mTOR Complex 2 Stabilizes Mcl-1 Protein by Suppressing Its Glycogen Synthase Kinase 3-Dependent and SCF-FBXW7-Mediated Degradation

mTOR complex 2 (mTORC2) regulates cell survival and growth through undefined mechanisms. Mcl-1, a Bcl-2 family protein, functions as an oncogenic protein. The connection between mTORC2 and Mcl-1 stability has not been established and was thus the focus of this study. Mcl-1 levels in cancer cells were decreased by mTOR kinase inhibitors (TORKinibs), which inhibit both mTORCs, by knocking down rictor and by knocking out rictor or Sin1 but not by silencing raptor. TORKinib treatment and rictor knockdown did not alter Mcl-1 mRNA levels but rather decreased its protein stability. Moreover, TORKinib-induced Mcl-1 reduction was rescued by proteasome inhibition. Consistently, TORKinib increased Mcl-1 ubiquitination. Hence, it is clear that inhibition of mTORC2 enhances Mcl-1 degradation, resulting in Mcl-1 reduction. Suppression of glycogen synthase kinase 3 (GSK3) or FBXW7 rescued Mcl-1 reduction induced by TORKinibs or rictor knockdown. Thus, mTORC2 inhibition apparently induces Mcl-1 degradation through a GSK3-dependent and SCF-FBXW7-mediated mechanism. Intriguingly, we detected a direct association between mTORC2 and SCF-FBXW7; this association could be inhibited by TORKinib treatment, suggesting that mTORC2 may directly associate with and inhibit the SCF-FBXW7 complex, resulting in delayed Mcl-1 degradation. Collectively, our findings highlight a novel mechanism by which mTORC2 regulates cell survival and growth by stabilizing Mcl-1.     

Stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells

Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post‐translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells. Pharmacologic Pim‐1 inhibition using the small molecule inhibitor CX‐6258 decreased steady state levels and half‐life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S‐proteasome, demonstrating that Pim‐1 protects NKX3.1 from proteasome‐mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim‐1 phospho‐acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N‐terminal PEST domain is essential for Pim‐1‐mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim‐1. Pim‐1‐mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells. J. Cell. Biochem. 114: 1050–1057, 2013. © 2012 Wiley Periodicals, Inc.     

Rictor Undergoes Glycogen Synthase Kinase 3 (GSK3)-dependent,FBXW7-mediated Ubiquitination and Proteasomal Degradation

Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation.     

Discovery of novel pyrazolo[1,5-a]pyrimidines as potent pan-Pim inhibitors by structure- and property-based drug design

Pim kinases are promising targets for the development of cancer therapeutics. Among the three Pim isoforms, Pim-2 is particularly important in multiple myeloma, yet is the most difficult to inhibit due to its high affinity for ATP. We identified compound 1 via high throughput screening. Using property-based drug design and co-crystal structures with Pim-1 kinase to guide analog design, we were able to improve potency against all three Pim isoforms including a significant 10,000-fold gain against Pim-2. Compound 17 is a novel lead with low picomolar potency on all three Pim kinase isoforms.     

ERK1/2 achieves sustained activation by stimulating MAPK phosphatase-1 degradation via the ubiquitin-proteasome pathway

Sustained extracellular signal-regulated kinase 1/2 (ERK1/2) activation does not always correlate with its upstream Ras-Raf-mitogen-activated protein kinase kinase 1/2 (MKK1/2) signal cascade in cancer cells, and the mechanism remains elusive. Here we report a novel mechanism by which sustained ERK1/2 activation is established. We demonstrate that Pb(II), a carcinogenic metal, persistently induces ERK1/2 activity in CL3 human lung cancer cells and that Ras-Raf-MKK1/2 signaling cannot fully account for such activation. It is intriguing that Pb(II) treatment reduces mitogen-activated protein kinase phosphatase 1 (MKP-1) protein levels in time- and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb(II)-treated cells, MKP-1 is polyubiquitinated, and proteasome inhibitors markedly alleviate the ubiquitination and degradation of MKP-1. Inhibiting the Pb(II)-induced ERK1/2 activation by PD98059 greatly suppresses MKP-1 ubiquitination and degradation. It is remarkable that constitutive activation of MKK1/2 triggers endogenous MKP-1 ubiquitination and degradation in various mammalian cell lines. Furthermore, expression of functional MKP-1 decreases ERK1/2 activation and the c-Fos protein level and enhances cytotoxicity under Pb(II) exposure. Taken together, these results demonstrate that activated ERK1/2 can trigger MKP-1 degradation via the ubiquitin-proteasome pathway, thus facilitating long-term activation of ERK1/2 against cytotoxicity.     

Ubiquitin Hydrolase UCH-L1 Destabilizes mTOR Complex 1 by Antagonizing DDB1-CUL4-Mediated Ubiquitination of Raptor

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates processes including mRNA translation, proliferation, and survival. By assembling with different cofactors, mTOR forms two complexes with distinct biological functions. Raptor-bound mTOR (mTORC1) governs cap-dependent mRNA translation, whereas mTOR, rictor, and mSin1 (mTORC2) activate the survival and proliferative kinase Akt. How the balance between the competing needs for mTORC1 and -2 is controlled in normal cells and deregulated in disease is poorly understood. Here, we show that the ubiquitin hydrolase UCH-L1 regulates the balance of mTOR signaling by disrupting mTORC1. We find that UCH-L1 impairs mTORC1 activity toward S6 kinase and 4EBP1 while increasing mTORC2 activity toward Akt. These effects are directly attributable to a dramatic rearrangement in mTOR complex assembly. UCH-L1 disrupts a complex between the DDB1-CUL4 ubiquitin ligase complex and raptor and counteracts DDB1-CUL4-mediated raptor ubiquitination. These events lead to mTORC1 dissolution and a secondary increase in mTORC2. Experiments in Uchl1-deficient and transgenic mice suggest that the balance between these pathways is important for preventing neurodegeneration and the development of malignancy. These data establish UCH-L1 as a key regulator of the dichotomy between mTORC1 and mTORC2 signaling.     

Alternate exon insertion controls selective ubiquitination and degradation of different AUF1 protein isoforms

The A+U-rich element (ARE) in the 3′ non-coding region (3′ NCR) of short-lived cytokine mRNAs binds several regulatory proteins, including hnRNP D/AUF1, which comprises four isoforms of 37, 40, 42 and 45 kDa. ARE-mRNA degradation involves ubiquitin–proteasome activity, and one or more AUF1 proteins are thought to be ubiquitinated. Here we have characterized the mechanism for differential ubiquitination and degradation of the different AUF1 protein isoforms. We demonstrate in an in vitro ubiquitination system that the p37, followed by the p40 protein, are strongly ubiquitinated, whereas the p42 and p45 forms are not. Over expression in cells of enzymes that control the ubiquitin cycle were found to control p37 and p40 AUF1 protein levels through ubiquitination and proteasome activity, but not p42 and p45 forms. The p42 and p45 AUF1 proteins share a C-terminal exon 7 that is not found in the p37/p40 isoforms. Our studies show that exon 7 blocks ubiquitination and rapid degradation of AUF1 proteins, whereas its deletion permits ubiquitination to occur and promotes rapid turnover of AUF1 proteins. Thus, the stabilities of AUF1 isoforms are differentially controlled by insertion of an alternate exon that regulates ubiquitin targeting activity.     

Ubiquitination and down-regulation of gap junction protein connexin-43 in response to 12-O-tetradecanoylphorbol 13-acetate treatment

Gap junctions are specialized plasma membrane domains enriched in connexin proteins that form channels between adjacent cells. Gap junctions are highly dynamic, and modulation of the connexin turnover rate is considered to play an important role in the regulation of gap junctional intercellular communication. In the present study, we show that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces ubiquitination of connexin-43 (Cx43) in IAR20 rat liver epithelial cells. The accelerated ubiquitination of Cx43 in response to TPA occurred concomitantly with Cx43 hyperphosphorylation and inhibition of cell-cell communication via gap junctions. The TPA-induced ubiquitination of Cx43 was mediated via protein kinase C and partly involved the mitogen-activated protein kinase pathway. Following ubiquitination, Cx43 was internalized and degraded. The loss of Cx43 protein was counteracted by ammonium chloride, indicating that acidification of internalized Cx43 gap junctions is a prerequisite for its degradation. Furthermore, the Cx43 degradation was partly counteracted by leupeptin, an inhibitor of cathepsin B, H, and L. Cx43 internalization and subsequent degradation were blocked by inhibitors of the proteasome. Evidence is provided that Cx43 is modified by multiple monoubiquitins rather than a polyubiquitin chain in response to TPA. Moreover, the TPA-induced ubiquitination of Cx43 was blocked by proteasomal inhibitors. Taken together, the data indicate that Cx43 ubiquitination is a highly regulated process. Moreover, the results suggest that the proteasome might play an indirect role in Cx43 degradation by affecting the level of monoubiquitin conjugation and trafficking of Cx43 to endosomal compartments.     

The ubiquitination of ribosomal S6 kinases is independent from the mitogen-induced phosphorylation/activation of the kinase

Ribosomal protein S6 kinase plays a critical role in the regulation of cell growth and energy metabolism. S6K belongs to the AGC family of serine/threonine kinases and is a downstream effector of the mTOR and PI3K signalling pathways. The activity and subcellular localisation of S6K are tightly controlled by phosphorylation/dephosphorylation events. We have recently demonstrated that steady-state levels of S6K isoforms, S6K1 and S6K2, are regulated by ubiquitination-mediated proteasomal degradation. In this study, we report for the first time that the ubiquitination status of S6K isoforms is coordinated by signalling pathways induced by mitogenic stimuli and extracellular stresses. The induction of signal transduction by serum and growth factors significantly increases the level of S6K ubiquitination, while the treatment of cells with UV and staurosporine has the opposite effect. Furthermore, we found that the phosphorylation/activation of S6Ks does not correlate directly with the induction of their ubiquitination in response to diverse cellular stimuli. This study suggests that the ubiquitination and subsequent proteasomal degradation of S6K are controlled by signalling pathways, which could possibly facilitate their association with the components of the ubiquitination machinery.     

Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.     

Activation of Protein Kinase C Triggers Its Ubiquitination and Degradation

Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms α, δ, and in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC δ. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC α, δ, and were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC α could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway.     

Regulation of stathmin phosphorylation in mouse liver progenitor‐29 cells during proteasome inhibition

Proteasome inhibitors are potential therapeutic agents in the treatment of hepatocarcinoma and other liver diseases. The analysis of alternative protein phosphorylation states might contribute to elucidate the underlying mechanisms of proteasome inhibitor‐induced apoptosis. We have investigated the response of mouse liver progenitor‐29 (MLP‐29) cells to MG132 using a combination of phosphoprotein affinity chromatography, DIGE, and nano LC‐MS/MS. Thirteen unique deregulated phosphoproteins involved in chaperone activity, stress response, mRNA processing and cell cycle control were unambiguously identified. Alterations in NDRG1 and stathmin suggest new mechanisms associated to proteasome inhibitor‐induced apoptosis in MLP‐29 cells. Particularly, a transient modification of the phosphorylation state of Ser¹⁶, Ser²⁵ and Ser³⁸, which are involved in the regulation of stathmin activity, was detected in three distinct isoforms upon proteasome inhibition. The parallel deregulation of calcium/calmodulin‐activated protein kinase II, extracellular regulated kinase‐1/2 and cyclin‐dependent kinase‐2, might explain the modified phosphorylation pattern of stathmin. Interestingly, stathmin phosphorylation profile was also modified in response to epoxomicin treatment, a more specific proteasome inhibitor. In summary, we report here data supporting that regulation of NDRG1 and stathmin by phosphorylation at specific Ser/Thr residues may participate in the cellular response induced by proteasome inhibitors.     

Rad23 and Rpn10: perennial wallflowers join the melee

Substrate ubiquitination is highly regulated; by contrast, substrate targeting to the proteasome has been considered a stochastic process that is mediated primarily by high-affinity interaction with multi-ubiquitin chains. However, recent findings have shown that substrate recognition by the proteasome is also regulated, and requires Rad23, Dsk2 and Rpn10. These studies suggest that the engagement of protein ubiquitination and translocation with degradation by the proteasome is coordinated by a series of regulated events.     

PKCdelta-dependent deubiquitination and stabilization of Gadd45 in A431 cells overexposed to EGF.

Epidermal growth factor (EGF) receptor-overexpressing p53-deficient A431 cells response to toxic dose of EGF by G1 arrest and apoptosis was studied. We previously reported an increased expression of growth arrest and DNA-damage-inducible gene, Gadd45, in EGF-overexposed A431 cells. The mechanism for this induction was increased half-lives of mRNA and protein. In this study, using phorbol ester (a PKC activator) and specific inhibitors of PKC isoforms, we showed that protein kinase C-delta (PKCdelta) was involved in the increase of Gadd45 protein stability. We further demonstrated that Gadd45 is ubiquitinated and is regulated by proteolysis. While EGF induced ubiquitination of total cellular proteins, there was a decrease in Gadd45 ubiquitination, which could be inhibited by Rottlerin, a PKCdelta-specific inhibitor. These results suggest that an increase in Gadd45 stability may involve PKCdelta-dependent ubiquitin-proteasome pathway.     

Identification of the Degradation Determinants of Insulin Receptor Substrate 1 for Signaling Cullin-RING E3 Ubiquitin Ligase 7-mediated Ubiquitination

Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase S6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain PI3K activity and plays critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser-307/Ser-312 and Ser-527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyperphosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1–260 led to substantial reduction of ubiquitination efficiency, suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and the N terminus of IRS1 for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.     

Regulation of RIPK3- and RHIM-dependent Necroptosis by the Proteasome

Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.