Characterization of the Interaction between Protein 4.1R and ZO-2

In red blood cells, protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. The picture is more complex in nucleated cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R, we have analyzed the role the exon 5-encoded sequence plays in 4.1R distribution. We show that exon 5 encodes a leucine-rich sequence that shares key features with nuclear export signals (NESs). This sequence adopts the topology employed for NESs of other proteins and conserves two hydrophobic residues that are shown to be critical for NES function. A 4.1R isoform expressing the leucine-rich sequence binds to the export receptor CRM1 in a RanGTP-dependent fashion, whereas this does not occur in a mutant whose two conserved hydrophobic residues are substituted. These two residues are also essential for 4.1R intracellular distribution, because the 4.1R protein containing the leucine-rich sequence localizes in the cytoplasm, whereas the mutant protein predominantly accumulates in the nucleus. We hypothesize that the leucine-rich sequence in 4.1R controls distribution and concomitantly function of a specific set of 4.1R isoforms.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Luque¹,

Pérez-Ferreiro²,

Pérez-González³

et al. 2003

“…4.1R isoforms appear to be critical components of many important supramolecular complexes. 4.1R isoforms have been shown to interact with interphase microtubules in human T cells (13), the components of the contractile apparatus in skeletal myofibers (14), and the tight junction proteins in epithelial cells (15). 4.1R isoforms have also been shown to be the components of the nuclear matrix (16,17) and may be involved in splicing processes (18).…”

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confidence: 99%

Protein 4.1R, a Microtubule-associated Protein Involved in Microtubule Aster Assembly in Mammalian Mitotic Extract

Huang

Jagadeeswaran

Liu

et al. 2004

Self Cite

Non-erythroid protein 4.1R (4.1R) consists of a complex family of isoforms. We have shown that 4.1R isoforms localize at the mitotic spindle/spindle poles and associate in a complex with the mitotic-spindle organization proteins Nuclear Mitotic Apparatus protein (NuMA), dynein, and dynactin. We addressed the mitotic function of 4.1R by investigating its association with microtubules, the main component of the mitotic spindles, and its role in mitotic aster assembly in vitro. 4.1R appears to partially co-localize with microtubules throughout the mitotic stages of the cell cycle. In vitro sedimentation assays showed that 4.1R isoforms directly interact with microtubules. Glutathione S-transferase (GST) pull-down assays using GST-4.1R fusions and mitotic cell extracts further showed that the association of 4.1R with tubulin results from both the membrane-binding domain and C-terminal domain of 4.1R. Moreover, 4.1R, but not actin, is a mitotic microtubuleassociated protein; 4.1R associates with microtubules in the microtubule pellet of the mitotic asters assembled in mammalian cell-free mitotic extract. The organization of microtubules into asters depends on 4.1R in that immunodepletion of 4.1R from the extract resulted in randomly dispersed microtubules. Furthermore, adding a 135-kDa recombinant 4.1R reconstituted the mitotic asters. Finally, we demonstrated that a mitotic 4.1R isoform appears to form a complex in vivo with tubulin and NuMA in highly synchronized mitotic HeLa extracts. Our results suggest that a 135-kDa non-erythroid 4.1R is important to cell division, because it participates in the formation of mitotic spindles and spindle poles through its interaction with mitotic microtubules.

“…The divergence in the C termini of ZO-1 like MAGUKs suggests that these proteins may have significant functional differences. Furthermore, ZOPs not only interact with occludin, claudins, and junctional adhesion molecule but also associate with a variety of cytoplasmic proteins of less defined function, including the myosin-binding protein cingulin (24), the Ras effector AF-6 (25), and the erythrocyte actin-binding protein 4.1 (26).…”

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confidence: 99%

The Tight Junction Protein ZO-2 Localizes to the Nucleus and Interacts with the Heterogeneous Nuclear Ribonucleoprotein Scaffold Attachment Factor-B

Traweger¹,

Fuchs²,

Krizbai³

et al. 2003

141

120

Zonula occludens proteins (ZOPs), currently comprising ZO-1, ZO-2, and ZO-3, belong to the family of membrane-associated guanylate kinase homologue (MAGUK) proteins that are involved in the organization of epithelial and endothelial intercellular junctions. ZOPs bind to the cytoplasmic C termini of junctional transmembrane proteins linking them to the actin cytoskeleton. They are characterized by several conserved modules, including three PDZ domains, one SH3 domain, and a guanylate kinase-like domain, elements indicating that ZOPs may serve multiple purposes. Interestingly, ZOPs contain some unique motifs not shared by other MAGUK family members, including nuclear localization and nuclear export signals and a leucine zipper-like sequence. Their potential involvement in cell growth and proliferation has been suggested earlier based on the observation that the N-terminal half of ZOPs displays significant similarity to the product of the Drosophila tumor suppressor gene lethal(1)discslarge (dlg). The nuclear targeting of ZOPs in subconfluent epithelial cell cultures is well documented, although the action of the junctional MAGUKs in the nucleus has remained elusive. Here we show for the first time that nuclear ZO-2 directly interacts with the DNA-binding protein scaffold attachment factor-B (SAF-B). Our results from two-hybrid assays and in vivo co-immunoprecipitation studies provide evidence to suggest that ZO-2 associates with the C-terminal portion of SAF-B via its PDZ-1 domain. We further demonstrate that enhanced green fluorescent protein (EGFP)-and DsRed-tagged ZO-2 and SAF-B fusion proteins partially co-localize in nuclei of transfected epithelial cells. As shown by laser confocal microscopy and epifluorescent analysis, nuclear ZO-2 is present in epithelial and endothelial cells, particularly in response to environmental stress conditions. Interestingly, no association of SAF-B with ZO-1 was found, which supports the notion that junctional MAGUKs serve nonredundant functions. Tight intercellular contacts (tight junctions (TJs))1 are responsible for the "barrier" function of epithelia and endothelia restricting the paracellular transport of solutes and molecules across epithelial and endothelial cell sheets (1-5). TJs consist of several transmembrane components that interact with a network of "peripheral" cytoplasmic proteins. So far, three types of TJ-related transmembrane proteins: occludin, claudins, and junctional adhesion molecule, have been described, all of them associating with at least one of the Zonula occludens proteins (ZOPs) (for review see Refs. 6 -8). ZOPs, in turn, establish a link between the junction site and the cytoskeleton by interacting directly with actin filaments (9 -11). The observation that ZOPs not only associate with each other but also with components of adherens junctions and gap junctions in cells lacking TJs (12-14) suggests a universal role for ZOPs at the cytoplasmic surface of the junctional plaque.ZOPs are structurally similar to the membrane-associated guanylate kinase h...