The effects of the polyglutamine repeat protein ataxin-1 on the UbL-UBA protein A1Up

The ataxin-1 interacting ubiquitin-like protein (A1Up) contains an amino-terminal ubiquitin-like (UbL) region, four stress-inducible, heat shock chaperonin-binding motifs (STI1), and an ubiquitin-associated domain (UBA) at the carboxyl terminus of A1Up. Although proteins that have both an UbL and UBA domain are thought to play a crucial role in proteasome-mediated activities, few are characterized, except for hHR23A/B. Similar to other UbL-containing proteins, the UbL of A1Up is essential for the interaction of A1Up with the S5a subunit of the 19S proteasome. Importantly, the interaction with the 19S proteasome was disrupted in the presence of the polyglutamine repeat protein, ataxin-1. The UbL domain of A1Up is ubiquitinated by both Lys(48)-linked and Lys(63)-linked chains. Intact A1Up is stable, suggesting that ubiquitination of A1Up is important for degradation-independent targeting of A1Up to the 19S proteasome. The UBA domain of A1Up binds polyubiquitin chains and has a role in the stability of A1Up and in the subcellular localization of A1Up. When the UBA domain was deleted, the localization of A1Up was entirely cytoplasmic, and it co-localized with the proteasome. Interestingly, the interaction between A1Up and mutant ataxin-1-(82Q) increased the half-life of A1Up, whereas nonpathogenic wild-type ataxin-1-(30Q) or ataxin-1-(82Q)-A776 did not.     

Structural and functional analysis of the Josephin domain of the polyglutamine protein ataxin-3

Ataxin-3 belongs to the family of polyglutamine proteins, which are associated with nine different neurodegenerative disorders. Relatively little is known about the structural and functional properties of ataxin-3, and only recently have these aspects of the protein begun to be explored. We have performed a preliminary investigation into the conserved N-terminal domain of ataxin-3, termed Josephin. We show that Josephin is a monomeric domain which folds into a globular conformation and possesses ubiquitin protease activity. In addition, we demonstrate that the presence of the polyglutamine region of the protein does not alter the structure of the protein. However, its presence destabilizes the Josephin domain. The implications of these data in the pathogenesis of polyglutamine repeat proteins are discussed.     

Autophagy mediates SUMO-induced degradation of a polyglutamine protein ataxin-3

Previously, we reported that small ubiquitin-like modifier-1 (SUMO-1) promotes the degradation of a polyglutamine (polyQ) protein ataxin-3 and proposed that proteasomes mediate the proteolysis. Here, we present evidence that autophagy is also responsible for SUMO-induced degradation of this polyQ protein. The autophagy inhibitor 3-MA increased the steady-state level of ataxin-3 and stabilized SUMO-modified ataxin-3 more prominently than the proteasome inhibitor MG132. Interestingly, SUMO-1 overexpression enhanced the co-localization of ataxin-3 and autophagy marker LC3 without increasing LC3 puncta formation suggesting that SUMO-1 is involved in the substrate recruitment rather than the induction of autophagy. To assess the importance of a putative SUMO-interacting motif (SIM) in ataxin-3 for SUMO-induced degradation, we constructed a SIM mutant of ataxin-3. Substitution of putative SIM (V165G) facilitated the degradation of polyQ-expanded ataxin-3, which is more resistant to SUMO-induced degradation than the normal ataxin-3. These results together indicate that SUMO-1 promotes the degradation of ataxin-3 via autophagy and the putative SIM of ataxin-3 plays a role in this process.     

Caspase-mediated proteolysis of the polyglutamine disease protein ataxin-3

Spinocerebellar ataxia type-3, also known as Machado-Joseph Disease, is one of many inherited neurodegenerative disorders caused by polyglutamine-encoding CAG repeat expansions in otherwise unrelated disease genes. Polyglutamine disorders are characterized by disease protein misfolding and aggregation; often within the nuclei of affected neurons. Although the precise mechanism of polyglutamine-mediated cell death remains elusive, evidence suggests that proteolysis of polyglutamine disease proteins by caspases contributes to pathogenesis. Using cellular models we now show that the endogenous spinocerebellar ataxia type-3 disease protein, ataxin-3, is proteolyzed in apoptotic paradigms, resulting in the loss of full-length ataxin-3 and the corresponding appearance of an approximately 28-kDa fragment containing the glutamine repeat. Broad-spectrum caspase inhibitors block ataxin-3 proteolysis and studies suggest that caspase-1 is a primary mediator of cleavage. Site-directed mutagenesis experiments eliminating three, six or nine potential caspase cleavage sites in the protein suggest redundancy in the site(s) at which cleavage can occur, as previously described for other disease proteins; but also map a major cleavage event to a cluster of aspartate residues within the ubiquitin-binding domain of ataxin-3 near the polyglutamine tract. Finally, caspase-mediated cleavage of expanded ataxin-3 resulted in increased ataxin-3 aggregation, suggesting a potential role for caspase-mediated proteolysis in spinocerebellar ataxia type-3 pathogenesis.     

A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1

RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.     

Cellular turnover of the polyglutamine disease protein ataxin-3 is regulated by its catalytic activity

Ataxin-3, a deubiquitinating enzyme, is the disease protein in spinocerebellar ataxia type 3, one of many neurodegenerative disorders caused by polyglutamine expansion. Little is known about the cellular regulation of ataxin-3. This is an important issue, since growing evidence links disease protein context to pathogenesis in polyglutamine disorders. Expanded ataxin-3, for example, is more neurotoxic in fruit fly models when its active site cysteine is mutated (1). We therefore sought to determine the influence of ataxin-3 enzymatic activity on various cellular properties. Here we present evidence that the catalytic activity of ataxin-3 regulates its cellular turnover, ubiquitination, and subcellular distribution. Cellular protein levels of catalytically inactive ataxin-3 were much higher than those of active ataxin-3, in part reflecting slower degradation. In vitro studies revealed that inactive ataxin-3 was more slowly degraded by the proteasome and that this degradation occurred independent of ubiquitination. Slower degradation of inactive ataxin-3 correlated with reduced interaction with the proteasome shuttle protein, VCP/p97. Enzymatically active ataxin-3 also showed a greater tendency to concentrate in the nucleus, where it colocalized with the proteasome in subnuclear foci. Taken together, these and other findings suggest that the catalytic activity of this disease-linked deubiquitinating enzyme regulates several of its cellular properties, which in turn may influence disease pathogenesis.     

Defining the role of ubiquitin-interacting motifs in the polyglutamine disease protein, ataxin-3

Polyglutamine (polyQ) expansions cause neurodegeneration that is associated with protein misfolding and influenced by functional properties of the host protein. The polyQ disease protein, ataxin-3, has predicted ubiquitin-specific protease and ubiquitin-binding domains, which suggest that ataxin-3 functions in ubiquitin-dependent protein surveillance. Here we investigate direct links between the ubiquitin-proteasome pathway and ataxin-3. In neural cells we show that, through its ubiquitin interaction motifs (UIMs), normal or expanded ataxin-3 binds a broad range of ubiquitinated proteins that accumulate when the proteasome is inhibited. The expression of a catalytically inactive ataxin-3 (normal or expanded) causes ubiquitinated proteins to accumulate in cells, even in the absence of proteasome inhibitor. This accumulation of ubiquitinated proteins occurs primarily in the cell nucleus in transfected cells and requires intact UIMs in ataxin-3. We further show that both normal and expanded ataxin-3 can undergo oligoubiquitination. Although this post-translational modification occurs in a UIM-dependent manner, it becomes independent of UIMs when the catalytic cysteine residue of ataxin-3 is mutated, suggesting that ataxin-3 ubiquitination is itself regulated in trans by its own de-ubiquitinating activity. Finally, pulse-chase labeling reveals that ataxin-3 is degraded by the proteasome, with expanded ataxin-3 being as efficiently degraded as normal ataxin-3. Mutating the UIMs does not alter degradation, suggesting that UIM-mediated oligoubiquitination of ataxin-3 modulates ataxin-3 function rather than stability. The function of ataxin-3 as a de-ubiquitinating enzyme, its post-translational modification by ubiquitin, and its degradation via the proteasome link this polyQ protein to ubiquitin-dependent pathways already implicated in disease pathogenesis.     

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.     

The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein

The tobacco mitogen-activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin-like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco-cultured BY-2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti-NPK1 antibodies and GFP-fused NPK1 were detected only in the nuclei of BY-2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl-terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY-2 cells, indicating that this sequence is functional in tobacco BY-2 cells. We also found that the 64-amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl-terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.     

A PY-NLS nuclear targeting signal is required for nuclear localization and function of the Saccharomyces cerevisiae mRNA-binding protein Hrp1

Proteins destined for import into the nucleus contain nuclear localization signals (NLSs) that are recognized by import receptors termed karyopherins or importins. Until recently, the only nuclear import sequence that had been well defined and characterized was the classical NLS (cNLS), which is recognized by importin alpha. However, Chook and coworkers (Lee, B. J., Cansizoglu, A. E., Süel, K. E., Louis, T. H., Zhang, Z., and Chook, Y. M. (2006) Cell 126, 543-558) have provided new insight into nuclear targeting with their identification of a novel NLS, termed the PY-NLS, that is recognized by the human karyopherin beta2/transportin (Kapbeta2) receptor. Here, we demonstrate that the PY-NLS is conserved in Saccharomyces cerevisiae and show for the first time that the PY-NLS is a functional nuclear targeting sequence in vivo. The apparent ortholog of Kapbeta2 in yeast, Kap104, has two known cargos, the mRNA-binding proteins Hrp1 and Nab2, which both contain putative PY-NLS-like sequences. We find that the PY-NLS-like sequence within Hrp1, which closely matches the PY-NLS consensus, is both necessary and sufficient for nuclear import and is also required for receptor binding and protein function. In contrast, the PY-NLS-like sequences in Nab2, which vary from the PY-NLS consensus, are not required for proper import or protein function, suggesting that Kap104 may interact with different cargos using multiple mechanisms. Dissection of the PY-NLS consensus reveals that the minimal PY-NLS in yeast consists of the C-terminal portion of the human consensus, R/H/KX(2-5)PY, with upstream basic or hydrophobic residues enhancing the targeting function. Finally, we apply this analysis to a bioinformatic search of the yeast proteome as a preliminary search for new potential Kap104 cargos.     

Intracellular distribution of a nuclear localization signal binding protein.

The transport of proteins into the nucleus requires the recognition of a nuclear localization signal sequence. Several proteins that interact with these sequences have been identified, including one of about 66 kDa. We have prepared antibodies that recognize the 66-kDa nuclear localization signal binding protein (NLSBP) and inhibit nuclear localization in vitro. By immunofluorescence, it is seen that the NLSBP is predominantly cytoplasmic and is distributed peripherally around the nucleus and the microtubule organizing center. There is also a weak punctate staining of the surface of the nucleus. Methanol-fixed cells can also be stained directly with fluorescently labeled karyophilic proteins. These stains reveal the same cytoplasmic structures as anti-NLSBP. The expression of the NLSBP is growth dependent. When cells grown to confluence are examined, the cytoplasmic staining is greatly reduced, leaving the punctate nuclear staining as the predominant feature. In serum-starved cells, very little staining of either the cytoplasm or the nucleus can be seen. Upon simulation by the addition of serum, the original cytoplasmic and nuclear envelope staining is restored. Cells grown in the presence of colchicine or taxol have an altered NLSBP distribution but apparently normal cytoplasmic nuclear transport.     

Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein

VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.     

Identification of a nuclear localization signal in mouse polycomb protein, M33

The mouse Polycomb group (PcG) protein M33 forms nuclear complexes with the products of other PcG members and maintains repressed states of developmentally important genes, including homeotic genes. In this context, nuclear localization is a prerequisite for M33 to exert its function. However, we previously found that M33 in mouse liver shuttles dynamically between the nucleus and the cytoplasm, depending on the proliferative states of cells, coupled with phosphorylation and dephosphorylation of M33 protein. To understand the mechanism and significance of this phenomenon, we identified the functional nuclear localization signal (NLS) of M33 protein. Deletion mutants that lack a particular one of three putative NLS motifs failed to localize in the nucleus. Green fluorescent protein (GFP) fused to this motif specifically localized in the nucleus. We conclude that this amino-acid stretch in M33 acts as the functional NLS for this protein.     

Nuclear localization of a non-caspase truncation product of atrophin-1, with an expanded polyglutamine repeat,increases cellular toxicity

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder similar to Huntington's disease, with clinical manifestations including chorea, incoordination, ataxia, and dementia. It is caused by an expansion of a CAG trinucleotide repeat encoding polyglutamine in the atrophin-1 gene. Both patients and DRPLA transgenic mice have nuclear accumulation of atrophin-1, especially an approximately 120-kDa fragment, which appears to represent a cleavage product. We now show that this is an N-terminal fragment that does not correspond to the previously described caspase-3 fragment, or any other known caspase cleavage product. The atrophin-1 sequence contains a putative nuclear localization signal in the N terminus of the protein and a putative nuclear export signal in the C terminus. We have tested the hypothesis that endogenous localization signals are functional in atrophin-1, and that nuclear localization and proteolytic cleavage contribute to atrophin-1 cell toxicity. In transient cell transfection experiments using a neuroblastoma cell line, full-length atrophin-1 with 26 (normal) or 65 (expanded) glutamines localized to both nucleus and cytoplasm, with no significant difference in toxicity between the normal and mutant proteins. A construct with 65 glutamine repeats encoding an N-terminal fragment (which removes an NES) of atrophin-1 similar in size to the truncation product in DRPLA patient tissue, showed increased nuclear labeling, and an increase in cellular toxicity, compared with a similar fragment with 26 glutamines. Full-length atrophin-1 with 65 polyglutamine repeats and mutations inactivating the NES also yielded increased nuclear localization and increased toxicity. These data suggest that truncation enhances cellular toxicity of the mutant protein, and that the NES is a relevant region deleted during truncation. Furthermore, mutating the NLS in the truncated protein shifted atrophin-1 more to the cytoplasm and eliminated the increased toxicity, consistent with the idea that nuclear localization enhances toxicity. In none of the experiments were inclusions visible in the nucleus or cytoplasm suggesting that inclusion formation is unrelated to cell death. These data indicate that truncation of atrophin-1 may alter its ability to shuttle between the nucleus and cytoplasm, leading to abnormal nuclear interactions and cell toxicity.     

A nuclear localization signal binding protein in the nucleolus

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine- labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.     

Intracellular localization of human cytidine deaminase. Identification of a functional nuclear localization signal.

The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product. They play a major role in pyrimidine nucleoside and nucleotide salvage. The intracellular distribution of cytidine deaminase and related enzymes has previously been considered to be cytosolic. Here we show that human cytidine deaminase (HCDA) is present in the nucleus. A highly specific, affinity purified polyclonal antibody against HCDA was used to analyze the intracellular localization of native HCDA in a variety of mammalian cells by in situ immunochemistry. Native HCDA was found to be present in the nucleus as well as the cytoplasm in several cell types. Indirect immunofluorescence microscopy indicated a predominantly nuclear localization of FLAG-tagged HCDA overexpressed in these cells. We have identified an amino-terminal bipartite nuclear localization signal that is both necessary and sufficient to direct HCDA and a non-nuclear reporter protein to the nucleus. We also show HCDA binding to the nuclear import receptor, importin alpha. Similar putative bipartite nuclear localization sequences are found in other cytidine/deoxycytidylate deaminases. The results presented here suggest that the pyrimidine nucleotide salvage pathway may operate in the nucleus. This localization may have implications in the regulation of nucleoside and nucleotide metabolism and nucleic acid biosynthesis.     

The C2A domain of double C2 protein gamma contains a functional nuclear localization signal

The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+ -dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Calpha functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+ -binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+ -independent C2A domain of double C2 protein gamma (Doc2gamma) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2gamma contains six Arg residues ((177)RLRRRRR(183)) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2gamma. Because of the presence of the basic cluster, the C2A domain of Doc2gamma did not show Ca2+ -dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif.