Activation-Induced Cytidine Deaminase Alters the Subcellular Localization of Tet Family Proteins

Activation-induced cytidine deminase (Aid), a unique enzyme that deaminates cytosine in DNA, shuttles between the nucleus and the cytoplasm. A recent study proposed a novel function of Aid in active DNA demethylation via deamination of 5-hydroxymethylcytosine, which is converted from 5-methylcytosine by the Ten-eleven translocation (Tet) family of enzymes. In this study, we examined the effect of simultaneous expression of Aid and Tet family proteins on the subcellular localization of each protein. We found that overexpressed Aid is mainly localized in the cytoplasm, whereas Tet1 and Tet2 are localized in the nucleus, and Tet3 is localized in both the cytoplasm and the nucleus. However, nuclear Tet proteins were gradually translocated to the cytoplasm when co-expressed with Aid. We also show that Aid-mediated translocation of Tet proteins is associated with Aid shuttling. Here we propose a possible role for Aid as a regulator of the subcellular localization of Tet family proteins.     

Nuclear shuttling of mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase (ERK) 2) was dynamically controlled by MAP/ERK kinase after antigen stimulation in RBL-2H3 cells

The mitogen-activated protein kinase (MAPK) cascade consists of the MAPK (extracellular signal-regulated kinase 2; ERK2) and its activator, MAPK kinase (MAP/ERK kinase; MEK). However, the mechanisms for activation of ERK2 have not been defined yet in cells. Here, we used fluorescent protein-tagged ERK2 and MEK to examine the localization of ERK2 and MEK in living rat basophilic leukemia (RBL-2H3) cells. ERK2 was mainly in the cytoplasm in resting cells but translocated into the nucleus after the ligation of IgE receptors. The import of ERK2 reached the maximum at 6--7 min, and then the imported ERK2 was exported from the nucleus. MEK mainly resided in the cytoplasm, and no significant MEK translocation was detected statically after ligation of IgE receptors. However, analysis of the dynamics of ERK2 and MEK suggested that both of them rapidly shuttle between the cytoplasm and the nucleus and that MEK regulates the nuclear shuttling of ERK2, whereas MEK remains mainly in the cytoplasm. In addition, the data suggested that the sustained calcium increase was required for the optimal translocation of ERK2 into the nucleus in RBL-2H3 cells. These results gave a new insight of the dynamics of ERK2 and MEK in the nuclear shuttling of RBL-2H3 cells after the ligation of IgE receptors.     

Stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70

Heat shock proteins of the hsp/hsc70 family are essential chaperones, implicated in the stress response, aging, and a growing number of human diseases. At the molecular level, hsc70s are required for the proper folding and intracellular targeting of polypeptides as well as the regulation of apoptosis. Cytoplasmic members of the hsp/hsc70 family are believed to shuttle between nuclei and cytoplasm; they are found in both compartments of unstressed cells. Our experiments demonstrate that actin filament-destabilizing drugs trigger the nuclear accumulation of hsc70s in unstressed and heat-shocked cells recovering from stress. Using human-mouse heterokaryons, we show that stress inhibits shuttling and sequesters the chaperone in nuclei. The inhibition of hsc70 shuttling upon heat shock is only transient, and transport is reestablished when cells recover from stress. Hsc70 shuttling is controlled by hsc70 retention in the nucleus, a process that is mediated by two distinct mechanisms, ATP-sensitive binding of hsc70s to chaperone substrates and, furthermore, the association with nucleoli. The nucleolar protein fibrillarin and ribosomal protein rpS6 were identified as components that show an increased association with hsc70s in the nucleus upon stress exposure. Together, our data suggest that stress abolishes the exit of hsc70s from the nucleus to the cytoplasm, thereby limiting their function to the nuclear compartment. We propose that during recovery from stress hsc70s are released from nuclear and nucleolar anchors, which is a prerequisite to restore shuttling. nuclear transport; chaperone; nuclear retention; nucleoli     

Proteins in nucleocytoplasmic interactions. 3. Redistributions of nuclear proteins during and following mitosis in Amoeba proteus

The behavior of nuclear proteins in Amoeba proteus was studied by tritiated amino acid labeling, nuclear transplantation, and cytoplasmic amputation. During prophase at least 77% (but probably over 95%) of the nuclear proteins is released to the cytoplasm. These same proteins return to the nucleus within the first 3 hr of interphase. When cytoplasm is amputated from an ameba in mitosis (shen the nuclear proteins are in the cytoplasm), the resultant daughter nuclei are depleted in the labeled nuclear proteins. The degree of depletion is less than proportional to the amount of cytoplasm removed because a portion of rapidly migrating protein (a nuclear protein that is normally shuttling between nucleus and cytoplasm and is thus also present in the cytoplasm) which would normally remain in the cytoplasm is taken up by the reconstituting daughter nuclei. Cytoplasmic fragments cut from mitotic cells are enriched in both major classes of nuclear proteins, i.e. rapidly migrating protein and slow turn-over protein. An interphase nucleus implanted into such an enucleated cell acquires from the cytoplasm essentially all of the excess nuclear proteins of both classes. The data indicate that there is a lack of binding sites in the cytoplasm for the rapidly migrating nuclear protein. The quantitative aspects of the distribution of rapidly migrating protein between the nucleus and the cytoplasm indicate that the distribution is governed primarily by factors within the nucleus.     

Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

Nuclear factor kappaB (NF-kappaB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-kappaB is controlled through its subcellular localization. Inactive NF-kappaB is sequestered in the cytoplasm by physical interaction with an inhibitor, IkappaBalpha. Signal-mediated IkappaBalpha degradation triggers the release and subsequent nuclear translocation of NF-kappaB. It remains unknown whether the NF-kappaB shuttling between the cytoplasm and nucleus is subjected to additional steps of regulation. In this study, we demonstrated that the RelA subunit of NF-kappaB exhibits strong cytoplasmic localization activity even in the absence of IkappaBalpha inhibition. The cytoplasmic distribution of RelA is largely mediated by a leucine-rich sequence homologous to the recently characterized nuclear export signal (NES). This putative NES is both required and sufficient to mediate cytoplasmic localization of RelA as well as that of heterologous proteins. Furthermore, the cytoplasmic distribution of RelA is sensitive to a nuclear export inhibitor, leptomycin B, suggesting that RelA undergoes continuous nuclear export. Interestingly, expression of p50 prevents the cytoplasmic expression of RelA, leading to the nuclear accumulation of both RelA and p50. Together, these results suggest that the nuclear and cytoplasmic shuttling of RelA is regulated by both an intrinsic NES-like sequence and the p50 subunit of NF-kappaB.