Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.     

A novel nuclear export signal and a REF interaction domain both promote mRNA export by the Epstein-Barr virus EB2 protein

A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.     

A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export

XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1-Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.     

Data augmentation algorithms for detecting conserved domains in protein sequences: a comparative study

Protein conserved domains are distinct units of molecular structure, usually associated with particular aspects of molecular function such as catalysis or binding. These conserved subsequences are often unobserved and thus in need of detection. Motif discovery methods can be used to find these unobserved domains given a set of sequences. This paper presents the data augmentation (DA) framework that unifies a suite of motif-finding algorithms through maximizing the same likelihood function by imputing the unobserved data. The data augmentation refers to those methods that formulate iterative optimization by exploiting the unobserved data. Two categories of maximum likelihood based motif-finding algorithms are illustrated under the DA framework. The first is the deterministic algorithms that are to maximize the likelihood function by performing an iteratively optimal local search in the alignment space. The second is the stochastic algorithms that are to iteratively draw motif location samples via Monte Carlo simulation and simultaneously keep track of the superior solution with the best likelihood. As a result, four DA motif discovery algorithms are described, evaluated, and compared by aligning real and simulated protein sequences.     

The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

The lamin B receptor (LBR) is a polytopic protein of the inner nuclear membrane. It is synthesized without a cleavable amino-terminal signal sequence and composed of a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. To identify a nuclear envelope targeting signal, we have examined the cellular localization by immunofluorescence microscopy of chicken LBR, its amino-terminal domain and chimeric proteins transiently expressed in transfected COS-7. Full- length LBR was targeted to the nuclear envelope. The amino-terminal domain, without any transmembrane segments, was transported to the nucleus but excluded from the nucleolus. When the amino-terminal domain of LBR was fused to the amino-terminal side of a transmembrane segment of a type II integral membrane protein of the ER/plasma membrane, the chimeric protein was targeted to the nuclear envelope, likely the inner nuclear membrane. When the amino-terminal domain was deleted from LBR and replaced by alpha-globin, the chimeric protein was retained in the ER. These findings demonstrate that the amino-terminal domain of LBR is targeted to the nucleus after synthesis in the cytoplasm and that this polypeptide can function as a nuclear envelope targeting signal when located at the amino terminus of a type II integral membrane protein synthesized on the ER.     

Four and a half LIM domain protein signaling and cardiomyopathy

Four and a half LIM domain (FHL) protein family members, FHL1 and FHL2, are multifunctional proteins that are enriched in cardiac muscle. Although they both localize within the cardiomyocyte sarcomere (titin N2B), they have been shown to have important yet unique functions within the context of cardiac hypertrophy and disease. Studies in FHL1-deficient mice have primarily uncovered mitogen-activated protein kinase (MAPK) scaffolding functions for FHL1 as part of a novel biomechanical stretch sensor within the cardiomyocyte sarcomere, which acts as a positive regulator of pressure overload-mediated cardiac hypertrophy. New data have highlighted a novel role for the serine/threonine protein phosphatase (PP5) as a deactivator of the FHL1-based biomechanical stretch sensor, which has implications in not only cardiac hypertrophy but also heart failure. In contrast, studies in FHL2-deficient mice have primarily uncovered an opposing role for FHL2 as a negative regulator of adrenergic-mediated signaling and cardiac hypertrophy, further suggesting unique functions targeted by FHL proteins in the “stressed” cardiomyocyte. In this review, we provide current knowledge of the role of FHL1 and FHL2 in cardiac muscle as it relates to their actions in cardiac hypertrophy and cardiomyopathy. A specific focus will be to dissect the pathways and protein-protein interactions that underlie FHLs’ signaling role in cardiac hypertrophy as well as provide a comprehensive list of FHL mutations linked to cardiac disease, using evidence gained from genetic mouse models and human genetic studies.     

Molecular cloning of a novel ubiquitin-like protein, UBIN, that binds to ER targeting signal sequences

To identify proteins that interact with HSP47, an endoplasmic reticulum (ER)-resident molecular chaperone, a yeast two-hybrid screening was performed using mouse full-length HSP47 including an N-terminal signal sequence as a bait. Analysis of several positive clones led to the identification and cloning of a novel gene, ubin, encoding a ubiquitin-like protein. Unlike other ubiquitin-like proteins, UBIN was shown to interact with signal sequences of various secretory and ER-luminal proteins, including HSP47, but not interact with signal sequences of mitochondrial targeting in two-hybrid system. The possible function of UBIN will be discussed with regards to novel characteristics of binding to signal sequences for ER targeting.