Regulation of the autophagy protein LC3 by phosphorylation

Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP⁺) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity.     

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.     

Activation of cellular Arf GTPases by poliovirus protein 3CD correlates with virus replication

We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.     

Activated CRH receptors inhibit autophagy by repressing conversion of LC3BI to LC3BII

Clinical studies have elucidated the negative correlation between microtubules-associated protein 1 light chain 3-B (LC3B) protein expression and overall survival of breast cancer patients. Our previous data demonstrated corticortropin-releasing hormone family (CRHs) suppressed migration of breast cancer cells via CRH receptors (CRHRs). Here, we showed that the activation of CRHRs (CRHR1 and CRHR2) remarkably reduced the conversion of LC3BI to LC3BII and hence repressed macroautophagy/autophagy, resulting in migration inhibition. By means of RT-4 cells (expressing higher CRHR1) with stable CRHR1 silence which was constructed by lentivirus with short hairpin RNA, we further confirmed CRH-inhibited LC3BII conversion. Using CRHRs agonists and antagonists, we found CRHRs triggered a marked reduction in the number of LC3B dots in both RT-4 and Hela cells(expressing higher CRHR2) which stably express RFP-GFP-LC3B. Of note, this decreased amount of autophagosome was associated with activation of Phospholipase C β (PLCβ)-Inositol triphosphate (IP₃)-mTOR signaling. Earle's Balanced Salt Solution (EBSS) decreased the expression of the key focal adhesion protein, paxillin, which was recovered by CRHRs ligands (CRH and UCN2). The effect of CRHRs ligands on paxillin resulted in the suppression of cell migration. Altogether, these data reveal a new link between CRHRs signaling and autophagy, and may help to envisage therapeutic strategies in cancer cell invasion.     

LC3 conjugation system in mammalian autophagy

Autophagy is the bulk degradation of proteins and organelles, a process essential for cellular maintenance, cell viability, differentiation and development in mammals. Autophagy has significant associations with neurodegenerative diseases, cardiomyopathies, cancer, programmed cell death, and bacterial and viral infections. During autophagy, a cup-shaped structure, the preautophagosome, engulfs cytosolic components, including organelles, and closes, forming an autophagosome, which subsequently fuses with a lysosome, leading to the proteolytic degradation of internal components of the autophagosome by lysosomal lytic enzymes. During the formation of mammalian autophagosomes, two ubiquitylation-like modifications are required, Atg12-conjugation and LC3-modification. LC3 is an autophagosomal ortholog of yeast Atg8. A lipidated form of LC3, LC3-II, has been shown to be an autophagosomal marker in mammals, and has been used to study autophagy in neurodegenerative and neuromuscular diseases, tumorigenesis, and bacterial and viral infections. The other Atg8 homologues, GABARAP and GATE-16, are also modified by the same mechanism. In non-starved rats, the tissue distribution of LC3-II differs from those of the lipidated forms of GABARAP and GATE-16, GABARAP-II and GATE-16-II, suggesting that there is a functional divergence among these three modified proteins. Delipidation of LC3-II and GABARAP-II is mediated by hAtg4B. We review the molecular mechanism of LC3-modification, the crosstalk between LC3-modification and mammalian Atg12-conjugation, and the cycle of LC3-lipidation and delipidation mediated by hAtg4B, as well as recent findings concerning the other two Atg8 homologues, GABARAP and GATE-16. We also highlight recent findings regarding the pathobiology of LC3-modification, including its role in microbial infection, cancer and neuromuscular diseases.     

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.     

MiR-204 regulates cardiomyocyte autophagy induced by ischemia-reperfusion through LC3-II

Background  

Autophagy plays a significant role in myocardial ischemia-reperfusion (IR) injury. So it is important to inhibit autophagy to protect cardiomyocytes besides anti-apoptosis. MiRNA has been demonstrated to protect cardiomyocytes against apoptosis during IR, while whether it has anti-autophagy effect has not been known. The aim of this study was to investigate whether miR-204 regulated autophagy by regulating LC3-II protein, which is the marker of autophagosome during myocardial IR injury.     

Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.     

Protein kinase C inhibits autophagy and phosphorylates LC3

During autophagy, the microtubule-associated protein light chain 3 (LC3), a specific autophagic marker in mammalian cells, is processed from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In HEK293 cells stably expressing FLAG-tagged LC3, activation of protein kinase C inhibited the autophagic processing of LC3-I to LC3-II induced by amino acid starvation or rapamycin. PKC inhibitors dramatically induced LC3 processing and autophagosome formation. Unlike autophagy induced by starvation or rapamycin, PKC inhibitor-induced autophagy was not blocked by the PI-3 kinase inhibitor wortmannin. Using orthophosphate metabolic labeling, we found that LC3 was phosphorylated in response to the PKC activator PMA or the protein phosphatase inhibitor calyculin A. Furthermore, bacterially expressed LC3 was directly phosphorylated by purified PKC in vitro. The sites of phosphorylation were mapped to T6 and T29 by nanoLC-coupled tandem mass spectrometry. Mutations of these residues significantly reduced LC3 phosphorylation by purified PKC in vitro. However, in HEK293 cells stably expressing LC3 with these sites mutated either singly or doubly to Ala, Asp or Glu, autophagy was not significantly affected, suggesting that PKC regulates autophagy through a mechanism independent of LC3 phosphorylation.     

Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells

Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth properties and characteristics very similar to those of the wild-type, untagged virus. Extracts prepared from the infected cells were subjected to immunoaffinity purification of the tagged proteins by adsorption to commercial antibody-linked beads and examined after elution for cellular and other viral proteins that remained bound to 3A sequences during purification. Viral proteins 2C, 2BC, 3D, and 3CD were detected in all three immunopurified 3A samples. Among the cellular proteins previously reported to interact with 3A either directly or indirectly, neither LIS1 nor phosphoinositol-4 kinase (PI4K) were detected in any of the purified tagged 3A samples. However, the guanine nucleotide exchange factor GBF1, which is a key regulator of membrane trafficking in the cellular protein secretory pathway and which has been shown previously to bind enteroviral protein 3A and to be required for viral RNA replication, was readily recovered along with immunoaffinity-purified 3A-FLAG. Surprisingly, we failed to cocapture GBF1 with 3A-HA or 3A-myc proteins. A model for variable binding of these 3A mutant proteins to GBF1 based on amino acid sequence motifs and the resulting practical and functional consequences thereof are discussed.     

PEBP1, a RAF kinase inhibitory protein,negatively regulates starvation-induced autophagy by direct interaction with LC3

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.     

LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization

Autophagy is an intracellular bulk degradation system, through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Microtuble-associated protein light chain 3 (LC3), a mammalian homolog of yeast Atg8, has been used as a specific marker to monitor autophagy. Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine and targeted to autophagic membranes. Therefore, changes in LC3 localization have been used to measure autophagy. However, this method has some limitations. In this report, we show that LC3 protein tends to aggregate in an autophagy-independent manner when it is transiently overexpressed by transfection. In addition, LC3 is easily incorporated into intracellular protein aggregates, such as inclusion bodies induced by polyQ expression or formed in autophagy-deficient hepatocytes, neurons, or senescent fibroblasts. These findings demonstrate that punctate dots containing LC3 do not always represent autophagic structures. Therefore, LC3 localization should be carefully interpreted, particularly if LC3 is overexpressed by transient transfection or if aggregates are formed within cells.     

Modification of the poliovirus capsid by ultraviolet light

Ultraviolet (UV) irradiation of type I poliovirus resulted in a modified (M) particle that had lost infectivity, lacked ability to adsorb to HeLa cells, lacked VP4, and reduced in S value. Additional irradiation resulted in the loss of VP2, further reduction in S value, and permeability of the capsid to RNAse, This particle (C) as well as M contain the genome. Acid pH (5.5-65) and sulfhydryl-reducing substances (dithiothreitol. reduced glutathione, and L-cysteine) inhibited UV-induced modification of the capsid. UV irradiation at alkaline pH (7.5-8.5) resulted in more extensive modification of the capsid than irradiation at neutral pH. Ionic compounds were found to inhibit the modifying reaction.     

LC3B is indispensable for selective autophagy of p62 but not basal autophagy

Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.     

Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. Bnip3 is an atypical BH3-only protein that is known to cause mitochondrial dysfunction and cell death. Interestingly, Bnip3 can also protect against cell death by inducing mitochondrial autophagy. The mechanism for this process, however, remains poorly understood. Bnip3 contains a C-terminal transmembrane domain that is essential for homodimerization and proapoptotic function. In this study, we show that homodimerization of Bnip3 is also a requirement for induction of autophagy. Several Bnip3 mutants that do not interfere with its mitochondrial localization but disrupt homodimerization failed to induce autophagy in cells. In addition, we discovered that endogenous Bnip3 is localized to both mitochondria and the endoplasmic reticulum (ER). To investigate the effects of Bnip3 at mitochondria or the ER on autophagy, Bnip3 was targeted specifically to each organelle by substituting the Bnip3 transmembrane domain with that of Acta or cytochrome b(5). We found that Bnip3 enhanced autophagy in cells from both sites. We also discovered that Bnip3 induced removal of both ER (ERphagy) and mitochondria (mitophagy) via autophagy. The clearance of these organelles was mediated in part via binding of Bnip3 to LC3 on the autophagosome. Although ablation of the Bnip3-LC3 interaction by mutating the LC3 binding site did not impair the prodeath activity of Bnip3, it significantly reduced both mitophagy and ERphagy. Our data indicate that Bnip3 regulates the apoptotic balance as an autophagy receptor that induces removal of both mitochondria and ER.     

Subversion of cellular autophagy by Anaplasma phagocytophilum

Anaplasma phagocytophilum , the causative agent of human granulocytic anaplasmosis, is an obligatory intracellular pathogen. After entry into host cells, the bacterium is diverted from the endosomal pathway and replicates in a membrane-bound compartment devoid of endosomal or lysosomal markers. Here, we show that several hallmarks of early autophagosomes can be identified in A. phagocytophilum replicative inclusions, including a double-lipid bilayer membrane and colocalization with GFP-tagged LC3 and Beclin 1, the human homologues of Saccharomyces cerevisiae autophagy-related proteins Atg8 and Atg6 respectively. While the membrane-associated form of LC3, LC3-II, increased during A. phagocytophilum infection, A. phagocytophilum -containing inclusions enveloped with punctate GFP-LC3 did not colocalize with a lysosomal marker. Stimulation of autophagy by rapamycin favoured A. phagocytophilum infection. Inhibition of the autophagosomal pathway by 3-methyladenine did not inhibit A. phagocytophilum internalization, but reversibly arrested its growth. Although autophagy is considered part of the innate immune system that clears a variety of intracellular pathogens, our study implies that A. phagocytophilum subverts this system to establish itself in an early autophagosome-like compartment segregated from lysosomes to facilitate its proliferation.     

p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related gamma-aminobutyrate receptor-associated protein and gamma-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 microm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.     

Selective MAP1LC3C (LC3C) autophagy requires noncanonical regulators and the C-terminal peptide

LC3s are canonical proteins necessary for the formation of autophagosomes. We have previously established that two paralogs, LC3B and LC3C, have opposite activities in renal cancer, with LC3B playing an oncogenic role and LC3C a tumor-suppressing role. LC3C is an evolutionary late gene present only in higher primates and humans. Its most distinct feature is a C-terminal 20-amino acid peptide cleaved in the process of glycine 126 lipidation. Here, we investigated mechanisms of LC3C-selective autophagy. LC3C autophagy requires noncanonical upstream regulatory complexes that include ULK3, UVRAG, RUBCN, PIK3C2A, and a member of ESCRT, TSG101. We established that postdivision midbody rings (PDMBs) implicated in cancer stem-cell regulation are direct targets of LC3C autophagy. LC3C C-terminal peptide is necessary and sufficient to mediate LC3C-dependent selective degradation of PDMBs. This work establishes a new noncanonical human-specific selective autophagic program relevant to cancer stem cells.