Importin 13 promotes NSCLC progression by mediating RFPL3 nuclear translocation and hTERT expression upregulation

Our previous studies have reported that RFPL3 protein exerts its unique function as a transcriptional factor of hTERT promoter after being transported into the lung cancer cell nucleus. However, the detailed mechanism by which RFPL3 undergoes nuclear transport has not been reported yet. Here, we identified RFPL3 as a potential import cargo for IPO13, which was found to be overexpressed in NSCLC cells and tissues. IPO13 interacted with RFPL3 in lung cancer cells, and the knockdown of IPO13 led to the cytoplasmic accumulation of RFPL3, the decreased anchoring of RFPL3 at hTERT promoter, and the downregulation of hTERT expression. Moreover, IPO13 silencing suppressed tumor growth in vitro and in vivo. IHC analysis confirmed the positive correlation between the expression levels of IPO13 and hTERT in the tumor tissues from patients with lung cancer. Furthermore, the mechanistic study revealed that IPO13 recognized RFPL3 via a functional nuclear localization signal (NLS), which is located in the B30.2 domain at the C-terminal region of RFPL3. Of note, the presence of EGFR mutations was significantly related to the increased IPO13 expression. The EGFR-TKI Osimertinib downregulated IPO13 expression level in NSCLC cell lines with EGFR mutations, but not in EGFR wild-type ones. In summary, our data suggest that inhibition of IPO13 transport activity itself might be an alternative and potential therapeutic strategy for NSCLC.

In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH

We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-ΔNLSΔCLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin.

Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1

Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989–1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair.

The Renpenning syndrome–associated protein PQBP1 facilitates the nuclear import of splicing factor TXNL4A through the karyopherin β2 receptor

Renpenning syndrome belongs to a group of X-linked intellectual disability disorders. The Renpenning syndrome–associated protein PQBP1 (polyglutamine-binding protein 1) is intrinsically disordered, associates with several splicing factors, and is involved in pre-mRNA splicing. PQBP1 uses its C-terminal YxxPxxVL motif for binding to the splicing factor TXNL4A (thioredoxin like 4A), but the biological function of this interaction has yet to be elucidated. In this study, using recombinant protein expression, in vitro binding assays, and immunofluorescence microscopy in HeLa cells, we found that a recently reported X-linked intellectual disability–associated missense mutation, resulting in the PQBP1-P244L variant, disrupts the interaction with TXNL4A. We further show that this interaction is critical for the subcellular location of TXNL4A. In combination with other PQBP1 variants lacking a functional nuclear localization signal required for recognition by the nuclear import receptor karyopherin β2, we demonstrate that PQBP1 facilitates the nuclear import of TXNL4A via a piggyback mechanism. These findings expand our understanding of the molecular basis of the PQBP1–TXNL4A interaction and of the etiology and pathogenesis of Renpenning syndrome and related disorders.