Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells

Matrin 3, a nuclear matrix protein has potential (1) to withhold promiscuously edited RNAs within the nucleus in cooperation with p54(nrb) and PSF, (2) to mediate NMDA-induced neuronal death, and (3) to modulate promoter activity of genes proximal to matrix/scaffold attachment region (MAR/SAR). We identified a bipartite nuclear localization signal (NLS) of chicken matrin 3 (cmatr3) at residues 583-602. By expressing green fluorescent protein (GFP) fused to the NLS mutant in chicken DT40 cells, we showed an essential role of the NLS for cell proliferation. Furthermore, we showed that both clusters of basic amino acids and a linker of the bipartite NLS were essential and sufficient for the nuclear import of GFP. Exogenous cmatr3 rescued the HeLa cells where human matrin 3 was suppressed by RNA interference, but cmatr3 containing deletions at either of the basic amino acid clusters or the linker could not.     

Regulation of MDR1 gene expression in multidrug-resistant cancer cells is independent from YB-1

The MDR1 gene encoded transmembrane ABC-transporter MDR1/P-glycoprotein can mediate the phenotype of multidrug resistance (MDR), a major obstacle in the clinical management of cancer patients. It was hypothesized that YB-1 is a fundamental regulatory factor of the MDR1 gene in tumor cells and can therewith enhance drug resistance. To analyze the potential impact of YB-1 in MDR cancer cells, two specific anti-YB-1 small interfering RNAs (siRNAs) were designed for transient triggering the gene-silencing RNA interference (RNAi) pathway in the MDR cell lines EPG85-257RDB and EPP85-181RDB as well as in their drug-sensitive counterparts EPG85-257P and EPP85-181P. Since both siRNAs showed biological activity, for stable inhibition of YB-1 corresponding tetracycline-inducible short hairpin RNA (shRNA)-encoding expression vectors were designed. By treatment of the cancer cells with these constructs, the expression of the targeted YB-1 encoding mRNA and protein was completely inhibited following tetracycline exposure. These gene-silencing effects were not accompanied by modulation of the MDR1 expression or by reversal of the drug-resistant phenotype. In conclusion, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that YB-1 is not involved in the regulation of the MDR1 gene or the development of the drug-resistant phenotype in MDR cancer cells.     

P-glycoprotein subcellular localization and cell morphotype in MDR1 gene-transfected human osteosarcoma cells.

Efflux of chemotherapy agents by P-glycoprotein at the plasma membrane is thought to be a major cause of cancer multidrug-resistance (MDR). However, the mechanism underlying the cellular accumulation and distribution of cytotoxic drugs is still poorly defined. We have recently found that P-glycoprotein is expressed also in the nucleus of MDR cell lines selected in doxorubicin (DXR), suggesting the possible involvement of this protein in the direct extrusion of the drug from the nucleus of resistant cells. In this study, we analyzed the subcellular localization of P-glycoprotein, in a series of U-2 OS osteosarcoma cell clones transfected with MDR1 gene in order to verify whether the nucleus is a constant site for the localization and functional activity of P-glycoprotein, and in which way some aspects of cell morphology related to MDR depend on the subcellular P-glycoprotein localization rather than on the exposure to the selective drug. Our results indicate that to achieve a subcellular drug distribution prevailing in the cytoplasm but not in the nucleus, a significant increase in the expression of P-glycoprotein at the different cellular compartments, including the plasma membrane, the cytoplasm, and the nucleus, is needed, although the in vitro drug resistance appears to be mainly dependent on the expression of P-glycoprotein at the cell surface. With regard to the morphological characteristics of MDR cells involving the cell surface and the chromatin arrangement, the influence of DXR appears to be prevalent, although P-glycoprotein overexpression cannot be excluded.     

CRM1 mediates nuclear export of HDAC7 independently of HDAC7 phosphorylation and association with 14-3-3s

CRM1, 14-3-3 proteins, and CaMK play important roles in trafficking of HDAC7, but the interplay between these proteins in this process is not clearly understood. Here, we show that CRM1 is capable of promoting cytoplasmic localization of wild-type and mutant HDAC7 (S178A/S344A/S479A), which is normally found in the nucleus. Using phospho-specific antibodies to HDAC7, we demonstrate that CaMK I promotes phosphorylation of S178, S344, and S479 of HDAC7. We also show that endogenous S178-phosphorylated HDAC7 is localized in both the nucleus and the cytoplasm, whereas S344- and S479-phosphorylated HDAC7 are exclusively localized in the nucleus. An HDAC7 mutant, S178E/S344E/S479E, which lost the ability to bind 14-3-3s, is localized in both the nucleus and the cytoplasm. Furthermore, the nuclear export of S178E/S344E/S479E is inhibited by LMB, but is enhanced by the CRM1. Taken together, these results strongly suggest that CRM1 mediated-nuclear export of HDAC7 is independent of HDAC7 phosphorylation and its association with 14-3-3s.     

Nuclear import of Pin1 is mediated by a novel sequence in the PPIase domain

Pin1 actively regulates diverse biological/pathological processes, but little is known about the regulatory mechanisms of its cellular localization. In this study, we report that the endogenous Pin1 is distributed in both nucleus and cytoplasm. We found that point mutations of several basic amino acids in the PPIase domain of Pin1 significantly compromise its nuclear localization. Such inhibition is independent of Pin1 enzymatic activity, and is mainly due to the defects in the nuclear import. A novel sequence harboring these residues was identified as a putative nuclear localization signal (NLS) of Pin1. Importin α5 of the nuclear import machinery was found to interact with Pin1.

Structured summary:

MINT-6803320: PIN1 (uniprotkb:Q13255) and importin alpha 5 (uniprotkb:P52294) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)MINT-6803333: importin alpha 3 (uniprotkb:O00505) and PIN1 (uniprotkb:Q13255) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)MINT-6803357: PIN1 (uniprotkb:Q13255) physically interacts (MI:0218) with importin alpha 5 (uniprotkb:P52294) by anti bait coimmunoprecipitation (MI:0006)MINT-6803345: St3 (uniprotkb:P40763) and importin alpha 5 (uniprotkb:P52294) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)     

Tubulin Beta3 Serves as a Target of HDAC3 and Mediates Resistance to Microtubule-Targeting Drugs

We investigated the role of HDAC3 in anti-cancer drug-resistance. The expression of HDAC3 was decreased in cancer cell lines resistant to anti-cancer drugs such as celastrol and taxol. HDAC3 conferred sensitivity to these anti-cancer drugs. HDAC3 activity was necessary for conferring sensitivity to these anti-cancer drugs. The down-regulation of HDAC3 increased the expression of MDR1 and conferred resistance to anti-cancer drugs. The expression of tubulin β3 was increased in drug-resistant cancer cell lines. ChIP assays showed the binding of HDAC3 to the promoter sequences of tubulin β3 and HDAC6. HDAC6 showed an interaction with tubulin β3. HDAC3 had a negative regulatory role in the expression of tubulin β3 and HDAC6. The down-regulation of HDAC6 decreased the expression of MDR1 and tubulin β3, but did not affect HDAC3 expression. The down-regulation of HDAC6 conferred sensitivity to taxol. The down-regulation of tubulin β3 did not affect the expression of HDAC6 or MDR1. The down-regulation of tubulin β3 conferred sensitivity to anti-cancer drugs. Our results showed that tubulin β3 serves as a downstream target of HDAC3 and mediates resistance to microtubule-targeting drugs. Thus, the HDAC3-HDAC6-Tubulin β axis can be employed for the development of anti-cancer drugs.     

Membrane localization of v-ErbB is required but not sufficient for ligand-independent transformation

The v-ErbB retroviral oncogene is a transduced, mutated copy of the avian EGF receptor gene, and its expression is sufficient to induce tumor formation in vivo. The structural alterations that release the oncogenic potential of the v-ErbB oncogene are similar to EGFR gene mutations described in human tumors. Thus, the study of v-ErbB tumor biology offers a useful model through which we can gain insight into the mechanism of EGFR-induced malignancies. Despite years of study, however, questions remain regarding the domains of v-ErbB required for oncogenicity. We sought to clarify the role of the transmembrane domain of v-ErbB during transformation using S3-v-ErbB, an acutely transforming retroviral oncogene isolated from avian sarcomas. Infection of primary fibroblasts with a retroviral vector containing S3-v-ErbB results in the formation of a transformation-associated phosphoprotein signaling complex, soft agar colony formation, and the rapid induction of highly vascularized sarcomas in vivo. To address contribution of the transmembrane domain of S3-v-ErbB during these processes, we constructed a mutant version of this oncogene with a precise deletion in this domain. Specifically, the S3-v-ErbB-TM- mutant was created through an in-frame deletion of the entire transmembrane domain. Primary fibroblasts expressing this S3-v-ErbB-TM- mutant fail to form a characteristic transformation-associated phosphoprotein complex and do not grow in an anchorage-independent manner. In addition, day-old chicks injected with a helper-independent retrovirus expressing the S3-v-ErbB-TM- mutant exhibit only limited tumor formation in vivo. These results demonstrate that the transmembrane domain and, consequently membrane localization, are essential for S3-v-ErbB-mediated transformation.     

Phosphoinositide binding by the disabled-1 PTB domain is necessary for membrane localization and Reelin signal transduction

Disabled-1 (Dab1) is an essential adaptor protein that functions in the Reelin signaling pathway and is required for the regulation of neuronal migration during embryonic development. Dab1 interacts with NPXY motifs in the cytoplasmic tails of the lipoprotein receptors ApoER2 and very low density lipoprotein receptor through an amino-terminal phosphotyrosine binding (PTB) domain. Binding of Reelin to these receptors leads to tyrosine phosphorylation of Dab1 and the initiation of a signaling cascade that results in remodeling of the cytoskeleton. Structural and biochemical studies of the Dab1 PTB domain have demonstrated that this domain binds to both the NPXY peptide motif in the lipoprotein receptor tails as well as to the head group of phosphoinositide 4,5-P2 through energetically independent mechanisms. Here we have investigated how phosphoinositide binding by the Dab1 PTB domain influences Reelin signal transduction. Our findings in cultured primary neurons that have been transduced with lentiviral constructs expressing mutant Dab1 forms reveal that phosphoinositide binding by the Dab1 PTB domain is necessary for proper membrane localization of Dab1 and for effective transduction of a Reelin signal.     

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.     

The nuclear localization signal of mitotic kinesin-like protein Mklp-1: effect on Mklp-1 function during cytokinesis

The mitotic kinesin-like protein (Mklp-1) localizes in the nucleus during interphase due to the presence of nuclear localization signal(s) [NLS(s)] within its sequence. Here, we mapped two NLSs to be 899SRKRRSST906 and 949KRKKP953 in the tail domain of Mklp-1, and showed that ectopic expression of a mutant Mklp-1 without the NLSs leads to cell cycle arrest at cytokinesis, indicating that the NLSs are necessary for Mklp-1 to execute its normal function during cell division. Furthermore, mutation of two serine residues in the first NLS to aspartic acid, which mimics phosphorylation, attenuated its nuclear localization function, suggesting that the function of this NLS might be regulated by phosphorylation.     

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.     

Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein

Sorcin, a 22 kDa Ca²⁺ binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between −716 and −709 bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR.