processes during development and in pathogenesis of diseases ranging from cancer to cerebrovascular disorders. Precise regulation of Notch signaling is essential for normal function and requires both timely activation and inactivation of the intracellular domain (ICD) of Notch receptors. In addition, inappropriate buildup of Notch3 ectodomain is a hallmark pathological feature of the stroke and dementia disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Thus, a clear understanding of mechanisms of Notch protein turnover is essential for understanding normal and pathological mechanisms of Notch function. Previous studies showed that the degradation of ICDs of Notch1 and Notch4 is controlled by the ubiquitin–proteasome system (UPS), though more recent work demonstrated that Notch1 ICD is also controlled by lysosomal degradation. The mechanism of degradation of Notch3 has not yet been identified. Here we report that the degradation of ICD of Notch3 (N3-ICD) is mediated by lysosomes. Lysosome inhibitors chloroquine and NH₄Cl led to the accumulation of transfected N3-ICD in 293 cells and endogenous N3-ICD in C2C12, H460, and HeLa cell lines; in addition, inhibition of lysosome function by chloroquine and NH₄Cl delayed the degradation of N3-ICD. In contrast, N3-ICD was not affected by proteasome inhibitors MG132 and lactacystin. Furthermore, we find that the Notch3 extracellular domain (N3-ECD) is also subjected to lysosome-dependent degradation. In sum, our experiments demonstrate a critical role for lysosomes in the degradation of Notch3, which distinguishes it from Notch1 and Notch4.     

Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 microM MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30-50%. Interestingly, administration of 1-10 microM MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 microM MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome c, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 microM MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.     

Identification and characterization of presenilin-independent Notch signaling.

Presenilin (PS) proteins control the proteolytic cleavage that precedes nuclear access of the Notch intracellular domain. Here we observe that a partial activation of the HES1 promoter can be detected in PS1/PS2 (PS1/2) double null cells using Notch1 Delta E constructs or following Delta 1 stimulation, despite an apparent abolition of the production and nuclear accumulation of the Notch intracellular domain. PS1/2-independent Notch activation is sensitive to Numblike, a physiological inhibitor of Notch. PS1/2-independent Notch signaling is also inhibited by an active gamma-secretase inhibitor in the low micromolar range and is not inhibited by an inactive analogue, similar to PS-dependent Notch signaling. However, experiments using a Notch1-Gal4-VP16 fusion protein indicate that the PS1/2-independent activity does not release Gal4-VP16 and is therefore unlikely to proceed via an intramembranous cleavage. These data reveal that a novel PS1/2-independent mechanism plays a partial role in Notch signal transduction.     

Two different stages of epidermal growth factor (EGF) receptor endocytosis are sensitive to free ubiquitin depletion produced by proteasome inhibitor MG132

Numerous studies implicate proteasomes in the regulation of EGF receptor (EGFR) endocytosis on the basis of the ability of inhibitors to decrease EGFR degradation, but the exact mechanisms remain obscure. We demonstrated that EGFR itself is not a direct target for proteasome, since it is delivered to lysosomes intact. Evidence is presented that the inhibitory effect of MG132 on EGF degradation is due mostly to free ubiquitin depletion resultant from the suppression of proteasomal functioning by MG132. By subcellular fractionation, we show two MG132-sensitive steps in the EGFR degradation pathway: sorting from early (EE) to late (LE) endosomes, and late stage of LE maturation. MG132 treatment resulted in stabilization of EGFR tyrosine phosphorylation and its association with c-Cbl. Nevertheless, ubiquitination of EGFR at late stages of endocytosis was significantly lower than that in control cells. Highly ubiquitinated forms of EGFR demonstrated more sensitivity to MG132 treatment.     

Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities

Notch signals are important for lymphocyte development but downstream events that follow Notch signaling are not well understood. Here, we report that signaling through Notch modulates the turnover of E2A proteins including E12 and E47, which are basic helix-loop-helix proteins crucial for B and T lymphocyte development. Notch-induced degradation requires phosphorylation of E47 by p42/p44 MAP kinases. Expression of the intracellular domain of Notch1 (N1-IC) enhances the association of E47 with the SCF(Skp2) E3 ubiquitin ligase and ubiquitination of E47, followed by proteasome-mediated degradation. Furthermore, N1-IC induces E2A degradation in B and T cells in the presence of activated MAP kinases. Activation of endogenous Notch receptors by treatment of splenocytes with anti-IgM or anti-CD3 plus anti-CD28 also leads to E2A degradation, which is blocked by the inhibitors of Notch activation or proteasome function. Notch-induced E2A degradation depends on the function of its downstream effector, RBP-Jkappa, probably to activate target genes involved in the ubiquitination of E2A proteins. Thus we propose that Notch regulates lymphocyte differentiation by controlling E2A protein turnover.     

Nerve growth factor-induced neurite outgrowth is potentiated by stabilization of TrkA receptors

Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK) and phosphatidylinositol- 3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the Ub(K63) chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors.     

Ras binding triggers ubiquitination of the Ras exchange factor Ras-GRF2

Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.     

Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry

Post-translational modification of proteins via the covalent attachment of Ubiquitin (Ub) plays an important role in the regulation of protein stability and function in eukaryotic cells. In the present study, we describe a novel method for identifying ubiquitinated proteins from a complex biological sample, such as a whole cell lysate, using a combination of immunoaffinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We have demonstrated the applicability of this approach by identifying 70 ubiquitinated proteins from the human MCF-7 breast cancer cell line after treatment with the proteasome inhibitor MG132. This method will aid the study of protein ubiquitination and may be used as a tool for the discovery of novel biomarkers that are associated with disease progression.     

Regulation of intracellular decorin via proteasome degradation in rat mesangial cells

Decorin (DCN) is a member of small leucine‐rich proteoglycan family that neutralizes the bioactivity of transforming growth factor‐beta1 (TGF‐β1). It has been proven to be a promising anti‐fibrotic agent to treat glomerulonephritis. But the underlining mechanism for regulating and degrading intracellular DCN is still not fully understood. In this study, we investigated the roles of ubiquitination in the regulation of cytoplasmic DCN metabolism in rat mesangial cells (MC) by immunoprecipitation and Western blot. The results showed that a proportion of cytoplasmic DCN was ubiquitinated in normal MC and was enhanced in N‐glycosylation inhibitor (tunicamycin)‐treated MC. After being treated with the proteasome inhibitor MG132, ubiquitinated DCN accumulated and displayed a prolonged half‐life, accompanied by decreased TGF‐β1 expression and reduced collagen IV mRNA level in MC. This study demonstrated that the stability and function of cytoplasmic DCN can be regulated by ubiquitin‐proteasome system (UPS) in MC, which implies that regulating the ubiquitination and degradation of DCN might be a novel approach for modulating MC bioactivity. J. Cell. Biochem. 111: 1010–1019, 2010. © 2010 Wiley‐Liss, Inc.     

A Proteasome Inhibitor-stimulated Nrf1 Protein-dependent Compensatory Increase in Proteasome Subunit Gene Expression Reduces Polycomb Group Protein Level

The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study, we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132. We show that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G₂, and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a prosurvival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells and confirm that PcG protein levels are regulated by proteasome activity.