hnRNPs H, H' and F behave differently with respect to posttranslational cleavage and subcellular localization.

hnRNPs H, H' and F belong to a subfamily of the hnRNPs sharing a high degree of sequence identity. Eukaryotic expression and specific C-terminal antibodies were used to demonstrate great variation in the intracellular fate of the proteins. hnRNPs H and H' become posttranslational cleaved into C-terminal 35 kDa proteins (H(C), H'(C)) and possibly into N-terminal 22 kDa proteins. No detectable cleavage was observed for hnRNP F. hnRNP H/H' is almost exclusively localized to the nucleus of many cell types while hnRNP F varies from a predominant nuclear localization in some cells to a predominant cytoplasmic localization in other cells. The different fates may reflect differences in functional roles that so far only have included nuclear functions. The presence of significant quantities of hnRNP F in the cytoplasm of many cells indicates that it also may have a functional role here.     

Endonuclease G mediates α‐synuclein cytotoxicity during Parkinson's disease

Malfunctioning of the protein α‐synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson‐diseased patients, while EndoG depletion largely reduces α‐synuclein‐induced cell death in human neuroblastoma cells. Xenogenic expression of human α‐synuclein in yeast cells triggers mitochondria‐nuclear translocation of EndoG and EndoG‐mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α‐synuclein‐driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α‐synuclein‐expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α‐synuclein cytotoxicity.     

Proteomic analysis of SP600125‐controlled TrkA‐dependent targets in SK‐N‐MC neuroblastoma cells: Inhibition of TrkA activity by SP600125

The c‐Jun N‐terminal kinase (JNK) is well known to play an important role in cell death signaling of the p75 neurotrophin receptor. However, little has been studied about a role of JNK in the signaling pathways of the tropomyosin‐related kinase A (TrkA) neurotrophin receptor. In this study, we investigated JNK inhibitor SP600125‐controlled TrkA‐dependent targets by proteomic analysis to better understand an involvement of JNK in TrkA‐mediated signaling pathways. PDQuest image analysis and protein identification results showed that hnRNP C1/C2, α‐tubulin, β‐tubulin homolog, actin homolog, and eIF‐5A‐1 protein spots were upregulated by ectopic expression of TrkA, whereas α‐enolase, peroxiredoxin‐6, PROS‐27, HSP70, PP1‐gamma, and PDH E1‐alpha were downregulated by TrkA, and these TrkA‐dependent upregulation and downregulation were significantly suppressed by SP600125. Notably, TrkA largely affected certain PTM(s) but not total protein amounts of the SP600125‐controlled TrkA‐dependent targets. Moreover, SP600125 strongly suppressed TrkA‐mediated tyrosine phosphorylation signaling pathways as well as JNK signaling, indicating that SP600125 could function as a TrkA inhibitor. Taken together, our results suggest that TrkA could play an important role in the cytoskeleton, cell death, cellular processing, and glucose metabolism through activation or inactivation of the SP600125‐controlled TrkA‐dependent targets.     

Functional dissection of hnRNP D suggests that nuclear import is required before hnRNP D can modulate mRNA turnover in the cytoplasm

Many shuttling proteins not only function in the nucleus but also control mRNA fates in the cytoplasm. We test whether a link exists between their nuclear association with mRNPs and their cytoplasmic functions using the p37 isoform of hnRNP D, which inhibits the rapid cytoplasmic mRNA decay in NIH3T3 cells. We showed that p37 shuttles between nucleus and cytoplasm, and narrowed down the nuclear import signal to a 50-amino-acid C-terminal domain. A p37 mutant missing this domain, still capable of associating with target mRNAs in vitro, was confined to the cytoplasm, where it was unable to block cytoplasmic mRNA turnover. Introducing heterologous shuttling domains to this mutant, thereby restoring its ability to enter the nucleus, concomitantly restored its cytoplasmic function. Association of p37 with its target mRNAs can only be detected when it can enter the nucleus. Our results suggest that nuclear import of hnRNP D is a prerequisite for it to exert its cytoplasmic function. This study provides a useful model system to elucidate the mechanisms by which "nuclear history" affects cytoplasmic mRNA fates.     

A tale of two α‐tubulin tails

Post‐translational modifications of tubulin, such as the removal of the C‐terminal tyrosine of α‐tubulin, have long been proposed to influence the ability of microtubule motors to walk along the microtubule surface. This hypothesis has now been tested for cytoplasmic dynein‐1 (dynein), revealing that active dynein–dynactin–adaptor complexes prefer to start moving on tyrosinated microtubules. This choice is governed by the p150 subunit of dynactin. Once moving, however, dynein is not choosy about whether the microtubule is tyrosinated or not.     

Identification of carbohydrate binding protein 35 in heterogeneous nuclear ribonucleoprotein complex

In previous studies, a lectin designated as carbohydrate binding protein 35 (CBP35) was identified in the nucleus and cytoplasm of cultured mouse 3T3 fibroblasts. In the present study, we observed that treatment of Triton X-100 permeabilized 3T3 cells with ribonuclease A released CBP35 from the nuclei, while parallel treatment with deoxyribonuclease I failed to do so. This conclusion was based on (a) immunofluorescence analysis of the nuclear residue after detergent and enzymatic treatments and (b) immunoblotting analysis of the supernatant fraction produced by these treatments. These results indicate that CBP35 may be associated with the ribonucleoprotein elements of the 3T3 cell nuclei. In corroboration with this conclusion, fractionation of the nucleoplasm derived from 3T3 cells on a cesium sulfate gradient (1.25-1.75 g/mL) localized CBP35 in fractions with densities of 1.30-1.32 g/mL, corresponding to the range of densities reported for heterogeneous nuclear ribonucleoprotein complex (hnRNP). Conversely, when nucleoplasm was fractionated on an affinity column of Sepharose derivatized with N-(epsilon-aminocaproyl)-D-galactosamine, the bound and eluted fraction contained RNA, as well as a set of polypeptides whose molecular weights matched those reported for the core particle of hnRNP. One of these polypeptides was identified as CBP35. These results suggest that CBP35 is a component of hnRNP.     

Tyrosination of α‐tubulin controls the initiation of processive dynein–dynactin motility

Post‐translational modifications (PTMs) of α/β‐tubulin are believed to regulate interactions with microtubule‐binding proteins. A well‐characterized PTM involves in the removal and re‐ligation of the C‐terminal tyrosine on α‐tubulin, but the purpose of this tyrosination–detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule‐binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α‐tubulin tyrosine facilitates initial motor–tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C‐terminal α‐tubulin tyrosine on dynein–dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein‐driven motility in cells.     

Inhibition of DNA synthesis by TGF-beta 1 coincides with inhibition of phosphorylation and cytoplasmic translocation of p53 protein.

Immunostaining demonstrated that p53 protein was localized in the cytoplasm of growing MCF-7 cells and in the nuclei of cells that were growth arrested by serum starvation. Serum stimulation of the arrested cells induced marked increases in DNA synthesis and p53 phosphorylation, and translocation of the protein from the nucleus to the cytoplasm at 20 h after the stimulation. This increase in the DNA synthesis that was significantly inhibited by TGF-beta 1 was coincident with the inhibition of phosphorylation and cytoplasmic translocation of the p53 protein.     

Activation of Smad‐mediated TGF‐β signaling triggers epithelial–mesenchymal transitions in murine cloned corneal progenitor cells

Epithelial–mesenchymal transition (EMT), via activation of Wnt signaling, is prevailing in embryogenesis, but postnatally it only occurs in pathological processes, such as in tissue fibrosis and tumor metastasis. Our prior studies led us to speculate that EMT might be involved in the loss of limbal epithelial stem cells in explant cultures. To examine this hypothesis, we successfully grew murine corneal/limbal epithelial progenitors by prolonging the culture time and by seeding at a low density in a serum‐free medium. Single cell‐derived clonal growth was accompanied by a gradient of Wnt signaling activity, from the center to the periphery, marked by a centrifugal loss of E‐cadherin and β‐catenin from intercellular junctions, coupled with nuclear translocation of β‐catenin and LEF‐1. Large‐colony‐forming efficiency at central location of colony was higher than peripheral location. Importantly, there was also progressive centrifugal differentiation, with positive K14 keratin expression and the loss of p63 and PCNA nuclear staining, and irreversible EMT, evidenced by cytoplasmic expression of α‐SMA and nuclear localization of S100A4; and by nuclear translocation of Smad4. Furthermore, cytoplasmic expression of α‐SMA was promoted by high‐density cultures and their conditioned media, which contained cell density‐dependent levels of TGF‐β1, TGF‐β2, GM‐CSF, and IL‐1α. Exogenous TGF‐β1 induced α‐SMA positive cells in a low‐density culture, while TGF‐β1 neutralizing antibody partially inhibited α‐SMA expression in a high‐density culture. Collectively, these results indicate that irreversible EMT emerges in the periphery of clonal expansion where differentiation and senescence of murine corneal/limbal epithelial progenitors occurs as a result of Smad‐mediated TGF‐β‐signaling. J. Cell. Physiol. 228: 225–234, 2013. © 2012 Wiley Periodicals, Inc.     

A nuclear localization domain in the hnRNP A1 protein

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.     

Baculovirus p33 Binds Human p53 and Enhances p53-Mediated Apoptosis

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.     

Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.