EHD2 shuttles to the nucleus and represses transcription

EHD2 shuttles to the nucleus and represses transcription EHD {EH (Eps15 (epidermal growth factor receptor substrate 15) homology)-domain-containing} proteins participate in several endocytic events, such as the internalization and the recycling processes. There are four EHD proteins in mammalian cells, EHD1–EHD4, each with diverse roles in the recycling pathway of endocytosis. EHD2 is a plasma-membrane-associated member of the EHD family that regulates internalization. Since several endocytic proteins have been shown to undergo nucleocytoplasmic shuttling and have been assigned roles in regulation of gene expression, we tested the possibility that EHD proteins also shuttle to the nucleus. Our results showed that, among the three EHD proteins (EHD1–EHD3) that were tested, only EHD2 accumulates in the nucleus under nuclear export inhibition treatment. Moreover, the presence of a NLS (nuclear localization signal) was essential for its entry into the nucleus. Nuclear exit of EHD2 depended partially on its NES (nuclear export signal). Elimination of a potential SUMOylation site in EHD2 resulted in a major accumulation of the protein in the nucleus, indicating the involvement of SUMOylation in the nuclear exit of EHD2. We confirmed the SUMOylation of EHD2 by employing co-immunoprecipitation and the yeast two-hybrid system. Using GAL4-based transactivation assay as well as a KLF7 (Krüppel-like factor 7)-dependent transcription assay of the p21WAF1/Cip1 ( CDKN1A (cyclin-dependent kinase inhibitor 1A)) gene, we showed that EHD2 represses transcription. qRT-PCR (quantitative real-time PCR) of RNA from cells overexpressing EHD2 or of RNA from cells knocked down for EHD2 confirmed that EHD2 represses transcription of the p21WAF1/Cip1 gene. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biochemical Journal Portland Press

EHD2 shuttles to the nucleus and represses transcription

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Publisher
Portland Press
Copyright
Copyright by Portland Press
ISSN
0264-6021
D.O.I.
10.1042/BJ20111268
Publisher site
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Abstract

EHD {EH (Eps15 (epidermal growth factor receptor substrate 15) homology)-domain-containing} proteins participate in several endocytic events, such as the internalization and the recycling processes. There are four EHD proteins in mammalian cells, EHD1–EHD4, each with diverse roles in the recycling pathway of endocytosis. EHD2 is a plasma-membrane-associated member of the EHD family that regulates internalization. Since several endocytic proteins have been shown to undergo nucleocytoplasmic shuttling and have been assigned roles in regulation of gene expression, we tested the possibility that EHD proteins also shuttle to the nucleus. Our results showed that, among the three EHD proteins (EHD1–EHD3) that were tested, only EHD2 accumulates in the nucleus under nuclear export inhibition treatment. Moreover, the presence of a NLS (nuclear localization signal) was essential for its entry into the nucleus. Nuclear exit of EHD2 depended partially on its NES (nuclear export signal). Elimination of a potential SUMOylation site in EHD2 resulted in a major accumulation of the protein in the nucleus, indicating the involvement of SUMOylation in the nuclear exit of EHD2. We confirmed the SUMOylation of EHD2 by employing co-immunoprecipitation and the yeast two-hybrid system. Using GAL4-based transactivation assay as well as a KLF7 (Krüppel-like factor 7)-dependent transcription assay of the p21WAF1/Cip1 ( CDKN1A (cyclin-dependent kinase inhibitor 1A)) gene, we showed that EHD2 represses transcription. qRT-PCR (quantitative real-time PCR) of RNA from cells overexpressing EHD2 or of RNA from cells knocked down for EHD2 confirmed that EHD2 represses transcription of the p21WAF1/Cip1 gene.

Journal

Biochemical JournalPortland Press

Published: Jun 15, 2012

Keywords: EH (Eps15 (epidermal growth factor receptor substrate 15) homology)-domain-containing 2 (EHD2), endocytosis, nucleocytoplasmic shuttling, SUMOylation

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