Autophagic proteolysis: control and specificity

The rate of proteolysis is an important determinant of the intracellular protein content. Part of the degradation of intracellular proteins occurs in the lysosomes and is mediated by macroautophagy. In liver, macroautophagy is very active and almost completely accounts for starvation-induced proteolysis. Factors inhibiting this process include amino acids, cell swelling and insulin. In the mechanisms controlling macroautophagy, protein phosphorylation plays an important role. Activation of a signal transduction pathway, ultimately leading to phosphorylation of ribosomal protein S6, accompanies inhibition of macroautophagy. Components of this pathway may include a heterotrimeric Gi3-protein, phosphatidylinositol 3- kinase and p70S6 kinase. Recent evidence indicates that lysosomal protein degradation can be selective and occurs via ubiquitin- dependent and -independent pathways. This revised version was published online in November 2006 with corrections to the Cover Date.     

Amino acids and insulin control autophagic proteolysis through different signaling pathways in relation to mTOR in isolated rat hepatocytes

Autophagy, a major bulk proteolytic pathway, contributes to intracellular protein turnover, together with protein synthesis. Both are subject to dynamic control by amino acids and insulin. The mechanisms of signaling and cross-talk of their physiological anabolic effects remain elusive. Recent studies established that amino acids and insulin induce p70 S6 kinase (p70(S6k)) phosphorylation by mTOR, involved in translational control of protein synthesis. Here, the signaling mechanisms of amino acids and insulin in macroautophagy in relation to mTOR were investigated. In isolated rat hepatocytes, both regulatory amino acids (RegAA) and insulin coordinately activated p70(S6k) phosphorylation, which was completely blocked by rapamycin, an mTOR inhibitor. However, rapamycin blocked proteolytic suppression by insulin, but did not block inhibition by RegAA. These contrasting results suggest that insulin controls autophagy through the mTOR pathway, but amino acids do not. Furthermore, micropermeabilization with Saccharomyces aureus alpha-toxin completely deprived hepatocytes of proteolytic responsiveness to RegAA and insulin, but still maintained p70(S6k) phosphorylation by RegAA. In contrast, Leu(8)-MAP, a non-transportable leucine analogue, did not mimic the effect of leucine on p70(S6k) phosphorylation, but maintained the activity on proteolysis. Finally, BCH, a System L-specific amino acid, did not affect proteolytic suppression or mTOR activation by leucine. All the results indicate that mTOR is not common to the signaling mechanisms of amino acids and insulin in autophagy, and that the amino acid signaling starts extracellularly with their "receptor(s)," probably other than transporters, and is mediated through a novel route distinct from the mTOR pathway employed by insulin.     

Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells

Activation of ERK1/2 stimulates macroautophagy in the human colon cancer cell line HT-29 by favoring the phosphorylation of the Galpha-interacting protein (GAIP) in an amino acid-dependent manner (Ogier-Denis, E., Pattingre, S., El Benna, J., and Codogno, P. (2000) J. Biol. Chem. 275, 39090-39095). Here we show that ERK1/2 activation by aurintricarboxylic acid (ATA) treatment induces the phosphorylation of GAIP in an amino acid-dependent manner. Accordingly, ATA challenge increased the rate of macroautophagy, whereas epidermal growth factor did not significantly affect macroautophagy and GAIP phosphorylation status. In fact, ATA activated the ERK1/2 signaling pathway, whereas epidermal growth factor stimulated both the ERK1/2 pathway and the class I phosphoinositide 3-kinase pathway, known to decrease the rate of macroautophagy. Amino acids interfered with the ATA-induced macroautophagy by inhibiting the activation of the kinase Raf-1. The role of the Ras/Raf-1/ERK1/2 signaling pathway in the GAIP- and amino acid-dependent control of macroautophagy was confirmed in HT-29 cells expressing the Ras(G12V,T35S) mutant. Similar to the protein phosphatase 2A inhibitor okadaic acid, amino acids sustained the phosphorylation of Ser(259), which is involved in the negative regulation of Raf-1. In conclusion, these results add a novel target to the amino acid signaling-dependent control of macroautophagy in intestinal cells.     

Immune surveillance via self digestion

The adaptive immune system is orchestrated by CD4+ T cells. These cells detect peptides presented on Major Histocompatibility Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4+ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4+ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.     

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.     

Human papillomavirus 16 E6 oncoprotein interferences with insulin signaling pathway by binding to tuberin

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either TSC1 or TSC2 tumor suppressor gene. TSC1 and TSC2 products, Harmatin and Tuberin, form the functional complex to serve as the negative regulator for insulin-induced phosphorylation of S6 kinase and elF4E-binding protein 1. High-risk human papillomavirus (HPV) infection is the necessary cause for cervical cancer. E6 oncoprotein encoded by HPV plays a pivotal role in carcinogenesis by interference with the host intracellular protein functions. In this study, we show that HPV16 E6 interacts with tumor suppressor gene TSC2 product, Tuberin, and results in the phosphorylation of S6 kinase and S6 even in the absence of insulin. The overexpression of Tuberin overcomes the effect of E6 on S6 kinase phosphorylation. Binding with HPV16 E6 causes the proteasome-mediated degradation of Tuberin. A DILG motif and an ELVG motif located in the carboxyl-terminal of Tuberin are required for E6 binding. In addition, the Tuberin interaction region in E6 has been mapped in the amino-terminal portion of HPV16 E6, which is different from the binding domain with p53. These results provide a possible link between E6-induced oncogenesis and the insulin-stimulated cell proliferation signaling pathway.     

Autophagy-mediated clearance of huntingtin aggregates triggered by the insulin-signaling pathway

Conditional mouse models of polyglutamine diseases, such as Huntington's disease (HD), have revealed that cells can clear accumulated pathogenic proteins if the continuous production of the mutant transgene is halted. Invariably, the clearance of the protein leads to regression of the disease symptoms in mice. In light of these findings, it is critical to determine the pathway responsible for alleviating this protein accumulation to define targets to fight these diseases. In a functional genetic screen of HD, we found that activation of insulin receptor substrate-2, which mediates the signaling cascades of insulin and insulin-like growth factor 1, leads to a macroautophagy-mediated clearance of the accumulated proteins. The macroautophagy is triggered despite activation of Akt, mammalian target of rapamycin (mTOR), and S6 kinase, but still requires proteins previously implicated in macroautophagy, such as Beclin1 and hVps34. These findings indicate that the accumulation of mutant protein can lead to mTOR-independent macroautophagy and that lysosome-mediated degradation of accumulated protein differs from degradation under conditions of starvation.     

Akt-dependent cell size regulation by the adhesion molecule on glia occurs independently of phosphatidylinositol 3-kinase and Rheb signaling

The role of cell adhesion molecules in mediating interactions with neighboring cells and the extracellular matrix has long been appreciated. More recently, these molecules have been shown to modulate intracellular signal transduction cascades critical for cell growth and proliferation. Expression of adhesion molecule on glia (AMOG) is downregulated in human and mouse gliomas, suggesting that AMOG may be important for growth regulation in the brain. In this report, we examined the role of AMOG expression on cell growth and intracellular signal transduction. We show that AMOG does not negatively regulate cell growth in vitro or in vivo. Instead, expression of AMOG in AMOG-deficient cells results in a dramatic increase in cell size associated with protein kinase B/Akt hyperactivation, which occurs independent of phosphatidylinositol 3-kinase activation. AMOG-mediated Akt phosphorylation specifically activates the mTOR/p70S6 kinase pathway previously implicated in cell size regulation, but it does not depend on tuberous sclerosis complex/Ras homolog enriched in brain (Rheb) signaling. These data support a novel role for a glial adhesion molecule in cell size regulation through selective activation of the Akt/mTOR/S6K signal transduction pathway.     

Phosphorylation and degradation of S6K1 (p70S6K1) in response to persistent JNK1 Activation

S6K (ribosomal S6 kinase p70, p70S6K) activation requires phosphorylation at two stages. The first phosphorylation is independent of insulin stimulation and mediated by an unknown kinase. The second phosphorylation is mediated by mTOR in insulin dependent manner. In this study, we identified JNK1 (c-Jun N-terminal kinase 1) as a kinase in the first phosphorylation. S6K protein was phosphorylated by JNK1 at S411 and S424 in the carboxyl terminal autoinhibitory domain. The phosphorylation was observed in kinase assay with purified S6K as a substrate, and in cells after JNK1 activation by TNF-α or MEKK1 expression. The phosphorylation was detected in JNK2 null cells, but not in JNK1 null cells after TNF-α treatment. When JNK1 activation was inhibited by MKK7 knockdown, the phosphorylation was blocked in cells. The phosphorylation led to S6K protein degradation in NF-κB deficient cells. The degradation was blocked by inhibition of proteasome activity with MG132. In wide type cells, the phosphorylation did not promote S6K degradation when IKK2 (IKKβ, IκB kinase beta) was activated. Instead, the phosphorylation allowed S6K activation by mTOR, which stabilizes S6K protein. In IKK2 null cells or cells treated by IKK2 inhibitor, the phosphorylation led to S6K degradation. These data suggest that S6K is phosphorylated by JNK1 and the phosphorylation makes S6K protein unstable in the absence of IKK2 activation. This study provides a mechanism for regulation of S6K protein stability.     

Regulation of the localization and stability of Cdc6 in living yeast cells

The Cdc6 protein is an essential regulator for initiation of DNA replication. Following the G1/S transition, Cdc6 is degraded through a ubiquitin-mediated proteolysis pathway. In this study, we tagged Cdc6 with green fluorescent protein (GFP) and used site-specific mutations to study the regulation of Cdc6 localization and degradation in living yeast cells. Our major findings are: (1). Cdc6-GFP distributes predominantly in the nucleus in all cell cycle stages, with a small increase in cytoplasmic localization in G2/M cells. (2). This nuclear localization is critical for Cdc6 degradation. When the N-terminal nuclear localization signal (NLS) was mutated, Cdc6-GFP no longer accumulated in the nucleus, and the mutant cdc6 was stabilized compared to wild type. (3). The putative CDK phosphorylation sites are not required for Cdc6 nuclear localization, but are important for protein stability. These observations suggest that the stability of Cdc6 protein is regulated by two factors: nuclear localization and phosphorylation by CDK1.     

Ligand-Stimulated VEGFR2 Signaling is Regulated by Co-Ordinated Trafficking and Proteolysis

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.     

Interaction between c-Abl and Arg tyrosine kinases and proteasome subunit PSMA7 regulates proteasome degradation

Proteasome-mediated proteolysis is a primary protein degradation pathway in cells. The present study demonstrates that c-Abl and Arg (abl-related gene) tyrosine kinases associate with and phosphorylate the proteasome PSMA7 (alpha4) subunit at Tyr-153. Consequently, proteasome-dependent proteolysis is compromised. Notably, cells expressing a phosphorylation mutant of PSMA7(Y153F) display impaired G1/S transition and S/G2 progression, highlighting the biological significance of tyrosine phosphorylation of a proteasome subunit as an important cellular regulatory control.     

Immune Surveillance via Self Digestion

The adaptive immune system is orchestrated by CD4⁺ T cells. These cells detect peptides presented on Major Histocompatiblity Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4⁺ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4⁺ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.

Addendum to:

MHC Class II Antigen Loading Compartments Continuously Receive Input from Autophagosomes

Dorothee Schmid, Marc Pypaert and Christian Münz

Immunity 2006; In press     

Protein phosphorylation and protein kinase activities in BC3H-1 myocytes. Differences between the effects of insulin and phorbol esters

To determine whether insulin activates protein kinase C in BC3H-1 myocytes, we evaluated changes in protein phosphorylation, protein kinase activities, and the intracellular translocation of protein kinase C activity in response to insulin and phorbol esters. Phorbol 12-myristate 13-acetate (PMA), but not insulin, stimulated the phosphorylation of an acidic Mr 80,000 protein which has been shown to be an apparently specific marker for protein kinase C activation. In addition, PMA, but not insulin, stimulated the rapid association of protein kinase C activity with a cellular particulate fraction. In contrast to these differences, both insulin and PMA stimulated the phosphorylation of ribosomal protein S6 and activated a ribosomal protein S6 kinase in cell-free extracts from cells exposed to these agents. In cells exposed to high concentrations of PMA for 16 h, protein kinase C activity and immunoreactivity were abolished, without changes in cellular morphology. Under these conditions, insulin, but not PMA, stimulated phosphorylation of the ribosomal protein S6 in intact cells and activated the S6 kinase in cell-free extracts derived from insulin-treated intact cells. We conclude that: insulin does not appear to activate protein kinase C in BC3H-1 myocytes, at least as assessed by phosphorylation of the Mr 80,000 protein; both insulin and PMA activate an S6 protein kinase in these cells; and insulin can promote S6 phosphorylation and activate the S6 kinase normally in protein kinase C-deficient cells. Activation of the S6 kinase by insulin and PMA, although apparently proceeding through different mechanisms, may explain some of the similar biological actions of these compounds in BC3H-1 myocytes.     

An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin

beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/betaTrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibited its association with FWD1. FWD1 facilitated ubiquitination and promoted degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability.     

Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells

Macroautophagy is a process by which cytoplasmic content and organelles are sequestered by double-membrane bound vesicles and subsequently delivered to lysosomes for degradation. Macroautophagy serves as a major intracellular pathway for protein degradation and as a pro-survival mechanism in time of stress by generating nutrients. In the present study, bafilomycin A₁, a vacuolar type H⁺-ATPase inhibitor, suppresses macroautophagy by preventing acidification of lysosomes in colon cancer cells. Diminished macroautophagy was evidenced by the accumulation of undegraded LC3 protein. Suppression of macroautophagy by bafilomycin A₁ induced G₀/G₁ cell cycle arrest and apoptosis which were accompanied by the down-regulation of cyclin D₁ and cyclin E, the up-regulation of p21^Cip1 as well as cleavages of caspases-3, -7, -8, and -9 and PARP. Further investigation revealed that bafilomycin A₁ increased the phosphorylation of ERK, JNK, and p38. In this regard, p38 inhibitor partially reversed the anti-proliferative effect of bafilomycin A₁. To conclude, inhibition of macroautophagy by bafilomycin A₁ lowers G₁-S transition and induces apoptosis in colon cancer cells. Our results not only indicate that inhibitors of macroautophagy may be used therapeutically to inhibit cancer growth, but also delineate the relationship between macroautophagy and apoptosis.     

S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover

A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR. signal transduction; atrophy; autophagy     

Regulation of protein stability by GSK3 mediated phosphorylation

Glycogen synthase kinase-3 (GSK3) plays important roles in numerous signaling pathways that regulate a variety of cellular processes including cell proliferation, differentiation, apoptosis and embryonic development. In the canonical Wnt signaling pathway, GSK3 phosphorylation mediates proteasomal targeting and degradation of β-catenin via the destruction complex. We recently reported a biochemical screen that discovered multiple additional protein substrates whose stability is regulated by Wnt signaling and/or GSK3 and these have important implications for Wnt/GSK3 regulation of different cellular processes.1 In this article, we also present a bio-informatics based screen for proteins whose stability may be controlled by GSK3 and β-Trcp, the SCF E3 ubiquitin ligase that is responsible for β-catenin degradation in the Wnt signaling pathway. Furthermore, we review various GSK3 regulated proteolysis substrates described in the literature. We propose that GSK3 phosphorylation dependent proteolysis is a widespread mechanism that the cell employs to regulate a variety of cell processes in response to signals.     

Involvement of phosphoinositide 3-kinase and Akt in the induction of muscle protein degradation by proteolysis-inducing factor

In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser(473) within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin-proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin-proteasome pathway through activation of NF-kappaB (nuclear factor kappaB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-kappaB and that this also allows for a specific synthesis of proteasome subunits.