Nuclear Localization of Chfr Is Crucial for Its Checkpoint Function

권영은,Ye Seul Kim,Young Mi Oh,Jae Hong Seol 한국분자세포생물학회 2009 Molecules and cells Vol.27 No.3

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

Nuclear Localization Signal in Human hnRNP L

Lee, So-Young,Lee, Hyune-Hwan,Choi, Mieyoung 한국유전학회 2002 Genes & Genomics Vol.24 No.4

The heterogeneous nuclear ribonucleoprotein (hnRNP) L is an abundant nuclear protein and is one of the major pre-mRNA binding proteins in human cells. The amino acid sequence of hnRNP L contains four loosely conserved RNP-concensus RNA-binding domains. In previous report, it was shown that the amino terminal 140 amino acids of the hnRNP L were necessary and sufficient for nuclear localization. In order to define the minimal region of NLS in hnRNP L, a series of amino terminal and carboxy terminal deletions of hnRNP L were fused to the 3' end of the myc epitope-tagged chicken muscle pyruvate kinase (PK) cDNA. The subcellular distribution of transiently expressed polypeptides was examined by immunofluorescence microscopy. Here we report that the nuclear localization signal (NLS) sequence of hnRNP L protein consists of 24-GRAPKRLKT-32 sequences and seems to be a member of classical NLS, the single basic domain. The construct with amino terminal 23 amino acids deletion is still able to confer complete nuclear localization onto PK, a cytoplasmic reporter protein. However, the shorter construct in which amino terminal 35 amino acids were deleted completely lost the capability of targeting of cytoplasmic PK to the nucleus.

The molecular mechanism for nuclear transport and its application

Yun Hak Kim,Myoung-Eun Han,Sae-Ock Oh 대한해부학회 2017 Anatomy & Cell Biology Vol.50 No.2

Transportation between the cytoplasm and the nucleoplasm is critical for many physiological and pathophysiological processes including gene expression, signal transduction, and oncogenesis. So, the molecular mechanism for the transportation needs to be studied not only to understand cell physiological processes but also to develop new diagnostic and therapeutic targets. Recent progress in the research of the nuclear transportation (import and export) via nuclear pore complex and four important factors affecting nuclear transport (nucleoporins, Ran, karyopherins, and nuclear localization signals/nuclear export signals) will be discussed. Moreover, the clinical significance of nuclear transport and its application will be reviewed. This review will provide some critical insight for the molecular design of therapeutics which need to be targeted inside the nucleus.

The nuclear localization of Yes Associated Protein (YAP) through the association with Karyopherin β2 (Kap β2)

Sung Hwa Shin, Eun Jeoung Lee, Sunghee Hyun Dowonkyoung Park, Sang Sun Kang 충북대학교 과학교육연구소 2017 과학교육연구논총 Vol.32 No.2

Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) are both found in the cytoplasm as well as the nucleus, where they regulate gene transcriptions. YAP/TAZ nuclear localization is a crucial dterminant of their function. Phosphorylation by protein kinases is another major factor responsible for the YAP/TAZ subcellular localization, resulting in retention of YAP/TAZ in the cytoplasm. However, it is still nuclear as to how YAP is transported into toe nucleus. Using site directed mutagenesis and confocal microscopic analysis, we demonstrated in this study that the motif(lqmekerlrlkqqellrqamrninpstanspkcqelalrsqlpt)inYAP plays a role as the putative PY-NLS to which Karyopherin β2 (Kapβ2) bind to facilitate its nuclear localization. Thus, our observations have provided new clues to understand YAP regulating in Hippo signal transduction pathway by Kapβ2 binding.

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor

Do, Hyun-Jin,Song, Hyuk,Yang, Heung-Mo,Kim, Dong-Ku,Kim, Nam-Hyung,Kim, Jin-Hoi,Cha, Kwang-Yul,Chung, Hyung-Min,Kim, Jae-Hwan Elsevier 2006 FEBS letters Vol.580 No.7

<P><B>Abstract</B></P><P>We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (<SUP>244</SUP>KRKR<SUP>247</SUP>) were critical for strong NLS activity, and two potent bipartite NLS motifs (236–252 and 249–267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (<SUP>318</SUP>KKK<SUP>320</SUP>) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.</P>

The hydrophobic amino acids involved in the interdomain association of phospholipase D1 regulate the shuttling of phospholipase D1 from vesicular organelles into the nucleus

장영훈,민도식 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.10

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-β, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the ydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.

A Conserved Motif Controls Nuclear Localization of Drosophila Muscleblind

Fernandez-Costa, Juan M.,Artero, Ruben Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.30 No.1

Human Muscleblind-like proteins are alternative splicing regulators that are functionally altered in the RNA-mediated disease myotonic dystrophy. There are different Muscleblind protein isoforms in Drosophila and we previously determined that these have different subcellular localizations in the COS-M6 cell line. Here, we describe the conservation of the sequence motif KRAEK in isoforms C and E and propose a specific function for this motif. Different Muscleblind isoforms localize to the peri-plasma membrane (MblA), cytoplasm (MblB), or show no preference for the nuclear or cytoplasmic compartment (MblC and MblD) in Drosophila S2 cells transiently transfected with Musclebind expression plasmids. Mutation of the KRAEK motif reduces MblC nuclear localization, whereas fusion of a single KRAEK motif to the heterologous protein ${\beta}$-galactosidase is sufficient to target the reporter protein to the nucleus of S2 cells. This motif is not exclusive to Muscleblind proteins and is detected in several other protein types. Taken together, these results suggest that the KRAEK motif regulates nuclear translocation of Muscleblind and may constitute a new class of nuclear localization signal.

A Conserved Motif Controls Nuclear Localization of Drosophila Muscleblind

Juan M. Fernandez-Costa,Ruben Artero 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.1

Human Muscleblind-like proteins are alternative splicing regulators that are functionally altered in the RNA-mediated disease myotonic dystrophy. There are different Muscleblind protein isoforms in Drosophila and we previously determined that these have different subcellular localizations in the COS-M6 cell line. Here, we describe the conservation of the sequence motif KRAEK in isoforms C and E and propose a specific function for this motif. Different Muscleblind isoforms localize to the peri-plasma membrane (MblA), cytoplasm (MblB), or show no preference for the nuclear or cytoplasmic compartment (MblC and MblD)in Drosophila S2 cells transiently transfected with Musclebind expression plasmids. Mutation of the KRAEK motif reduces MblC nuclear localization, whereas fusion of a single KRAEK motif to the heterologous protein β-galactosidase is sufficient to target the reporter protein to the nucleus of S2 cells. This motif is not exclusive to Muscleblind proteins and is detected in several other protein types. Taken together, these results suggest that the KRAEK motif regulates nuclear translocation of Muscleblind and may constitute a new class of nuclear localization signal.

Four Consecutive Arginine Residues at Positions 836-839 of EBV gp110 Determine Intracellular Localization of gp110

Lee, Suk-Kyeong 가톨릭대학교 2000 Bulletin of The Catholic Research Institutes of Me Vol.28 No.-

Epstein-Barr virus (EBV) glycoprotein 110 (GP110) has sequence homology with herpes simplex virus-1 (HSV-1) gB, however the role of gp110 in EBVs life cycle differs from that of gB. Unlike HSV-1 gB, which is essential for HSV-1 infection but dispensable for virus production, gp110 is required for assembly and egress of EBV. EBV gp110 is found mainly in the endoplasmic reticulum (ER)/nuclear membrane, while little or no gp110 is detected in the plasma membrane or a mature viral particle. Conversely, HSV-1 gB is abundant in the envelope of mature virions and in the plasma membrane as well as in the ER/nuclear membrane of HSV-1 infected cells. Interestingly, there are four consecutive arginine residues (at positions 836-839 of gp110) in the C-terminal domain previously shown to be important for gp110's intracellular localization. To determine if these arginines function as an ER/nuclear localization signal, point mutants were constructed differentially substituting the four arginines. The glycosylation pattern and intracellular localization of the mutants were investigated by assessing sensitivity to endoglycosidase H (endo H) digestion and performing indirect immunofluorscence assays. Substitution of part of the four arginines changed the glycosylation profile and targeting of gp110. In addition, mutations preserving the net charge of the four arginines as well as those causing net charge shift resulted in the changed intracellular localization and altered glycosylation pattern. These results suggest that not only the net charge but also the conformation of the four arginines are important for gp110's processing and sub-cellular localization. (Virology 264:650-658, 1999)

Nuclear Localization Signals in Prototype Foamy Viral Integrase for Successive Infection and Replication in Dividing Cells

Hossain, Md. Alamgir,Ali, Md. Khadem,Shin, Cha-Gyun Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.2

We identified four basic amino acid residues as nuclear localization signals (NLS) in the C-terminal domain of the prototype foamy viral (PFV) integrase (IN) protein that were essential for viral replication. We constructed seven point mutants in the C-terminal domain by changing the lysine and arginine at residues 305, 308, 313, 315, 318, 324, and 329 to threonine or proline, respectively, to identify residues conferring NLS activity. Our results showed that mutation of these residues had no effect on expression assembly, release of viral particles, or in vitro recombinant IN enzymatic activity. However, mutations at residues 305 (R ${\rightarrow}$ T), 313(R ${\rightarrow}$ T), 315(R ${\rightarrow}$ P), and 329(R ${\rightarrow}$ T) lead to the production of defective viral particles with loss of infectivity, whereas non-defective mutations at residues 308(R ${\rightarrow}$ T), 318(K ${\rightarrow}$ T), and 324(K ${\rightarrow}$ T) did not show any adverse effects on subsequent production or release of viral particles. Sub-cellular fractionation and immunostaining for viral protein PFV-IN and PFV-Gag localization revealed predominant cytoplasmic localization of PFV-IN in defective mutants, whereas cytoplasmic and nuclear localization of PFV-IN was observed in wild type and non-defective mutants. However sub-cellular localization of PFV-Gag resulted in predominant nuclear localization and less presence in the cytoplasm of the wild type and non-defective mutants. But defective mutants showed only nuclear localization of Gag. Therefore, we postulate that four basic arginine residues at 305, 313, 315 and 329 confer the karyoplilic properties of PFV-IN and are essential for successful viral integration and replication.