Two wobble-splicing events affect ING4 protein subnuclear localization and degradation

ING4 (inhibitor of growth 4) is a candidate tumor suppressor gene that is implicated as a repressor of cell growth, angiogenesis, cell spreading and cell migration and can suppress loss of contact inhibition in vitro. Another group and we identified four wobble-splicing isoforms of ING4 generated by alternative splicing at two tandem splice sites, GC(N)₇GT and NAGNAG, which caused canonical (GT-AG) and non-canonical (GC-AG) splice site wobbling selection. Expression of the four ING4 wobble-splicing isoforms did not vary significantly in any of the cell lines examined. Here we show that ING4_v1 is translocated to the nucleolus, indicating that ING4 contains an intrinsic nucleolar localization signal. We further demonstrate that the subcellular localization of ING4 is modulated by two wobble-splicing events at the exon 4-5 boundary, causing displacement from the nucleolus to the nucleus. We also observed that ING4 is degraded through the ubiquitin-proteasome pathway and that it is subjected to N-terminal ubiquitination. We demonstrate that nucleolar accumulation of ING4 prolongs its half-life, but lack of nucleolar targeting potentially increases ING4 degradation. Taken together, our data suggest that the two wobble-splicing events at the exon 4-5 boundary influence subnuclear localization and degradation of ING4.     

Characterization of nuclear localization and nuclear export signals of yeast actin-binding protein Pan1

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.     

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.     

灰盖拟鬼伞核定位蛋白重组表达系统的构建

为构建灰盖拟鬼伞Coprinopsis cinerea的核定位蛋白重组表达系统,本研究通过蛋白序列比对和信息学分析,预测了灰盖拟鬼伞组蛋白H2B的核定位序列,构建了融合组蛋白H2B核定位序列的绿色荧光蛋白(green fluorescent protein,GFP)重组表达载体,将该载体转入灰盖拟鬼伞AmutBmut菌...     

Sequence requirement for nuclear localization and growth inhibition of p27Kip1R,a degradation-resistant isoform of p27Kip1

p27(Kip1R) is an isoform of p27(Kip1), having a distinct C-terminus. The sequences of p27(Kip1R) required for nuclear localization and growth inhibition were determined in HeLa cells using a green fluorescence protein (GFP) as a reporter molecule. Region 153-168 and residues K168 and I169 were determined to play a critical role in the nuclear localization of p27(Kip1R). Aliphatic amino acid was found to be a substitute for the basic residue in the typical nuclear localization signal, while its functional substitution was incomplete, thereby causing a significant cytoplasmic retention of p27(Kip1R). p27(Kip1R) is thus the first example of an atypical bipartite nuclear localization signal with aliphatic amino acid as a functional residue. Despite cytoplasmic retention, p27(Kip1R) inhibited the cell growth as well as p27(Kip1), while GFP alone had no effect. The mutants lacking an N-terminus containing the binding regions for cyclins and cyclin-dependent kinases also showed a significant degree of nuclear localization, but failed to inhibit cell growth. The growth inhibition by p27(Kip1R) as well as p27(Kip1) was thus suggested to originate in the common N-terminal region.     

Stability of folding structure of Zic zinc finger proteins

Zic family proteins have five C₂H₂-type zinc finger (ZF) motifs. We physicochemically characterized the folding properties of Zic ZFs. Alteration of chelation with zinc ions and of hydrophobic interactions changed circular dichroism spectra, suggesting that they caused structural changes. The motifs were heat stable, but electrostatic interactions had little effect on structural stability. These results highlight the importance of chelating interactions and hydrophobic interactions for the stability of the folding structure of Zic ZF proteins.     

Nuclear transport of Ras-associated tumor suppressor proteins: different transport receptor binding specificities for arginine-rich nuclear targeting signals

Ras proteins regulate a wide range of biological processes by interacting with a variety of effector proteins. In addition to the known role in tumorigensis, the activated form of Ras exhibits growth-inhibitory effects by unknown mechanisms. Several Ras effector proteins identified as mediators of apoptosis and cell-cycle arrest also exhibit properties normally associated with tumor suppressor proteins. Here, we show that Ras effector RASSF5/NORE-1 binds strongly to K-Ras but weakly to both N-Ras and H-Ras. RASSF5 was found to localize both in the nucleus and the nucleolus in contrast to other Ras effector proteins, RASSF1C and RASSF2, which are localized in the nucleus and excluded from nucleolus. A 50 amino acid residue transferable arginine-rich nucleolar localization signal (NoLS) identified in RASSF5 is capable of interacting with importin-beta and transporting the cargo into the nucleolus. Surprisingly, similar arginine-rich signals identified in RASSF1C and RASSF2 interact with importin-alpha and transport the heterologous cytoplasmic proteins to the nucleus. Interestingly, mutation of arginine residues within these nuclear targeting signals prevented interaction of Ras effector proteins with respective transport receptors and abolished their nuclear translocation. These results provide evidence for the first time that arginine-rich signals are able to recognize different nuclear import receptors and transport the RASSF proteins into distinct sub-cellular compartments. In addition, our data suggest that the nuclear localization of RASSF5 is critical for its cell growth control activity. Together, these data suggest that the transport of Ras effector superfamily proteins into the nucleus/nucleolus may play a vital role in modulating Ras-mediated cell proliferation during tumorigenesis.     

Characterization of a nuclear compartment shared by nuclear bodies applying ectopic protein expression and correlative light and electron microscopy

To investigate the accessibility of interphase nuclei for nuclear body-sized particles, we analyzed in cultured cells from human origin by correlative fluorescence and electron microscopy (EM) the bundle-formation of Xenopus-vimentin targeted to the nucleus via a nuclear localization signal (NLS). Moreover, we investigated the spatial relationship of speckles, Cajal bodies, and crystalline particles formed by Mx1 fused to yellow fluorescent protein (YFP), with respect to these bundle arrays. At 37 degrees C, the nucleus-targeted, temperature-sensitive Xenopus vimentin was deposited in focal accumulations. Upon shift to 28 degrees C, polymerization was induced and filament arrays became visible. Within 2 h after temperature shift, arrays were found to be composed of filaments loosely embedded in the nucleoplasm. The filaments were restricted to limited areas of the nucleus between focal accumulations. Upon incubation at 28 degrees C for several hours, NLS vimentin filaments formed bundles looping throughout the nuclei. Speckles and Cajal bodies frequently localized in direct neighborhood to vimentin bundles. Similarly, small crystalline particles formed by YFP-tagged Mx1 also located next to vimentin bundles. Taking into account that nuclear targeted vimentin locates in the interchromosomal domain (ICD), we conclude that nuclear body-sized particles share a common nuclear space which is controlled by higher order chromatin organization.     

Acidic domain is indispensable for MDM2 to negatively regulate the acetylation of p53

MDM2 is the most important negative regulator of tumor suppressor p53. Both RING finger domain and acidic domain of MDM2 contribute to the ubiquitination of p53. The crosstalk between ubiquitination and acetylation of p53 prompts us to examine whether acidic domain is essential for MDM2 to regulate the acetylation of p53. We find that the acidic domain of MDM2 is necessary to inhibit p300-mediated acetylation of p53 as well as to mediate the deacetylation of p53. Our results indicate that acidic domain of MDM2 provides essential information for acetyltransferase p300 and deacetylase HDAC1 and is indispensable for MDM2 to negatively regulate the acetylation of p53.     

Characterization of the nuclear localization signal of the mouse TET3 protein

DNA demethylation is associated with gene activation and is mediated by a family of ten-eleven translocation (TET) dioxygenase. The TET3 protein is a 1668-amino-acid DNA demethylase that is predicted to possess five nuclear localization signals (NLSs). In this paper, we used a series of green fluorescent protein-tagged and mutation constructs to identify a conserved NLS (KKRK) embedded between amino acid 1615 and 1618 of mouse TET3. The KKRK sequence facilitates the cytoplasmic protein’s translocation into the nucleus. Additionally TET3 may be imported into the nucleus by importin-α and importin-β.