The effect of hypusine modification on the intracellular localization of eIF5A

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential for eukaryotic cell proliferation and is the only protein containing hypusine, [N^ε-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine. eIF5A also undergoes an acetylation at specific Lys residue(s). In this study, we have investigated the effect of hypusine modification and acetylation on the subcellular localization of eIF5A. Immunocytochemical analyses showed differences in the distribution of non-hypusinated eIF5A precursor and the hypusine-containing mature eIF5A. While the precursor is found in both cytoplasm and nucleus, the hypusinated eIF5A is primarily localized in cytoplasm. eIF5A mutant proteins, defective in hypusine modification (K50A, K50R) were localized in a similar manner to the eIF5A precursor, whereas hypusine-modified mutant proteins (K47A, K47R, K68A) were localized mainly in the cytoplasm. These findings provide strong evidence that the hypusine modification of eIF5A dictates its localization in the cytoplasmic compartment where it is required for protein synthesis.     

Charge modification at multiple C-terminal lysine residues regulates p53 oligomerization and its nucleus-cytoplasm trafficking

The basal level of the tumor suppressor p53 is regulated by MDM2-mediated ubiquitination at specific lysines, which leads to p53 nuclear export and degradation. Upon p53 activation, however, these lysines become acetylated by p300/CREB-binding protein. Here we have reported an unexpected finding that p300-mediated acetylation also regulates p53 subcellular localization and can promote cytoplasmic localization of p53. This activity is independent of MDM2 but requires a p53 nuclear export signal and acetylation of multiple lysines by p300. Mechanistically, we showed that conversion of a minimal four of these lysines to alanines but not arginines mimics p300-mediated p53 nuclear export, and these lysine-neutralizing mutations effectively prevent p53 tetramerization, thus exposing the oligomerization-regulated nuclear export signal. Our study suggested a threshold mechanism whereby the degree of acetylation regulates p53 nucleus-cytoplasm trafficking by neutralizing a lysine-dependent charge patch, which in turn, controls oligomerization-dependent p53 nuclear export.     

核孔蛋白p62促进非受体酪氨酸激酶c-Abl进入细胞核

非受体酪氨酸激酶c-Abl广泛表达于各组织细胞中,其序列高度保守,它的亚细胞定位与其功能密切相关。c-Abl借助其C端的3个核定位信号（NLS）和1个核输出信号（NES）完成细胞核一细胞质问的穿梭过程。关于c-Abl核-质穿梭的详细机制还不清楚。通过酵母双杂交系统,以人类Ib型c-Abl作为诱饵蛋白进行HeLa细胞eDNA文库的筛选,获得了可能在c-Abl核-质穿梭过程中具有调控怍用核孔蛋白p62。核孔复合物（NPC）是大分子物质进行核-质运输的惟一通道,p62是NPC的重要组成部分,它位于中央通道内侧,在许多物质的核-质穿梭过程中具有调节作用。免疫共沉淀和体外结合实验证实,c-Abl和p62之间具有相互作用,而且这种相互作用是通过c-Abl的SH3结构域与p62的P299位点之间的结合实现的;p62可被c-Abl部分磷酸化此外,在293和DKO细胞株中共转染c-Abl和p62,发现核内的c-Abl分布增多。以上结果表明,p62具有促进e-Abl进入细咆核的作用。     

Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis

Besides its role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) initiates a cell death cascade. Diverse apoptotic stimuli activate inducible nitric oxide synthase (iNOS) or neuronal NOS (nNOS), with the generated nitric oxide (NO) S-nitrosylating GAPDH, abolishing its catalytic activity and conferring on it the ability to bind to Siah1, an E3-ubiquitin-ligase with a nuclear localization signal (NLS). The GAPDH-Siah1 protein complex, in turn, translocates to the nucleus and mediates cell death; these processes are blocked by procedures that interfere with GAPDH-Siah1 binding. Nuclear events induced by GAPDH to kill cells have been obscure. Here we show that nuclear GAPDH is acetylated at Lys 160 by the acetyltransferase p300/CREB binding protein (CBP) through direct protein interaction, which in turn stimulates the acetylation and catalytic activity of p300/CBP. Consequently, downstream targets of p300/CBP, such as p53 (Refs 10,11,12,13,14,15), are activated and cause cell death. A dominant-negative mutant GAPDH with the substitution of Lys 160 to Arg (GAPDH-K160R) prevents activation of p300/CBP, blocks induction of apoptotic genes and decreases cell death. Our findings reveal a pathway in which NO-induced nuclear GAPDH mediates cell death through p300/CBP.     

Subcellular localization of different regions of porcine Six1 gene and its expression analysis in C2C12 myoblasts

Six1 protein belongs to the Six homeoproteins family, exposing typical domain structure. Although the functions of Six1 have been drawn much attention, the roles of its individual domains are not completely elucidated. Here, we first detected the expression patterns of myogenin, MyoD, Myf5, and Six1 genes using real-time PCR in differentiating C2C12 cells cultured in differentiation medium for 2 or 6?days. The results showed that Six1 gene had the similar expression pattern with myogenin, MyoD, and Myf5 genes, which suggests that it may affect the myogenic differentiation. In order to evaluate the role of distinct domains of Six1 protein in subcellular localization, we constructed a series of truncated vectors tagged with green fluorescent proteins expressing various regions of porcine Six1 protein for subcellular localization analysis. Fluorescence confocal microscopy analysis showed that the different regions of Six1 protein displayed discrete distributions throughout the nucleus and the cytoplasm. The full-length CDS was exclusively localized in the nucleus and the individual HD domain was preferentially distributed to the nucleus both in C2C12 cells and in PK cells. However, the SD domain was diffusely distributed to the cytoplasm and the nucleus, and the localization of SD domain was biased to cytoplasm in C2C12 cells. Taken together, we conclude that the HD domain is important for the nuclear localization of porcine Six1 protein.     

MAP kinase subcellular localization controls both pattern and proliferation in the developing Drosophila wing

Mitogen-activated protein kinases (MAPKs) phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK phosphorylation was always followed by rapid nuclear translocation. However, we and others have found that MAPK phosphorylation is not always sufficient for nuclear translocation in vivo. In the developing Drosophila wing, MAPK-mediated signaling is required both for patterning and for cell proliferation, although the mechanism of this differential control is not fully understood. Here, we show that phosphorylated MAPK (pMAPK) is held in the cytoplasm in differentiating larval and pupal wing vein cells, and we show that this cytoplasmic hold is required for vein cell fate. At the same time, we show that MAPK does move into the nucleus of other wing cells where it promotes cell proliferation. We propose a novel Ras pathway bifurcation in Drosophila and our results suggest a mechanism by which MAPK phosphorylation can signal two different cellular outcomes (differentiation versus proliferation) based on the subcellular localization of MAPK.