Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein

The activity and stability of the tumor suppressor p53 are regulated by interactions with key cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53 contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the full-length p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as evidenced by the observed increase in helicity and increased level of dispersion of the amide proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which extend from Phe19 to Leu25 and from Pro47 to Trp53, respectively. In the complex, the NCBD forms a bundle of three helices (Cα1, residues 2066-2075; Cα2, residues 2081-2092; and Cα3, residues 2095-2105) with a hydrophobic groove into which p53 helices Pα1 and Pα2 dock. The polypeptide chain between the p53 helices remains flexible and makes no detectable intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53 and R2105 of NCBD may contribute to the binding specificity. The structure provides the first insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.     

Targeting of the mammalian nucleoporin p62 to the nuclear envelope in the yeast Saccharomyces cerevisiae and HeLa cells.

We have analyzed the sorting of the mammalian nucleoporin p62 in human culture cells and in the yeast Saccharomyces cerevisiae. To this end, gene fusions were generated that carry Aequorea victoria green fluorescence protein and defined portions of p62. Upon transient gene expression fluorescent fusion proteins were localized in HeLa cells. Likewise, fusion proteins were studied in S. cerevisiae using wild-type as well as mutant cells that cluster nuclear pore complexes. Our results demonstrate that evolutionarily distant organisms, such as humans and yeasts, recognize the same sequence elements of p62 for sorting to the nuclear envelope. Specifically, the entire sequence of p62 or its complete C-terminal domain targeted fusion proteins to the nuclear membranes. In contrast, truncations of the C-terminal domain or the N-terminal segment of p62 failed to associate with the nuclear envelope in either organism. In HeLa cells overexpression of several p62-containing fusion proteins resulted in nuclear fragmentation. The C-terminal domain of p62 caused this effect, and amino acid residues 477 to 525 were sufficient to induce aberrant nuclei. Thus, overexpression of 49 amino acid residues located at the C-terminal tail of p62 interferes with the nuclear integrity in human culture cells.     

The N-terminal domain of the mammalian nucleoporin p62 interacts with other nucleoporins of the FXFG family during interphase

Nuclear pore complexes (NPCs) provide the only sites for macromolecular transport between nucleus and cytoplasm. The nucleoporin p62, a component of higher eukaryotic NPCs, is located at the central gated channel and involved in nuclear trafficking of various cargos. p62 is organized into an N-terminal segment that contains FXFG repeats and binds the soluble transport factor NTF2, whereas the C-terminal portion associates with other nucleoporins and importin-beta1. We have now identified new components that interact specifically with the p62 N-terminal domain. Using the p62 N-terminal segment as bait, we affinity-purified nucleoporins Nup358, Nup214 and Nup153 from crude cell extracts. In ligand binding assays, the N-terminal p62 segment associated with Nup358 and p62, suggesting their direct binding to the p62 N-terminal portion. Furthermore, p62 was isolated in complex with Nup358, Nup214 and Nup153 from growing HeLa cells, indicating that the interactions Nup358/p62, Nup214/p62 and p62/Nup153 also occur in vivo. The formation of Nup358/p62 and p62/Nup153 complexes was restricted to interphase cells, whereas Nup214/p62 binding was detected in interphase as well as during mitosis. Our results support a model of complex interactions between FXFG containing nucleoporins, and we propose that some of these interactions may contribute to the movement of cargo across the NPC.     

Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.     

Domain topology of the p62 complex within the 3-D architecture of the nuclear pore complex

The nuclear pore complex (NPC) is the only known gateway for exchange of macromolecules between the cytoplasm and nucleus of eukaryotic cells. One key compound of the NPC is the p62 subcomplex, which consists of the nucleoporins p62, p54, and p58/p45 and is supposed to be involved in nuclear protein import and export. Here we show the localization of distinct domains of the p62 complex by immuno-electron microscopy using isolated nuclei from Xenopus oocytes. To determine the exact position of the p62 complex, we examined the localization of the C and N-terminal domains of p62 by immunogold-labeling using domain-specific antibodies against p62. In addition we expressed epitope-tagged versions of p62, p54, and p58 in Xenopus oocytes and localized the domains with antibodies against the tags. This first systematic analysis of the domain topology of the p62 complex within the NPC revealed that the p62 complex is anchored to the cytoplasmic face of the NPC most likely by the coiled-coil domains of the three nucleoporins. Furthermore, we found the phenylalanine-glycine (FG)-repeat domain of p62, but not of p58 and p54, to be of mobile and flexible nature.     

Two structurally distinct domains of the nucleoporin Nup170 cooperate to tether a subset of nucleoporins to nuclear pores

How individual nucleoporins (Nups) perform their role in nuclear pore structure and function is largely unknown. In this study, we examined the structure of purified Nup170 to obtain clues about its function. We show that Nup170 adopts a crescent moon shape with two structurally distinct and separable domains, a β-propeller N terminus and an α-solenoid C terminus. To address the individual roles of each domain, we expressed these domains separately in yeast. Notably, overexpression of the Nup170 C domain was toxic in nup170Δ cells and caused accumulation of several Nups in cytoplasmic foci. Further experiments indicated that the C-terminal domain anchors Nup170 to nuclear pores, whereas the N-terminal domain functions to recruit or retain a subset of Nups, including Nup159, Nup188, and Pom34, at nuclear pores. We conclude that Nup170 performs its role as a structural adapter between cytoplasmically oriented Nups and the nuclear pore membrane.     

Domain topology of nucleoporin Nup98 within the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.     

A vinculin binding domain from the talin rod unfolds to form a complex with the vinculin head

The cytoskeletal protein talin plays a key role in activating integrins and in coupling them to the actin cytoskeleton. Its N-terminal globular head, which binds beta integrins, is linked to an extended rod having a C-terminal actin binding site and several vinculin binding sites (VBSs). The NMR structure of residues 755-889 of the rod (containing a VBS) is shown to be an amphipathic four-helix bundle with a left-handed topology. A talin peptide corresponding to the VBS binds the vinculin head; the X-ray crystallographic structure of this complex shows that the residues which interact with vinculin are buried in the hydrophobic core of the talin fragment. NMR shows that the interaction involves a major structural change in the talin fragment, including unfolding of one of its helices, making the VBS accessible to vinculin. Interestingly, the talin 755-889 fragment binds more than one vinculin head molecule, suggesting that the talin rod may contain additional as yet unrecognized VBSs.     

Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins

Macromolecular trafficking across the nuclear envelope involves interactions between cytosolic transport factors and nuclear pore complex proteins. The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins-localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carried out cDNA cloning of rat p58, p54, and p45. We found that p58 contains regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its NH2 and COOH termini separated by a predicted alpha-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a COOH-terminal predicted coiled-coil region. p45 and p58 appear to be generated by alternative splicing, with p45 containing the NH2-terminal FG repeat region and the coiled-coil region of p58. Using immunogold electron microscopy, we found that p58/p45 and p54 are localized on both sides of the nuclear pore complex, like p62. Previous studies have shown that immobilized recombinant p62 can bind the cytosolic nuclear import factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can deplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF2. At least in the case of p58, this involves FG repeat regions. Moreover, p58 can bind to a complex containing transport ligand, the nuclear localization sequence receptor (Srp1 alpha) and p97. These data support a model in which the p62 complex binds to a multicomponent particle consisting of transport ligand and cytosolic factors to achieve accumulation of ligand near the central gated channel of the nuclear pore complex.     

Characterization of nuclear factors binding to AT-rich element in the rat p53 promoter

In this study, we identified AT-rich element located at positions -504 to -516 in the rat p53 promoter by DNase I foot printing assay. This region was previously identified as a positive regulatory element in the murine p53 promoter and designated as PBF1 (p53 binding factor 1) binding site. However, the proteins binding to this AT-rich element have not been identified yet. Therefore, we characterized the binding protein by various biochemical methods. First, we confirmed that by the oligonucleotide competition assay, nuclear factors bound to the AT-rich element in a sequence-specific manner. Two binding proteins were identified in southwestern blotting analysis and the molecular masses of the proteins were 60 and 40 kDa, respectively. The proteins were stable to denaturants or ionic strength. Treatment of chelators showed that the binding proteins did not require divalent cation for DNA-binding activity. In addition, the binding proteins were labile to protease treatment. This study showed that 60 and 40 kDa proteins bound to AT-rich element and the physico-chemical properties provided new insights into the binding proteins.     

The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.     

Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr

Background  

Tpr is a large protein with an extended coiled-coil domain that is localized within the nuclear basket of the nuclear pore complex. Previous studies [1] involving antibody microinjection into mammalian cells suggested a role for Tpr in nuclear export of proteins via the CRM1 export receptor. In addition, Tpr was found to co-immunoprecipitate with importins α and β from Xenopus laevis egg extracts [2], although the function of this is unresolved. Yeast Mlp1p and Mlp2p, which are homologous to vertebrate Tpr, have been implicated in mRNA surveillance to retain unspliced mRNAs in the nucleus[3, 4]. To augment an understanding of the role of Tpr in nucleocytoplasmic trafficking, we explored the interactions of recombinant Tpr with the karyopherins CRM1, importin β and importin α by solid phase binding assays. We also investigated the conditions required for nuclear import of Tpr using an in vitro assay.     

Versatility at the nuclear pore complex: lessons learned from the nucleoporin Nup153

The vertebrate pore protein Nup153 plays pivotal roles in nuclear pore function. In addition to being important to pore architecture, Nup153 is a key participant in both import and export. The scope of Nup153 function also extends beyond the canonical view of the pore as a trafficking gateway. During the transition into mitosis, Nup153 directs proteins involved in membrane remodeling to the nuclear envelope. As cells exit mitosis, Nup153 is recruited to the chromosomal surface, where nuclear pores are formed anew in a complicated process still under much experimental scrutiny. In addition, Nup153 is targeted for protease cleavage during apoptosis and in response to certain viral infections, providing molecular insight into pore reconfiguration during cell response. Overall, the versatile nature of Nup153 underscores an emerging view of the nuclear pore at the nexus of many key cellular processes.     

The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear pore complex

We have isolated a major protein constituent from a highly enriched fraction of yeast nuclear pore complexes (NPCs). The gene encoding this protein, Nup188p, was cloned, sequenced, and found to be nonessential upon deletion. Nup188p cofractionates with yeast NPCs and gives an immunofluorescent staining pattern typical of nucleoporins. Using immunoelectron microscopy, Nup188p was shown to localize to both the cytoplasmic and nucleoplasmic faces of the NPC core. There, Nup188p interacts with an integral protein of the pore membrane domain, Pom152p, and another abundant nucleoporin, Nic96p. The effects of various mutations in the NUP188 gene on the structure of the nuclear envelope and the function of the NPC were examined. While null mutants of NUP188 appear normal, other mutants allelic to NUP188 exhibit a dominant effect leading to the formation of NPC-associated nuclear envelope herniations and growth inhibition at 37 degrees C. In addition, depletion of the interacting protein Pom152p in cells lacking Nup188p resulted in severe deformations of the nuclear envelope. We suggest that Nup188p is one of a group of proteins that form the octagonal core structure of the NPC and thus functions in the structural organization of the NPC and nuclear envelope.     

PKA phosphorylation of p62/SQSTM1 regulates PB1 domain interaction partner binding

p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic–basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by cAMP-dependent protein kinase (PKA). Such phosphorylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein.     

Utrophin lacks the rod domain actin binding activity of dystrophin

We previously identified a cluster of basic spectrin-like repeats in the dystrophin rod domain that binds F-actin through electrostatic interactions (Amann, K. J., Renley, B. A., and Ervasti, J. M. (1998) J. Biol. Chem. 273, 28419-28423). Because of the importance of actin binding to the presumed physiological role of dystrophin, we sought to determine whether the autosomal homologue of dystrophin, utrophin, shared this rod domain actin binding activity. We therefore produced recombinant proteins representing the cluster of basic repeats of the dystrophin rod domain (DYSR11-17) or the homologous region of the utrophin rod domain (UTROR11-16). Although UTROR11-16 is 64% similar and 41% identical to DYSR11-17, UTROR11-16 (pI = 4. 86) lacks the basic character of the repeats found in DYSR11-17 (pI = 7.44). By circular dichroism, gel filtration, and sedimentation velocity analysis, we determined that each purified recombinant protein had adopted a stable, predominantly alpha-helical fold and existed as a highly soluble monomer. DYSR11-17 bound F-actin with an apparent K(d) of 7.3 +/- 1.3 microM and a molar stoichiometry of 1:5. Significantly, UTROR11-16 failed to bind F-actin at concentrations as high as 100 microM. We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin.     

The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex

All transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). Despite their enormous size, ∼60 MD in vertebrates, they are comprised of only ∼30 distinct proteins (nucleoporins or Nups), many of which form subcomplexes that act as building blocks for NPC assembly. One of these evolutionarily conserved subcomplexes, the Nup93 complex, is a major structural component linking the NPC to the membranes of the NE. Using in vitro nuclear assembly assays, we show that two components of the Nup93 complex, Nup188 and Nup205, are dispensable for NPC formation. However, nuclei lacking Nup188 increase in size by several fold compared with wild type. We demonstrate that this phenotype is caused by an accelerated translocation of integral membrane proteins through NPCs, suggesting that Nup188 confines the passage of membrane proteins and is thus crucial for the homeostasis of the different nuclear membranes.