Search for KPNA7 cargo proteins in human cells reveals MVP and ZNF414 as novel regulators of cancer cell growth

Abstract

Karyopherin alpha 7 (KPNA7) belongs to a family of nuclear import proteins that recognize and bind nuclear localization signals (NLSs) in proteins to be transported to the nucleus. Previously we found that KPNA7 is overexpressed in a subset of pancreatic cancer cell lines and acts as a critical regulator of growth in these cells. This characteristic of KPNA7 is likely to be mediated by its cargo proteins that are still mainly unknown. Here, we used protein affinity chromatography in Hs700T and MIA PaCa-2 pancreatic cancer cell lines and identified 377 putative KPNA7 cargo proteins, most of which were known or predicted to localize to the nucleus. The interaction was confirmed for two of the candidates, MVP and ZNF414, using co-immunoprecipitation, and their transport to the nucleus was hindered by siRNA based KPNA7 silencing. Most importantly, silencing of MVP and ZNF414 resulted in marked reduction in Hs700T cell growth. In conclusion, these data uncover two previously unknown human KPNA7 cargo proteins with distinct roles as novel regulators of pancreatic cancer cell growth, thus deepening our understanding on the contribution of nuclear transport in cancer pathogenesis.

Importin α: functions as a nuclear transport factor and beyond

“…104,105) Knockdown and knockout studies showed that KPNA7 was required for early embryogenesis. 72,84) Of note, mouse KPNA7 is localized in the nucleus or spindles in oocytes, depending on the maturation stage.…”

An epilepsy-associated mutation in the nuclear import receptor KPNA7 reduces nuclear localization signal binding

“…Several studies have identified NLS-containing proteins that bind KPNA7, and in a few cases, demonstrated these are cargoes for KPNA7-dependent transport 34,35 . Reasoning that epilepsy reflects changes in neuronal activity, we used SILAC labeling and a straightforward MS/MS pipeline to identify KPNA7-interacting proteins in hiPSC-derived neurons.…”

“…Given that KPNA7 mutations are associated with epilepsy 23 , we set out to identify KPNA7-interacting proteins using extracts of neuronal origin. Previous studies have used tandem mass spectrometry (MS/MS) to identify KPNA7-interacting proteins in HEK293 34 and pancreatic cancer cell lines 35 . Given the E344Q KPNA7 mutant shows a reduction in NLS binding, identification of KPNA7 cargo proteins could generate hypotheses for how mutation of KPNA7 affects neuronal differentiation.…”

“…This result indicates that the bipartite NLS in hnRNP R (inclusive of the two basic elements identified) is responsible for nuclear localization of hnRNP R. We then evaluated the ability of the NLSs to bind import receptors. Each was expressed as a GST-GFP fusion protein, immobilized on beads, and used in pull-down binding assays with KPNA proteins labeled by in vitro translation with 35 S-methionine ( Fig. 4e).…”

“…3c). Previous studies investigating KPNA7 interacting proteins by MS using HEK293 and pancreatic cancer cell lines also reported enrichment for proteins involved in RNA processing 34,35 .Scientific RepoRtS | (2020) 10:4844 | https://doi.…”