Dataset Information

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

ABSTRACT: KPNA7 is a member of the Importin-? family of nuclear import receptors. KPNA7 forms a complex with Importin-? and facilitates the translocation of signal-containing proteins from the cytoplasm to the nucleus. Exome sequencing of siblings with severe neurodevelopmental defects and clinical features of epilepsy identified two amino acid-altering mutations in KPNA7. Here, we show that the E344Q substitution reduces KPNA7 binding to nuclear localization signals, and that this limits KPNA7 nuclear import activity. The P339A substitution, by contrast, has little effect on KPNA7 binding to nuclear localization signals. Given the neuronal phenotype described in the two patients, we used SILAC labeling, affinity enrichment, and mass spectrometry to identify KPNA7-interacting proteins in human induced pluripotent stem cell-derived neurons. We identified heterogeneous nuclear ribonucleoproteins hnRNP R and hnRNP U as KPNA7-interacting proteins. The E344Q substitution reduced binding and KPNA7-mediated import of these cargoes. The c.1030G > C allele which generates E344Q is within a predicted CTCF binding site, and we found that it reduces CTCF binding by approximately 40-fold. Our data support a role for altered neuronal expression and activity of KPNA7 in a rare type of pediatric epilepsy.

SUBMITTER: Oostdyk LT

PROVIDER: S-EPMC7076015 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Oostdyk Luke T LT Wang Zhenjia Z Zang Chongzhi C Li Hui H McConnell Michael J MJ Paschal Bryce M BM

Scientific reports 20200316 1

KPNA7 is a member of the Importin-α family of nuclear import receptors. KPNA7 forms a complex with Importin-β and facilitates the translocation of signal-containing proteins from the cytoplasm to the nucleus. Exome sequencing of siblings with severe neurodevelopmental defects and clinical features of epilepsy identified two amino acid-altering mutations in KPNA7. Here, we show that the E344Q substitution reduces KPNA7 binding to nuclear localization signals, and that this limits KPNA7 nuclear im ...[more]

PMID: 32179771

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Project description:Histone deacetylase 5 (HDAC5), a class IIa deacetylase, is a prominent regulator of cellular and epigenetic processes that underlie the progression of human disease, ranging from cardiac hypertrophy to cancer. Although it is established that phosphorylation mediates 14-3-3 protein binding and provides the essential link between HDAC5 nucleo-cytoplasmic shuttling and transcriptional repression, thus far only four phospho-acceptor sites have been functionally characterized. Here, using a combinatorial proteomics approach and phosphomutant screening, we present the first evidence that HDAC5 has at least 17 in vivo phosphorylation sites within functional domains, including Ser278 and Ser279 within the nuclear localization signal (NLS), Ser1108 within the nuclear export signal, and Ser755 in deacetylase domain. Global and targeted MS/MS analyses of NLS peptides demonstrated the presence of single (Ser278 and Ser279) and double (Ser278/Ser279) phosphorylations. The double S278/279A mutation showed reduced association with HDAC3, slightly decreased deacetylation activity, and significantly increased cytoplasmic localization compared with wild type HDAC5, whereas the S278A and S1108A phosphomutants were not altered. Live cell imaging revealed a deficiency in nuclear import of S278/279A HDAC5. Phosphomutant stable cell lines confirmed the cellular redistribution of NLS mutants and revealed a more pronounced cytoplasmic localization for the single S279A mutant. Proteomic analysis of immunoisolated S278/279A, S279A, and S259/498A mutants linked altered cellular localization to changes in protein interactions. S278/279A and S279A HDAC5 showed reduced association with the NCoR-HDAC3 nuclear corepressor complex as well as protein kinase D enzymes, which were potentiated in the S259/498A mutant. These results provide the first link between phosphorylation outside the known 14-3-3 sites and downstream changes in protein interactions. Together these studies identify Ser279 as a critical phosphorylation within the NLS involved in the nuclear import of HDAC5, providing a regulatory point in nucleo-cytoplasmic shuttling that may be conserved in other class IIa HDACs-HDAC4 and HDAC9.

Dataset Information

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

Publications

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

Similar Datasets

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