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.     

Tumor-exosomes and leukocyte activation: an ambivalent crosstalk

Background

ADP-ribosylation is a posttranslational modification catalyzed in cells by ADP-ribosyltransferases (ARTD or PARP enzymes). The ARTD family consists of 17 members. Some ARTDs modify their substrates by adding ADP-ribose in an iterative process, thereby synthesizing ADP-ribose polymers, the best-studied example being ARTD1/PARP1. Other ARTDs appear to mono-ADP-ribosylate their substrates and are unable to form polymers. The founding member of this latter subclass is ARTD10/PARP10, which we identified as an interaction partner of the nuclear oncoprotein MYC. Biochemically ARTD10 uses substrate-assisted catalysis to modify its substrates. Our previous studies indicated that ARTD10 may shuttle between the nuclear and cytoplasmic compartments. We have now addressed this in more detail.

Results

We have characterized the subcellular localization of ARTD10 using live-cell imaging techniques. ARTD10 shuttles between the cytoplasmic and nuclear compartments. When nuclear, ARTD10 can interact with MYC as measured by bimolecular fluorescence complementation. The shuttling is controlled by a Crm1-dependent nuclear export sequence and a central ARTD10 region that promotes nuclear localization. The latter lacks a classical nuclear localization sequence and does not promote full nuclear localization. Rather this non-conventional nuclear localization sequence results in an equal distribution of ARTD10 between the cytoplasmic and the nuclear compartments. ARTD10 forms discrete and dynamic bodies primarily in the cytoplasm but also in the nucleus. These contain poly-ubiquitin and co-localize in part with structures containing the poly-ubiquitin receptor p62/SQSTM1. The co-localization depends on the ubiquitin-associated domain of p62, which mediates interaction with poly-ubiquitin.

Conclusions

Our findings demonstrate that ARTD10 is a highly dynamic protein. It shuttles between the nuclear and cytosolic compartments dependent on a classical nuclear export sequence and a domain that mediates nuclear uptake. Moreover ARTD10 forms discrete bodies that exchange subunits rapidly. These bodies associate at least in part with the poly-ubiquitin receptor p62. Because this protein is involved in the uptake of cargo into autophagosomes, our results suggest a link between the formation of ARTD10 bodies and autophagy.

Lay abstract

Post-translational modifications refer to changes in the chemical appearance of proteins and occur, as the name implies, after proteins have been synthesized. These modifications frequently affect the behavior of proteins, including alterations in their activity or their subcellular localization. One of these modifications is the addition of ADP-ribose to a substrate from the cofactor NAD⁺. The enzymes responsible for this reaction are ADP-ribosyltransferases (ARTDs or previously named PARPs). Presently we know very little about the role of mono-ADP-ribosylation of proteins that occurs in cells. We identified ARTD10, a mono-ADP-ribosyltransferase, as an interaction partner of the oncoprotein MYC. In this study we have analyzed how ARTD10 moves within a cell. By using different live-cell imaging technologies that allow us to follow the position of ARTD10 molecules over time, we found that ARTD10 shuttles constantly in and out of the nucleus. In the cytosol ARTD10 forms distinct structures or bodies that themselves are moving within the cell and that exchange ARTD10 subunits rapidly. We have identified the regions within ARTD10 that are required for these movements. Moreover we defined these bodies as structures that interact with p62. This protein is known to play a role in bringing other proteins to a structure referred to as the autophagosome, which is involved in eliminating debris in cells. Thus our work suggests that ARTD10 might be involved in and is regulated by ADP-riboslyation autophagosomal processes.     

核孔蛋白p62促进非受体酪氨酸激酶c-Abl进入细胞核

非受体酪氨酸激酶c-Abl广泛表达于各组织细胞中,其序列高度保守,它的亚细胞定位与其功能密切相关。c-Abl借助其C端的3个核定位信号（NLS）和1个核输出信号（NES）完成细胞核一细胞质问的穿梭过程。关于c-Abl核-质穿梭的详细机制还不清楚。通过酵母双杂交系统,以人类Ib型c-Abl作为诱饵蛋白进行HeLa细胞eDNA文库的筛选,获得了可能在c-Abl核-质穿梭过程中具有调控怍用核孔蛋白p62。核孔复合物（NPC）是大分子物质进行核-质运输的惟一通道,p62是NPC的重要组成部分,它位于中央通道内侧,在许多物质的核-质穿梭过程中具有调节作用。免疫共沉淀和体外结合实验证实,c-Abl和p62之间具有相互作用,而且这种相互作用是通过c-Abl的SH3结构域与p62的P299位点之间的结合实现的;p62可被c-Abl部分磷酸化此外,在293和DKO细胞株中共转染c-Abl和p62,发现核内的c-Abl分布增多。以上结果表明,p62具有促进e-Abl进入细咆核的作用。     

A novel GTPase, CRAG, mediates promyelocytic leukemia protein-associated nuclear body formation and degradation of expanded polyglutamine protein

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG-polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin-proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.     

Evidence for cytosolic p27(Kip1) ubiquitylation and degradation during adipocyte hyperplasia

Objective: Subcellular localization has been shown to play an important role in determining activity and accumulation of p27 protein during cell cycle progression. The purpose of this study was to examine p27 localization and ubiquitylation in relation to E3 ligase expression during adipocyte hyperplasia. Research Methods and Procedures: This study used the murine 3T3‐L1 preadipocyte model to examine p27 regulation during synchronous cell cycle progression. Cell lysates were isolated over time after hormonal stimulation, fractionated to cytosolic and nuclear compartments, and immunoblotted for relative protein determinations. Results: Data presented in this study show that p27 was present in the cytosol and nucleus in density‐arrested preadipocytes and that abundance in both compartments decreased in a phase‐specific manner as preadipocytes synchronously re‐entered the cell cycle during early phases of adipocyte differentiation. Blocking CRM1‐mediated nuclear export did not prevent degradation, nor did it cause nuclear accumulation of p27, suggesting that distinct mechanisms mediating cytosolic and nuclear p27 degradation were involved. Treating preadipocytes with a potent and specific proteasome inhibitor during hormonal stimulation prevented Skp2 accumulation and p27¹⁸⁷ phosphorylation, which are essential events for SCF^Skp2 E3 ligase activity and nuclear p27 ubiquitylation during S/G₂ phase progression. Proteasome blockade also resulted in the first evidence of cytosolic p27 ubiquitylation during late G₁ phase as preadipocytes undergo the transition from quiescence to proliferation. Discussion: These data are consistent with the postulate that p27 is ubiquitylated and targeted for degradation by the 26S proteasome in a phase‐specific manner by distinct ubiquitin E3 ligases localized to the cytosol and nucleus during adipocyte hyperplasia.     

Nuclear and cytoplasmic shuttling of TRADD induces apoptosis via different mechanisms

The adapter protein tumor necrosis factor receptor (TNFR)1-associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.     

Caspase-activated PAK-2 is regulated by subcellular targeting and proteasomal degradation

p21-activated protein kinases (PAKs) are a family of serine/threonine protein kinases that are activated by binding of the p21 G proteins Cdc42 or Rac. The ubiquitous PAK-2 (gamma-PAK) is unique among the PAK isoforms because it is also activated through proteolytic cleavage by caspases or caspase-like proteases. In response to stress stimulants such as tumor necrosis factor alpha or growth factor withdrawal, PAK-2 is activated as a full-length enzyme and as a proteolytic PAK-2p34 fragment. Activation of full-length PAK-2 stimulates cell survival, whereas proteolytic activation of PAK-2p34 is involved in programmed cell death. Here we provide evidence that the proapoptotic effect of PAK-2p34 is regulated by subcellular targeting and degradation by the proteasome. Full-length PAK-2 is localized in the cytoplasm, whereas the proteolytic PAK-2p34 fragment translocates to the nucleus. Subcellular localization of PAK-2 is regulated by nuclear localization and nuclear export signal motifs. A nuclear export signal motif within the regulatory domain prevents nuclear localization of full-length PAK-2. Proteolytic activation removes most of the regulatory domain and disrupts the nuclear export signal. The activated PAK-2p34 fragment contains a nuclear localization signal and translocates to the nucleus. However, levels of activated PAK-2p34 are tightly regulated through ubiquitination and degradation by the proteasome. Inhibition of degradation by blocking polyubiquitination results in significantly increased levels of PAK-2p34 and as a consequence, in stimulation of programmed cell death. Therefore, nuclear targeting and inhibition of degradation appear to be critical for stimulation of the cell death response by PAK-2p34.     

Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called 'aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.     

Charge modification at multiple C-terminal lysine residues regulates p53 oligomerization and its nucleus-cytoplasm trafficking

The basal level of the tumor suppressor p53 is regulated by MDM2-mediated ubiquitination at specific lysines, which leads to p53 nuclear export and degradation. Upon p53 activation, however, these lysines become acetylated by p300/CREB-binding protein. Here we have reported an unexpected finding that p300-mediated acetylation also regulates p53 subcellular localization and can promote cytoplasmic localization of p53. This activity is independent of MDM2 but requires a p53 nuclear export signal and acetylation of multiple lysines by p300. Mechanistically, we showed that conversion of a minimal four of these lysines to alanines but not arginines mimics p300-mediated p53 nuclear export, and these lysine-neutralizing mutations effectively prevent p53 tetramerization, thus exposing the oligomerization-regulated nuclear export signal. Our study suggested a threshold mechanism whereby the degree of acetylation regulates p53 nucleus-cytoplasm trafficking by neutralizing a lysine-dependent charge patch, which in turn, controls oligomerization-dependent p53 nuclear export.     

Control of the localization and function of a miRNA silencing component TNRC6A by Argonaute protein

GW182 family proteins play important roles in microRNA (miRNA)-mediated RNA silencing. They directly interact with Argonaute (Ago) proteins in processing bodies (P bodies), cytoplasmic foci involved in mRNA degradation and storage. Recently, we revealed that a human GW182 family protein, TNRC6A, is a nuclear-cytoplasmic shuttling protein, and its subcellular localization is regulated by its own nuclear localization signal and nuclear export signal. Regarding the further controlling mechanism of TNRC6A subcellular localization, we found that TNRC6A protein is tethered in P bodies by direct interaction with Ago2 under Ago2 overexpression condition in HeLa cells. Furthermore, it was revealed that such Ago proteins might be strongly tethered in the P bodies through Ago-bound small RNAs. Thus, our results indicate that TNRC6A subcellular localization is substantially controlled by the interaction with Ago proteins. Furthermore, it was also revealed that the TNRC6A subcellular localization affects the RNA silencing activity.     

The MDM2 RING-finger domain is required to promote p53 nuclear export

MDM2 can bind to p53 and promote its ubiquitination and subsequent degradation by the proteasome. Current models propose that nuclear export of p53 is required for MDM2-mediated degradation, although the function of MDM2 in p53 nuclear export has not been clarified. Here we show that MDM2 can promote the nuclear export of p53 in transiently transfected cells. This activity requires the nuclear-export signal (NES) of p53, but not the NES of MDM2. A mutation within the MDM2 RING-finger domain that inhibits p53 ubiquitination also inhibits the ability of MDM2 to promote p53 nuclear export. Finally, inhibition of nuclear export stabilizes wild-type p53 and leads to accumulation of ubiquitinated p53 in the nucleus. Our results indicate that MDM2-mediated ubiquitination, or other activities associated with the RING-finger domain, can stimulate the export of p53 to the cytoplasm through the activity of the p53 NES.     

Arsenic-induced SUMO-dependent recruitment of RNF4 into PML nuclear bodies

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML.     

The nuclear dot protein sp100, characterization of domains necessary for dimerization, subcellular localization, and modification by small ubiquitin-like modifiers

The Sp100 and promyelocytic leukemia proteins (PML) are constituents of nuclear domains, known as nuclear dots (NDs) or PML bodies, and are both covalently modified by the small ubiquitin-related protein SUMO-1. NDs play a role in autoimmunity, virus infections, and in the etiology of acute promyelocytic leukemia. To date, little is known about the function of the Sp100 protein. Here we analyzed Sp100 domains that determine its subcellular localization, dimerization, and SUMOylation. A functional nuclear localization signal and an ND-targeting region that coincides with an Sp100 homodimerization domain were mapped. Sequences similar to the Sp100 homodimerization/ND-targeting region occur in several other proteins and constitute a novel protein motif, termed HSR domain. The lysine residue of the Sp100 protein, to which SUMO-1 is covalently linked, was mapped within and may therefore modulate the previously described HP1 protein-binding site. A consensus sequence for SUMOylation of proteins in general is suggested. SUMOylation strictly depended on a functional nuclear localization signal but was not necessary for nuclear import or ND targeting. A three-dimensional structure of Sp100, which supports the mapping data and provides additional information on Sp100 structure/function relationships, was generated by computer modeling. Taken together, our studies indicate the existence of well defined Sp100 domains with functions in ND targeting, nuclear import, nuclear SUMOylation, and protein-protein interaction.     

Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus.

The PML protein, identified first as part of the oncogenic PML-RARalpha chimera in acute promyelocytic leukemia (APL), concentrates within discrete subnuclear structures, corresponding to some types of nuclear bodies. These structures are disrupted in APL cells, and retinoic acid (RA) can trigger their reorganization, correlating with its therapeutic effect in this type of leukemia. Recently, arsenic trioxide (As2O3) was identified as a potent antileukemic agent which, similarly to RA, induces complete remissions in APL patients. Here we show that, in APL cells, As2O3 triggers rapid degradation of PML-RARalpha and provokes the restoration of intact nuclear bodies. In non-APL cells, the ubiquitin-like protein SUMO-1 is covalently attached to a subset of wild-type PML in a reversible and phosphorylation-dependent manner. The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO-1-polymodified forms of PML are compartmentalized exclusively in the PML nuclear bodies. As2O3 administration strikingly increases the pool of SUMO-1-PML conjugates that, subsequently, accumulate in enlarged nuclear bodies. In contrast to PML-RARalpha, the overall amount of PML seems to remain unaltered up to 36 h following As2O3 treatment. These findings indicate that the conjugation of PML with SUMO-1 modulates its intracellular localization and suggest that post-translational modification by SUMO-1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures. They provide additional evidence that the role of 'ubiquitin-like' post-translational modification is not limited to a degradation signal.     

Competition between sumoylation and ubiquitination of serine hydroxymethyltransferase 1 determines its nuclear localization and its accumulation in the nucleus

Serine hydroxymethyltransferase 1 (SHMT1) expression limits rates of de novo dTMP synthesis in the nucleus. Here we report that SHMT1 is ubiquitinated at the small ubiquitin-like modifier (SUMO) consensus motif and that ubiquitination at that site is required for SHMT1 degradation. SHMT1 protein levels are cell cycle-regulated, and Ub-SHMT1 levels are lowest at S phase when SHMT1 undergoes SUMO modification and nuclear transport. Mutation of the SUMO consensus motif increases SHMT1 stability. SHMT1 interacts with components of the proteasome in both the nucleus and cytoplasm, indicating that degradation occurs in both compartments. Ubc13-mediated ubiquitination is required for SHMT1 nuclear export and increases stability of SHMT1 within the nucleus, whereas Ubc9-mediated modification with Sumo2/3 is involved in nuclear degradation. These data demonstrate that SUMO and ubiquitin modification of SHMT1 occurs on the same lysine residue and determine the localization and accumulation of SHMT1 in the nucleus.     

NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets

Autophagy is an evolutionary conserved cell survival process for degradation of long-lived proteins, damaged organelles and protein aggregates. The mammalian proteins p62 and NBR1 are selectively degraded by autophagy and can act as cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates. Despite differing in size and primary sequence, both proteins share a similar domain architecture containing an N-terminal PB1 domain, a LIR motif interacting with ATG8 family proteins, and a C-terminal UBA domain interacting with ubiquitin. The LIR motif is essential for their autophagic degradation, indicating that ATG8 family proteins are responsible for the docking of p62 and NBR1 to nucleating autophagosomes. p62 and NBR1 co-operate in the sequestration of misfolded and ubiquitinated proteins in p62 bodies and are both required for their degradation by autophagy. Here we discuss the role of p62 and NBR1 in degradation of ubiquitinated cargoes and the putative role of LIR as a general motif for docking of proteins to ATG8 family proteins.