An Alternative Domain Containing a Leucine-rich Sequence Regulates Nuclear Cytoplasmic Localization of Protein 4.1R

Luque, Carlos M.; Pérez-Ferreiro, Carmen M.; Pérez-González, Alicia; Englmeier, Ludwig; Koffa, Maria; Correas, Isabel

doi:10.1074/jbc.m201521200

Cited by 18 publications

(15 citation statements)

References 67 publications

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“…Actin contains two nuclear export signals but no nuclear localization signal, and thus it is unclear how actin becomes nuclear. Several 4.1 family members have actinbinding domains, and 4.1R has sites regulating nuclear localization distinct from its actin-binding site (40)(41)(42). It is possible that one regulated means for actin to become nuclear is via 4.1 binding, a hypothesis to be tested in the future.…”

Section: Discussionmentioning

confidence: 99%

Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

Krauss

Chen

Penman

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Structural protein 4.1, which has crucial interactions within the spectrin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higherresolution detergent-extracted cell whole-mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical. W e are investigating functions of the protein 4.1 family in nuclei, centrosomes, and mitotic spindles in eukaryotic cells in relation to cell division (1-3). Protein 4.1 is a superfamily of multifunctional structural proteins widely expressed in nucleated cells (4), the prototypical member of which has well defined interactions with spectrin, actin, and integral membrane proteins in the human red cell membrane skeleton (reviewed by refs. 5 and 6). We recently showed that protein 4.1 is essential for proper assembly of functional nuclei in vitro in Xenopus egg extracts and identified the 4.1 spectrin-actin binding domain (SABD) as one of the 4.1 domains critical for this process. Furthermore, using a mutant 4.1 SABD unable to bind actin, we demonstrated that 4.1-actin binding capacity is necessary for nuclear reconstitution (3). These observations prompted us to investigate further the roles of actin in nuclear assembly.Actin is widely recognized as a major cytoskeletal component involved in dynamic processes such as cell motility and shape changes, cytoplasmic vesicle transport, and muscle contraction. Actin also was reported for several decades to be intranuclear, in particular to be associated with nuclear matrix (7-9), although many of these reports initially met with skepticism. Objections included possible cytoplasmic conta...

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Section: Discussionmentioning

confidence: 99%

Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

Krauss

Chen

Penman

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been reported that proteins containing an NLS often have casein kinase II sites 10 -30 amino acids from the NLS (Schmucker et al, 1999). Nuclear localization signals have also been reported for 4.1R (Correas et al, 2001;Luque et al, 2003) and NF2 (Schmucker et al, 1999).…”

Section: Identification Of Exon-intron Boundaries Of Dal-1/41bmentioning

confidence: 92%

Allele‐specific loss of heterozygosity at the DAL‐1/4.1B (EPB41L3) tumor‐suppressor gene locus in the absence of mutation

Kittiniyom

Mastronardi

Roemer

et al. 2004

Genes Chromosomes & Cancer

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DAL-1/4.1B (EPB41L3)is a member of the protein 4.1 superfamily, which encompasses structural proteins that play important roles in membrane processes via interactions with actin, spectrin, and the cytoplasmic domains of integral membrane proteins. DAL-1/4.1B localizes within chromosomal region 18p11.3, which is affected by loss of heterozygosity (LOH) in various adult tumors. Reintroduction of this protein into DAL-1/4.1B-null lung and breast tumor cell lines significantly reduced the number of cells, providing functional evidence that this protein possesses a growth suppressor function not confined to a single cell type. For characterization of the mutational mechanisms responsible for loss of DAL-1/4.1B function in tumors, the exon-intron structure of DAL-1/4.1B was examined for mutations in 15 normal/tumor pairs of non-small cell lung carcinoma by single-strand conformation polymorphism analysis. These studies revealed that small intragenic mutations are uncommon in DAL-1/4.1B. Furthermore, LOH analysis on 129 informative early-stage breast tumors utilizing a new intragenic C/T single-nucleotide polymorphism in exon 14 revealed that LOH resulted in preferential retention of the C-containing allele, suggesting that allele-specific loss is occurring. These studies indicate that mechanisms such as imprinting or monoallelic expression in combination with loss of heterozygosity may be responsible for loss of the DAL-1/4.1B protein in early breast disease.

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“…(Luque and Correas, 2000;Luque et al, 1999). The 4.1R 60 ∆16,18-GFP cDNA was constructed as detailed elsewhere (Luque et al, 2003). The glutathione-S-transferase (GST), GST C-terminus (GST-Cter), 60 ∆16,18 and GST-4.1R 80 ∆16 proteins were prepared as described elsewhere (Perez-Ferreiro et al, 2001).…”

Section: Methodsmentioning

confidence: 99%

Protein 4.1R regulates interphase microtubule organization at the centrosome

Pérez-Ferreiro

Vernos

Correas

2004

Journal of Cell Science

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In human red blood cells, protein 4.1 (4.1R) stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles of 4.1R in nonerythroid cells, we analysed the effect of ectopic expression of 4.1R isoforms on interphase microtubules in fibroblastic cells. We found that specific 4.1R isoforms disturbed the microtubule architecture but not the actin cytoskeleton. Biochemical sedimentation and/or confocal microscopy analyses showed that the pericentriolar components γ-tubulin and pericentrin remained at centrosomes, whereas the distributions of proteins p150Glued and the dynein intermediate chain were altered. Remarkably, 4.1R was displaced from the centrosome. In microtubule depolymerizing-repolymerizing assays, 4.1R-transfected cells showed an ability to depolymerize and nucleate microtubules that was similar to that of untransfected cells; however, microtubules became disorganized soon after regrowth. In microtubule-depolymerized transfected cells and during the initial steps of microtubule regrowth, centrosomal 4.1R localized with γ-tubulin but did not when microtubules became disorganized. To learn more about centrosomal 4.1R function, isolated centrosomes were examined by confocal microscopy, western blot and in vitro microtubule aster-assembly assays. The experiments showed that 4.1R was present in isolated centrosome preparations, that it remained in the center of in-vitro-assembled microtubule asters and that more asters were assembled by the addition of protein 4.1R fused to glutathione-S-transferase. Together, these results indicate that 4.1R plays a key role at the centrosome, contributing to the maintenance of a radial microtubule organization.

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An Alternative Domain Containing a Leucine-rich Sequence Regulates Nuclear Cytoplasmic Localization of Protein 4.1R

Cited by 18 publications

References 67 publications

Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

Allele‐specific loss of heterozygosity at the DAL‐1/4.1B (EPB41L3) tumor‐suppressor gene locus in the absence of mutation

Protein 4.1R regulates interphase microtubule organization at the centrosome

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