Regulation Of Intracellular Localization Research Articles

The N-terminal intrinsically disordered region mediates intracellular localization and self-oligomerization of ALS2

ALS2, a product of the causative gene for familial amyotrophic lateral sclerosis (ALS) type 2, plays a pivotal role in the regulation of endosome dynamics by activating small GTPase Rab5 via its intrinsic guanine nucleotide-exchange factor activity. Previously, we have reported that the N-terminal region of ALS2 has crucial roles in its endosomal localization and self-oligomerization, both of which are indispensable for the cellular function of ALS2. The N-terminus of ALS2 contains the regulator of chromosome condensation 1-like domain (RLD), which is predicted to form a seven-bladed β-propeller structure. Interestingly, the RLD is interrupted by the intrinsically disordered region (IDR), within which there are several amino acid residues which undergo phosphorylation. In this study, we sought to investigate as to whether and how the IDR as well as phosphorylation at either Ser483, Ser492 or Thr510 affect the intracellular localization and self-oligomerization of ALS2. All phospho- and dephospho-mimetic ALS2 mutants that were transiently expressed in HeLa cells were diffusely distributed throughout the cytosol with a partial localization to early endosomes. When expressed under Rac1-activating conditions, these mutants were localized to membrane ruffles as well as enlarged endosomes. Further, gel-filtration analysis revealed that these mutants primarily existed as a tetramer in cells. However, all these phenotypes were indistinguishable from those of wild-type ALS2. On the other hand, IDR-deleted ALS2 mutant was exclusively present in perinuclear aggregates colocalizing with the autophagy-related protein SQSTM1. Moreover, IDR-deleted ALS2 mutant formed an abnormally high molecular weight complex compared to wild-type ALS2. These results indicate that the IDR of ALS2 plays a crucial role not only in the regulation of intracellular localization but also in the self-oligomerization of ALS2 in cells, whereas phosphorylation of certain residues within the IDR exerts limited effects on such phenotypes.

Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

AbstractCationic guanidinium and phosphonium functionalized cytosine morpholino tetramer (G3) and trimer (P3) have been reported for the first time. They show efficient cellular uptake properties in full growth medium, quantified by fluorescence activated cell sorting (FACS) and visualized by fluorescence microscopy. An interesting feature of these transporters is their localization in the nucleolus or the rest of nucleus, determined by the nature of cationic moieties attached with cytosine base. Co‐staining with MitoTracker Deep Red depicts their ability to penetrate mitochondrial membrane. Fluorescence imaging displays their uniform distribution throughout zebrafish larvae with no significant toxicity. FACS analysis at 4°C indicated that transfection of G3 transporter is energy dependent whereas P3 is energy independent. When covalently conjugated, G3 is capable of transporting a 25‐mer phosphorodiamidate morpholino oligomer (PMO) into the cells. It exhibits antisense effect against Gli1 gene in hedgehog pathway, confirmed in preliminary β‐glactosidase assay performed in Ptch1–/– cells. Further antisense effect was confirmed when G3‐conjugated zebrafish Gli1‐PMO was injected into embryo to obtain Gli1‐dependent phenotypes. To the best of our knowledge, herein, we first report that molecular transporters with a minimum number of three cationic groups are capable of efficiently passing through plasma, nuclear and mitochondrial membranes and show distribution in zebrafish larvae.

Decreased TIP30 promotes Snail-mediated epithelial–mesenchymal transition and tumor-initiating properties in hepatocellular carcinoma

The poor prognosis of hepatocellular carcinoma (HCC) is mainly due to tumor recurrence and metastases. Recently, epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis. However, the underlying molecular mechanisms are yet to be elucidated. Here, we show that 30-kDa Tat-interacting protein (TIP30), also called CC3, is significantly downregulated during transforming growth factor-β-induced EMT. In our in vitro and in vivo studies, we show that decreased TIP30 expression leads to EMT, as well as enhanced motility and invasion of HCC cells. Also, increased self-renewal ability and chemotherapeutic resistance are observed with TIP30 depletion. Moreover, Snail is one of the key transcription factors promoting EMT, and overexpression of TIP30 greatly decreased nucleic accumulation in Snail through the regulation of intracellular localization. Small interfering RNAs targeting Snail attenuated EMT and tumor-initiating properties induced by TIP30 deficiency. We further confirmed that TIP30 competitively interrupted the interaction of Snail with importin-β2 to block the nuclear import of Snail. Consistently, TIP30 expression significantly correlates with E-cadherin expression in HCC patients. TIP30 or combination of E-cadherin is a powerful marker in predicting the prognosis of HCC. Taken together, our results suggest a novel and critical role of TIP30 involved in HCC progression and aggressiveness.

Regulation of Intracellular Localization and Transcriptional Activity of FOXO4 by Protein Kinase B through Phosphorylation at the Motif Sites Conserved among the FOXO Family

FOXO4 transcription factor, also referred to AFX, contains three putative phosphorylation motif sites for protein kinase B (PKB), Thr32, Ser197, and Ser262, and it is proposed that phosphorylated FOXO4 stays in the cytosol and is imported to the nucleus through dephosphorylation to induce target gene expression. These three sites were revealed to be phosphorylated by PKB in vitro on phosphopeptide analysis, and in cultured cells on immunoblotting with phosphorylation-site specific antibodies. The mutants with either Thr32 or Ser197 replaced by Ala were found mostly in the nuclear but not the cytosol fraction, and treatment with platelet-derived growth factor did not change their distributions in the cells. FOXO4 proteins mutated at these two sites showed 3- to 5-fold higher transcriptional activity than that of the wild type. In contrast, the replacement of Ser262 did not alter the localization or transcriptional activity. These results indicate that phosphorylation at Thr32 and Ser197 is indispensable, whereas that at Ser262 is not critical, for regulation of the nuclear localization and transcriptional activity of FOXO4. These properties are similar to those of FOXO1 and FOXO3, and thus FOXO transcription factors seem to be regulated through a common mechanism by PKB in the growth factor signaling pathway.

Methylation of Xenopus CIRP2 regulates its arginine- and glycine-rich region-mediated nucleocytoplasmic distribution.

Cold-inducible RNA-binding protein (CIRP) was originally found in mammalian cells as a protein that is overexpressed upon a temperature downshift. Recently, we identified a Xenopus homolog of CIRP, termed xCIRP2, as a major cytoplasmic RNA-binding protein in oocytes. In this study we found by yeast two-hybrid screening that the Xenopus homolog of protein arginine N-methyltransferase 1 (xPRMT1) interacted with xCIRP2. We found that an arginine- and glycine-rich region of xCIRP2, termed the RG4 domain, was a target of xPRMT1 for methylation in vitro. xCIRP2 expressed in cultured cells accumulated in the nucleus as does mammalian CIRP. Interestingly, the RG4 domain was necessary for nuclear localization of xCIRP2. RG4-mediated nuclear accumulation of xCIRP2 was diminished in the presence of transcription inhibitors, suggesting that nuclear localization of xCIRP2 was dependent on ongoing transcription with RNA polymerase II. Analysis of interspecies heterokaryons revealed that xCIRP2 was capable of nucleocytoplasmic shuttling and the RG4 domain functioned as a nucleocytoplasmic shuttling signal. Methylation by overexpressed xPRMT1 caused cytoplasmic accumulation of xCIRP2. Possible implications of the relationship between regulation of intracellular localization and multiple functions of xCIRP2 will be discussed.