Full-length P62 Research Articles

New Target of Oxidative Stress Regulation in Cochleae: Alternative Splicing of the p62/Sqstm1 Gene.

We investigated oxidative stress and antioxidant response in the p62/Sqstm1-Keap1-Nrf2 pathway in C57BL/6 mice cochleae during age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL), and the function of full-length and variant p62 in the regulation of Nrf2 activation. Groups of young (2months), old (13-14months), control, and acoustic trauma (AT) mice were examined cochlear damage and oxidative stress as follows: auditory brainstem response and hair cell counts; malondialdehyde (MDA) levels measured by assay kit and 7,8-dihydro-8-oxoguanine (8-oxoG) detected by immunohistochemistry. Full-length and variant p62 were examined for expression in cochleae, hippocampus (HIP), and auditory cortex (AC) using immunoblotting. Keap1-Nrf2 pathway activation was based on immunoblotting of nuclear Nrf2 and quantitative real-time PCR of Nrf2 target genes HO-1/NQO-1. The oxidative function of full-length and variant p62 was examined in HEI-OC-1 cells by flow cytometry. The results showed hearing loss, and cochlear hair cell loss was associated with MDA accumulation and 8-oxoG expression during ARHL and NIHL. Nrf2 showed no obvious changes in nuclear protein. Expression levels mRNA for HO-1 and NQO1 were lower in old mice and mildly greater in AT Mice. The expression of p62 splicing variant lacking the Keap1-interacting region was greater than full-length p62 in cochleae. However, the expression of p62 splicing variant was lesser than full-length p62 in HIP and AC. For HEI-OC-1 cells, overexpression of full-length p62 decreased ROS levels induced by H2O2. Oxidative stress is closely related to ARHL and NIHL. Changing the ratio of full-length to variant p62 protein expression may be a new target to reduce the level of oxidative stress in cochleae.

Cleaving a different function for p62

Cell Biology The scaffold protein p62 has a critical role in autophagy, the regulated intracellular degradation of proteins and organelles. Sanchez-Garrido et al. identified a proteolytic fragment of human p62, called p62TRM, that had a distinct function from full-length p62 (see the Focus by Martens). Instead of promoting autophagy, p62TRM regulated responses to nutrient availability. This function was lost in p62 variants with disease-associated mutations in the cleavage site, suggesting that defective nutrient sensing may account for some of the symptoms of patients with p62 mutations. Sci. Signal. 11 , eaat6903, eaav3530 (2018).

PB1 and UBA domains of p62 are essential for aggresome-like induced structure formation

ALIS are large, transient, cytosolic aggregates that serve as storage compartments for ubiquitin-tagged defective ribosomal products. We determined the importance of the protein p62 in the formation of ALIS and demonstrated that two domains of p62—PB1 and UBA—are essential for ALIS assembly. Those two major binding domains of p62, also known as sequestosome 1, were shown to play a critical role in the formation of autophagosomes or cytoplasmic aggregates. Specifically, the PB1 domain is essential for self-oligomerization, and the UBA domain allows p62 to bind to polyubiquitin chains or ubiquitinated proteins. After stimulation of RAW 264.7 macrophages with lipopolysaccharide, we observed a significant decrease in the number of cells with ALIS. Importantly, cells overexpressing either a PB1 mutant or UBA-deleted p62 construct also exhibited a substantially diminished number of cells containing ALIS. Since both p62 and ubiquitin are found in ALIS, we evaluated the dynamics of YFP-tagged p62 in ALIS. In contrast to the findings of a previous study that evaluated GFP-tagged ubiquitin motility in ALIS, we determined that YFP-tagged p62 has very limited mobility. Lastly, we determined that GST–tagged full-length p62 binds to Lys-63-linked polyubiquitin chains but not to Lys-48-linked chains. Overall, our findings provide insight on the essential role that p62, particularly its PB1 and UBA domains, has in the formation of ALIS.

Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant.

ABSTRACTA key antioxidant pathway, the Keap1-Nrf2 system, is regulated by p62/Sqstm1 via multiple mechanisms, including gene expression, posttranslational modifications (such as ubiquitination and phosphorylation), and autophagic degradation of p62/Sqstm1 and Keap1. Here we demonstrate a novel mode of regulation of the Keap1-Nrf2 system, mediated by a splicing variant of p62/Sqstm1 pre-mRNA. Ensembl database searches and subsequent biochemical analyses of mice revealed the presence of an mRNA that encodes a p62/Sqstm1 protein lacking the Keap1-interacting region (KIR), which is essential for the interaction with Keap1. Like full-length p62, the variant was induced under conditions in which Nrf2 was activated (e.g., impairment of autophagy), formed oligomers with itself and/or the full-length protein, and was degraded by autophagy. However, the variant failed to interact with Keap1 and sequester it in variant-positive aggregates. Remarkably, while full-length p62 stabilized Nrf2 and induced the gene expression of Nrf2 targets, the variant increased the amount of Keap1 and enhanced ubiquitination of Nrf2, thereby suppressing the induction of Nrf2 targets. Hepatocytes isolated from genetically modified mice that express full-length p62, but not the variant, were susceptible to activation of Nrf2 in response to stress. Collectively, our results suggest that splicing of p62/Sqstm1 pre-mRNA negatively regulates the Keap1-Nrf2 pathway.

The right and wrong of DOKing the nuclear receptor

In this issue, Burgermeister et al., present very interesting data on the function of DOK1, an enigmatic multifunctional adaptor protein (Friedrich et al., 2016). Previous studies nicely summarized by the authors, have shown that DOK1 functions as a tumor suppressor by negatively regulating signaling via its interaction with p120 RAS GTPase-activating protein (GAP) to dampen MAPK signaling cascades downstream of receptor and non-receptor kinases in various cellular contexts (Songyang et al., 2001, Shinohara et al., 2004, Zhao et al., 2006). Remarkably, Dok-1 knockout studies demonstrated hematopoietic defects (Yasuda et al., 2004) and development of sarcomas with the loss of other DOK members (Mashima et al., 2010). The studies of DOK1 function were not only complicated by the presence of several protein isoforms encoded by the same DOK1, but also dynamic shifts of the protein between the nucleus and the cytosol in resting and proliferating cells, respectively. Regarding the docking of DOK1, the full-length p62 DOK1 has an N-terminal plextrin homology (PH) domain for membrane binding, and a phospho-tyrosine-binding (PTB) domain thought to be involved in interactions with other protein partners. Unexpectedly, the authors convincingly demonstrate that the C-terminal PTB domain has yet another partner which directly regulates cell growth. And this is when peroxisome proliferator-activated receptor gamma (PPARg) enters the stage. Detailed experiments demonstrate not only the physical interactions between the two novel partners, but also a sensitivity of common cancer cell lines to PPARg agonists in a DOK1-dependent manner. The potential for repurposing of PPARg agonists, an established class of anti-diabetic medications, is one of the practical implications of the results of the Burgermeister et al. paper. As for the clinical relevance, the authors demonstrate that the loss of DOK1 expression portends worse survival in patients with colorectal cancer.

P62-ZZ Domain Inhibition Prevents MM Cell-Induced Epigenetic Changes at the Runx2 and C/EBPb Promoters

Multiple myeloma bone disease (MMBD) is a paradigm for uncoupled bone remodeling and is characterized by non-healing lytic bone lesions. Osteoblast (OB) function is highly suppressed or absent, persists in the absence of MM cells, and remains a significant cause of skeletal-related events. The persistence of OB suppression in MMBD suggests that MM cells induce repressive epigenetic changes at the Runx2 gene, the key transcription factor required for OB differentiation of bone marrow stromal cells (BMSC), which are preOB. We reported that TNFα is a major suppressor of OB in MMBD that reduces Runx2 levels by inducing Gfi1, a transcriptional repressor of Runx2. Using ChIP analysis of MM-exposed BMSC, we showed that Gfi1 directly binds the Runx2 promoter and recruits the chromatin corepressor HDAC1 to the Runx2 promoter in MM-exposed BMSC, reducing transcriptionally permissive euchromatin marks such as H3K9ac.Recently, we reported that p62 (sequestosome-1) in BMSC is critical for the formation of MM cell-induced signaling complexes that mediate OB suppression and IL-6 production. We found that XRK3F2, a novel inhibitor of the p62 ZZ domain (p62-ZZ), blunted MM cell-induced repression of Runx2 and induction of Gfi1 in BMSC, and induced new bone formation and remodeling in mice with established MMBD, but did not alter normal bone. In the current study, we further evaluated the specificity of XRK3F2's inhibition of p62-ZZ interactions and investigated the mechanism by which blocking p62-ZZ prevents MM-induced suppression of key osteogenic transcription factors in BMSC.We previously showed that TNFα and MM cell-induced IL-6 production by BMSC are both p62 dependent and independent. XRK3F2 blocked TNFα-induced NFκB signaling in BMSC and MM cells, and partially inhibited TNFα-induced IL-6 production by BMSC. We now report that XRK3F2 blunted the TNFα upregulation observed in BMSC after MM cell coculture but did not alter TNFα production in p62 knockout (p62KO) BMSC following coculture with MM cells. TNFα treatment of p62KO BMSCs resulted in minimal induction of IL-6, which was not altered by XRK3F2. Transfection of p62KO BMSC with full-length p62 constructs restored TNFα-induced IL-6 production and XRK3F2's capacity to reduce TNFα-induced IL-6. In contrast, XRK3F2 had no effect on TNFα-induced IL-6 production by p62KO BMSC transfected with p62 constructs lacking p62-ZZ.To further investigate XRK3F2's mechanism of action, we tested if XRK3F2 prevents Gfi1-induced epigenetic suppression of Runx2 by preventing Gfi1's upregulation and binding to Runx2. ChIP analysis of MM exposed BMSC treated with XRK3F2 demonstrated that XRK3F2 reduced MM-induced Gfi1 occupancy of the Runx2 promoter and prevented MM-induced reduction of H3K9ac. Since the region proximal to the Runx2 promoter contains putative C/EBPβ binding sites, we tested if MM-induced p62-ZZ signaling activates C/EBPβ, a transcription factor that regulates OB lineage maturation and IL-6 production in BMSC and plays a role in the upregulation of Gfi1 expression. MM-exposed BMSC had increased enrichment of C/EBPβ at both the Gfi1 gene and the C/EBPβ-regulated Il6 gene, and XRK3F2 reduced upregulation of MM-induced C/EBPβ binding at both the Gfi1 and Il6 genes. We conclude that XRK3F2's reduction of the MM-induced C/EBPβ binding in BMSC results in decreased Gfi1 mRNA expression, and thereby reduces MM-induced Gfi1 occupancy and HDAC1 recruitment at the Runx2 promoter. These results suggest that targeting p62-ZZ in MMBD may reverse C/EBPβ mediated Gfi1 activation, resulting in rescue of the MM cell-induced epigenetic suppression of Runx2 in BMSC, and that next-generation derivatives of XRK3F2 should impact BMSC-supported MM cell survival. DisclosuresSilbermann:Celgene: Research Funding; Amgen: Consultancy. Xie:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Roodman:Eli Lilly: Research Funding; Amgen: Consultancy.

Impact of p62/SQSTM1 UBA Domain Mutations Linked to Paget’s Disease of Bone on Ubiquitin Recognition

The scaffold protein p62/SQSTM1 acts as a hub in regulating a diverse range of signaling pathways which are dependent upon a functional ubiquitin-binding C-terminal UBA domain. Mutations linked to Paget's disease of bone (PDB) commonly cluster within the UBA domain. The p62 UBA domain is unique in forming a highly stable dimer which regulates ubiquitin recognition by using overlapping surface patches in both dimerization and ubiquitin binding, making the two association events competitive. NMR structural analysis and biophysical methods show that some PDB mutations modulated the ubiquitin binding affinity by both direct and indirect mechanisms that affect UBA structural integrity, dimer stability, and contacts at the UBA-ubiquitin interface. In other cases, common PDB mutations (P392L in particular) result in no significant change in ubiquitin binding affinity for the UBA domain in isolation; however, all PDB UBA mutations lead to loss of function with respect to ubiquitin binding in the context of full-length p62, suggesting a more complex underlying mechanism.

P62 as a Therapeutic Target for Myeloma Cell Growth and Osteoclast Formation.

Abstract 2857Poster Board II-833We reported that sequestosome 1 (p62) plays a critical role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with multiple myeloma (MM), and that p62 is a potential therapeutic target for MM. In contrast to treating patients with inhibitors of each of the multiple signaling pathways activated in marrow stromal cells by MM cells (e.g. NF-kB or p38 MAPK), blocking the function of p62 should inhibit the activation of the multiple pathways mediated by p62 and have a broader effect on the bone marrow microenvironment. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling in marrow stromal cells when they interact with myeloma cells, and develop inhibitory peptides as potential therapeutic agents that interfere with p62's role in these signaling complexes. To pursue this objective, we transfected p62−/− stromal cells with p62 deletion constructs and assessed their effects on NF-kB and p38 MAPK signaling induced by MM cells or TNF-a. p62−/− stromal cells support of MM growth or OCL formation was significantly decreased compared to WT stromal cells. We made a series of 5' deletion constructs of p62 that lacked specific p62 domains: ΔPB1 (Δ1) lacks homodimerization domain and binding to PKCz, ΔPB1, ZZ (Δ2) lacks PB1 and RIP1 binding domains, and ΔPB1, ZZ, TBS, (Δ3) the PB1, RIP1, p38 and TRAF6 binding domains have been deleted. These constructs were tested for their capacity to restore p62 function in p62−/−stromal cells and support MM cell growth and OCL formation. GFP-labeled MM1.S myeloma cells were cocultured with p62−/− and WT marrow stromal cells transduced with the different p62 deletion constructs. Transduction of p62−/− stromal cells with the full-length p62 construct restored the capacity of p62−/− stromal cells to enhance the growth of MM cells to levels induced by WT stromal cells. Transduction of p62−/− stromal cells with the Δ1 construct also restored stromal cell support of MM growth. Therefore, the PB1 domain is not important for this function. Transduction of p62−/− stromal cells with the Δ2 construct, resulted in an inability of the stromal cells to support MM cell growth. Additional loss of the p38 and TRAF6 binding domains did not further impair p62−/− stromal cells support of MM cell growth. These results suggest that the RIP1 binding domain plays a critical role in supporting the growth of MM cells by marrow stromal cells. We then examined the capacity of p62−/− stromal cells transduced with various p62 deletion constructs to support OCL formation. Normal CFU-GM, a source of OCL precursors, were cocultured with p62−/− stromal cells transfected with the different p62 cDNA deletion constructs. The Δ1 construct completely rescued p62−/− support of OCL formation. However, Δ2 construct transduced p62−/− stromal cells only partly restored stromal cell support of OCL formation. Transduction of the Δ3 construct did not restore the capacity of the p62−/− stromal cells to support OCL formation. Similarly, transduction of the Δ2 and Δ3 construction decreased WT stromal cell support of MM cell growth. We then tested the feasibility of using transduction domain (PTD) fusion peptides as a potential means of delivering dominant negative p62 constructs into stromal cells in vitro and in vivo to block MM cell growth and VCAM-1 expression induced by marrow stromal cells. PTD binding domain fusion peptides containing NEMO binding protein that blocks NF-kB activity was used as a model system to determine the feasibility of transducing marrow stromal cells with p62 constructs. Addition of PTD-NEMO fusion peptides to stromal cells significantly inhibited WT stromal cell to enhance MM cell growth and VCAM-1 expression on stromal cells, which is the capacity of dependent, in part, on NF-kB signaling. These results demonstrate that the ZZ, p38 MAPK and TRAF-6 domains of p62 together are required for stromal cell support of MM cell growth and OCL formation and suggest that PTD constructs containing dominant negatives for p62 may be a feasible method for blocking p62 function in the MM marrow microenvironment. Disclosures:Roodman:Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy.

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.

Loss of Ubiquitin-Binding Associated With Paget's Disease of Bone p62 (SQSTM1) Mutations

We have studied the effects of various PDB-causing mutations of SQSTM1 on the in vitro ubiquitin-binding properties of the p62 protein. All mutations caused loss of monoubiquitin-binding and impaired K48-linked polyubiquitin-binding, which was only evident at physiological temperature. This suggests that SQSTM1 mutations predispose to PDB through a common mechanism that depends on loss of ubiquitin-binding by p62. Mutations in the SQSTM1 gene, which affect the ubiquitin-associated (UBA) domain of the p62 protein, are a common cause of Paget's disease of bone (PDB). We previously showed that the isolated UBA domain of p62 binds K48-linked polyubiquitin chains in vitro and that PDB-causing mutations in the UBA domain can be resolved in to those which retain (P392L and G411S) or lose (M404V and G425R) the ability to bind K48-linked polyubiquitin. To further clarify the mechanisms by which these mutations predispose to PDB, we have extended these analyses to study the ubiquitin-binding properties of the PDB-causing mutations in the context of the full-length p62 protein. We studied the effects of various PDB-causing mutations on the interaction between glutathione S-transferase (GST)-tagged p62 proteins and monoubiquitin, as well as K48-linked polyubiquitin chains, using in vitro ubiquitin-binding assays. All of the PDB-causing mutations assessed (P392L, E396X, M404V, G411S, and G425R) caused loss of monoubiquitin binding and impaired K48-linked polyubiquitin-binding when introduced into the full-length p62 protein. However, these effects were only observed when the binding experiments were conducted at physiological temperature (37 degrees C); they were not seen at room temperature or at 4 degrees C. Our in vitro findings suggest that PDB-causing mutations of SQSTM1 could predispose to disease through a common mechanism that is dependent on impaired binding of p62 to a ubiquitylated target and show that 5q35-linked PDB is the first example of a human disorder caused by loss of function mutations in a UBA domain.

N-terminal protein acylation confers localization to cholesterol, sphingolipid-enriched membranes but not to lipid rafts/caveolae.

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.

C‐terminal domain of the mitotic apparatus protein p62 targets the protein to the nucleolus during interphase

Mitotic apparatuses from sea urchin embryos contain a protein (p62), previously shown to be required for mitotic progression. This protein localizes to the mitotic apparatus during cell division in urchin embryos and mammalian tissue culture cells. We show here by immunofluorescence that p62 is localized to the nucleus of mammalian cells during interphase and is highly concentrated in nucleoli. In addition, a fusion protein composed of full-length p62 and green fluorescent protein also localizes to nucleoli when expressed in COS-7 cells in culture. Analysis of the primary sequence of p62 reveals three distinct domains of the protein based on amino acid charge distribution: the acidic N-terminal domain, the basic C-terminal domain, and the central, M-domain, which contains alternating subdomains of clusters of acidic and basic residues. To identify the domain important for nucleolar localization during interphase, specific domains of p62 alone, or in combination with each other or with β-galactosidase were fused to green fluorescent protein. Following confirmation of the fusion constructs by sequence analysis, the constructs were expressed in mammalian cells, expression was confirmed by immunoblotting, and the fusion proteins were localized via fluorescence microscopy. The data demonstrate that the C-terminal domain of p62 is both necessary and sufficient for the nuclear localization and nucleolar binding of p62 that is observed during interphase. Cell Motil. Cytoskeleton 44:68–80, 1999. © 1999 Wiley-Liss, Inc.

The transmembrane domains of Sindbis virus envelope glycoproteins induce cell death.

Sindbis virus, the prototype alphavirus, kills cells by inducing apoptosis. To investigate potential mechanisms by which Sindbis virus induces apoptosis, we examined whether specific viral gene products were able to induce cell death. Genes encoding the three structural proteins--capsid, the precursor E1 (6K plus E1), and the precursor E2 (P62 or E3 plus E2)--were cotransfected with a beta-galactosidase reporter plasmid in transient-transfection assays in rat prostate adenocarcinoma AT3 cells. Cell death, as determined by measuring the loss of blue cells, was observed in AT3 cells transfected with 6K plus E1 and with P62 but not in cells transfected with capsid. Deletion mutagenesis of P62 indicated that large regions of the cytoplasmic domain and extracellular domain were not essential for the induction of cell death. However, constructs containing the minimal E3 signal sequence fused to the E2 transmembrane domain and the minimal E3 signal sequence fused to the E1 transmembrane domain induced death as efficiently as full-length P62 and 6K plus E1, whereas no cell death was observed after transfection with a control construct containing the E3 signal sequence linked to the transmembrane domain of murine CD4. These data demonstrate that intracellular expression of the transmembrane domains of the Sindbis virus envelope glycoproteins can kill AT3 cells.

Molecular Characterization of p62, a Mitotic Apparatus Protein Required for Mitotic Progression

A 62-kDa (p62) mitotic apparatus-associated protein is important for the proper progression of mitosis in sea urchin embryos (Dinsmore, J. H., and Sloboda, R. D. (1989) Cell 53, 769-780). We have isolated and characterized a full-length p62 cDNA of 3374 base pairs which encodes an extremely acidic polypeptide of 411 amino acids having a calculated Mr of 46,388 and a pI of 4.01; p62 is a unique protein with no significant identity to any known proteins. Southern and Northern blot analyses demonstrate that the gene for p62 is present once in the sea urchin genome and the corresponding mRNA is present in unfertilized eggs and in early embryos through and up to the gastrula stage. Sequence analysis suggests certain regions may participate in chromatin association and microtubule binding, an observation that is consistent with previous immunological data (Ye, X., and Sloboda, R. D. (1995) Cell Motil. Cytoskeleton 30, 310-323) as well as data reported herein. Confocal microscopy reveals that during interphase the protein binds to chromatin in the nuclei of sea urchin eggs. In the germinal vesicles of clam oocytes at prophase of meiosis I, p62 binds to the condensed chromosomes. Currently, truncated clones of p62 are being used to identify the tubulin and chromatin binding domains.

The gene encoding rat nuclear pore glycoprotein p62 is intronless

Glycoproteins of the nuclear pore complex are thought to play an important role in the transport of regulatory proteins and ribonucleoproteins across the nuclear envelope. However, the genetic elements and signals that control the expression of nuclear pore glycoproteins are poorly understood. To study the transcriptional regulation of mammalian nuclear pore glycoprotein biosynthesis, we have isolated the gene coding for the major rat nuclear pore glycoprotein p62. The p62 gene consists of a 2941-base pair region that is linear with the full length p62 cDNA with no intervening sequences. Quantitative Southern analysis revealed that the gene is present in single copy. The p62 gene encodes a 525-amino acid open reading frame that directs the synthesis of the 62-kDa pore glycoprotein in vitro and in transfected cultured cells. The 5'-flanking region contains two potential transcription start sites; primer extension analysis revealed that the furthest upstream site is preferentially used in vivo. When linked to a reporter gene, the 5'-flanking region of the p62 gene serves as an active promoter.