Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome.

Mimori, Tsuneyo; Ohosone, Yasuo; Hama, Nobuaki; Suwa, Akira; Akizuki, Masashi; Homma, Mitsuo; Griffith, Andrew J.; Hardin, John A.

doi:10.1073/pnas.87.5.1777

Cited by 145 publications

(81 citation statements)

References 21 publications

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“…Thus, we examined the cellular localization of the Ku70 and Ku80 proteins in HeLa-S3 cells using confocal laser scanning microscopy ( Figure 1A). Double staining using antibodies speci®c for the Ku70 and Ku80 subunits, respectively, resulted in uorescence mainly in the nucleoplasm of almost all the interphase cells screened, as reported in previous studies (Reeves, 1985;Yaneva et al, 1985;Mimori et al, 1990;Koike et al, 1998). However, in the course of our study, we found that the staining pattern of Ku80 did not completely coincide with that of Ku70 in some cells ( Figure 1A).…”

Section: Resultssupporting

confidence: 86%

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Koike¹,

Ikuta

Miyasaka³

et al. 1999

Oncogene

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Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation. Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations. Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70. Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle. Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku. The nuclear translocation of Ku80 started during the late telophase/ early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70. Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70. To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and de®ned to a region spanning nine amino acid residues (positions 561 ± 569). The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97. All these ®ndings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70.

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Section: Resultssupporting

confidence: 86%

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Koike¹,

Ikuta

Miyasaka³

et al. 1999

Oncogene

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“…The Ku proteins were originally identified as autoantigens, recognized by the sera of patients with autoimmune diseases such as systemic lupus erythematosus and scleroderma (41). The two Ku proteins, Ku70 and Ku80, have been well demonstrated to dimerize and function in repair of DNA double strand breaks, DNA telomere length maintenance, transcription regulation, and V(D)J recombination (42)(43)(44)(45).…”

Section: Discussionmentioning

confidence: 99%

Ku80 as a Novel Receptor for Thymosin β4 That Mediates Its Intracellular Activity Different from G-actin Sequestering

Bednarek

Boncela

Smolarczyk

et al. 2008

Journal of Biological Chemistry

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Our data demonstrate that increased intracellular expression of thymosin ␤4 (T␤4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced T␤4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of T␤4 (AcSDKPT/4A) , T␤4 (KLKKTET/7A) , and T␤4 (K16A) mutants fused with green fluorescent protein did not differ significantly from those of wild-type T␤4. Overexpression of T␤4, T␤4 (AcSDKPT/4A) , and T␤4 (K16A) affected intracellular formation of actin filaments. As expected, T␤4 (K16A) uptake by nuclei was impaired. On the other hand, overexpression of T␤4 (KLKKTET/7A) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular T␤4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both T␤4 and T␤4 (AcSDKPT/4A) induced the PAI-1 gene to the same extent, whereas mutants T␤4 (KLKKTET/7A) and T␤4 (K16A) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATPdependent DNA helicase II was found to be associated with T␤4. Ku80 and T␤4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and T␤4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for T␤4 and mediates its intracellular activity.2 is the most abundant member of the highly conserved family of acidic polypeptides called ␤-thymosins. Although it is a typical intracellular polypeptide, it plays numerous roles, both intracellularly and extracellularly. Numerous observations indicate that T␤4 is involved in adhesion and spreading of fibroblasts (1, 2), differentiation of endothelial cells (3, 4), directional migration of endothelial cells and keratinocytes (5-8), angiogenesis (3-6, 9, 10), wound healing (6, 7, 11), hair follicle growth (8), and apoptosis (12, 13), and has been described to possess anti-inflammatory properties (11,14). In addition, elevated T␤4 expression has been observed in various malignant cell lines and tumors (15-17) and its levels seem to be associated with increased tumorigenicity and metastatic potential (10, 13, 18 -20). The increased expression of T␤4 correlates with the invasive capability of the cells, the degree of morphologic transformation, and disintegration of actin filaments (21). Recent studies have also correlated increased T␤4 expression with potentiated cell growth (13, 22) but this observation seems not to be universal (2, 10).T␤4 is conside...

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“…Interaction of Ku80 Deletion Mutants with Full-length Ku70 -A number of potential functional domains have been identified in Ku80 based on sequence homology (17)(18)(19)36). These include leucine zipper-like domains that could promote protein-protein interaction as well as clusters of basic amino acid domains that could be involved in DNA recognition.…”

Section: Table Imentioning

confidence: 99%

Defining the Minimal Domain of Ku80 for Interaction with Ku70

Osipovich

Durum

Muegge

1997

Journal of Biological Chemistry

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The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination. Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80-and 70-kDa subunits. We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku. Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners. Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another. Ku80 and Ku70 formed only heterodimers and did not homodimerize. Ku80 was restricted to interacting with just one Ku70 molecule at a time. The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined. It consisted of a 28-amino acid region extending from amino acid 449 to 477. This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70. We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies. This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions.

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Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome.

Abstract: Anti-Ku (p70/p80) autoantibodies in patients with scleroderma-polymyositis overlap syndrome recognize a 70-kDa/80-kDa protein heterodimer which binds to terminal regions of double-stranded DNA. In the present study, we isolated full-length cDNAs that encode the 80-kDa Ku subunit.

Cited by 145 publications

References 21 publications

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Ku80 as a Novel Receptor for Thymosin β4 That Mediates Its Intracellular Activity Different from G-actin Sequestering

Defining the Minimal Domain of Ku80 for Interaction with Ku70

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