EHD2 shuttles to the nucleus and represses transcription

Pekar, Olga; Benjamin, Sigi; Weidberg, Hilla; Smaldone, Silvia; Ramirez, Francesco; Horowitz, Mia

doi:10.1042/bj20111268

Cited by 26 publications

(38 citation statements)

References 57 publications

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“…This is an important question because a stabilized repressor may increase the chromatin resident time of its associated chromatin modifiers. In some cases, temporal control of stability is achieved by mechanisms that shuttle the repressors between the nucleus and cytoplasm (29)(30)(31). Although REST has been observed in some cellular contexts in the cytoplasm (28,31,32), its half-life in this cell compartment has not been measured, and whether its cytoplasmic complexes contain kinases or phosphatases has not been determined.…”

Section: Discussionmentioning

confidence: 99%

C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation

Nesti

Corson

McCleskey

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The repressor element 1 (RE1) silencing transcription factor (REST) in stem cells represses hundreds of genes essential to neuronal function. During neurogenesis, REST is degraded in neural progenitors to promote subsequent elaboration of a mature neuronal phenotype. Prior studies indicate that part of the degradation mechanism involves phosphorylation of two sites in the C terminus of REST that require activity of beta-transducin repeat containing E3 ubiquitin protein ligase, βTrCP. We identify a proline-directed phosphorylation motif, at serines 861/864 upstream of these sites, which is a substrate for the peptidylprolyl cis/trans isomerase, Pin1, as well as the ERK1/2 kinases. Mutation at S861/864 stabilizes REST, as does inhibition of Pin1 activity. Interestingly, we find that C-terminal domain small phosphatase 1 (CTDSP1), which is recruited by REST to neuronal genes, is present in REST immunocomplexes, dephosphorylates S861/864, and stabilizes REST. Expression of a REST peptide containing S861/864 in neural progenitors inhibits terminal neuronal differentiation. Together with previous work indicating that both REST and CTDSP1 are expressed to high levels in stem cells and down-regulated during neurogenesis, our results suggest that CTDSP1 activity stabilizes REST in stem cells and that ERK-dependent phosphorylation combined with Pin1 activity promotes REST degradation in neural progenitors.T he repressor element 1 (RE1) silencing transcription factor (REST) is a transcriptional repressor that suppresses neuronal gene expression in nonneural cells, such as fibroblasts, as well as in neural progenitors (1-3). Its targets represent genes required for the terminally differentiated neuronal cell phenotype, including genes encoding voltage and ligand-dependent ion channels, receptors, growth factors, and axonal-guidance proteins (4-7). Thus, during neurogenesis, REST is progressively down-regulated to allow elaboration of the mature neuronal phenotype (3). Nonetheless, precisely how REST itself is regulated still remains an open question. Relatively little is known about either its transcriptional or posttranscriptional regulation (3,8,9). In contrast, several studies have focused on posttranslational regulation of REST (3, 10, 11), but the identity of the signaling molecules involved has received little attention.In neural progenitors and human embryonic kidney (HEK) cells, rapid REST turnover is mediated by targeting to a proteasomal pathway (3, 10, 11). REST degradation during neuronal differentiation in culture requires interaction with beta-transducin repeat containing E3 ubiquitin protein ligase (βTrCP) for targeting to the proteasome (11). βTrCP was also required for cell-cycle-dependent degradation of REST in HEK cells (10). Two adjacent phosphorylated peptides in the C-terminal domain of REST were identified as βTrCP substrates in these studies, and function as degrons. One kinase responsible for the phosphorylation and degron activity in hippocampus is casein kinase 1, CK1 (12), but whether it function...

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Section: Discussionmentioning

confidence: 99%

C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation

Nesti

Corson

McCleskey

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Entry of EHD2 into the nucleus depends on a nuclear localization sequence (NLS) present in its helical domain. We also showed that its exit from the nucleus depends mainly on its SUMOylation (SUMO-small ubiquitin like modifier) [ 30 ].…”

Section: Introductionmentioning

confidence: 99%

“…At the molecular level, this posttranslational modification changes the surface of a target protein, enabling/disabling interactions with other proteins [ 32 ]. Although a number of endocytic proteins have been shown to undergo SUMOylation, EHD2 is the only EHD member shown to be modified by SUMOylation [ 30 ].…”

Section: Introductionmentioning

confidence: 99%

SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment

2015

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Endocytosis defines the entry of molecules or macromolecules through the plasma membrane as well as membrane trafficking in the cell. It depends on a large number of proteins that undergo protein-protein and protein-phospholipid interactions. EH Domain containing (EHDs) proteins formulate a family, whose members participate in different stages of endocytosis. Of the four mammalian EHDs (EHD1-EHD4) EHD1 and EHD3 control traffic to the endocytic recycling compartment (ERC) and from the ERC to the plasma membrane, while EHD2 modulates internalization. Recently, we have shown that EHD2 undergoes SUMOylation, which facilitates its exit from the nucleus, where it serves as a co-repressor. In the present study, we tested whether EHD3 undergoes SUMOylation and what is its role in endocytic recycling. We show, both in-vitro and in cell culture, that EHD3 undergoes SUMOylation. Localization of EHD3 to the tubular structures of the ERC depends on its SUMOylation on lysines 315 and 511. Absence of SUMOylation of EHD3 has no effect on its dimerization, an important factor in membrane localization of EHD3, but has a dominant negative effect on its appearance in tubular ERC structures. Non-SUMOylated EHD3 delays transferrin recycling from the ERC to the cell surface. Our findings indicate that SUMOylation of EHD3 is involved in tubulation of the ERC membranes, which is important for efficient recycling.

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“…and thus fail to insert properly into the plasma membrane. We rationalize that under such conditions where membrane binding is partially impaired, the lysine residues in the EHD2 helical domain, which act both as membrane contact sites [ 9 ] and a bipartite nuclear localization sequence [ 27 ], are now free to act in the latter capacity and promote EHD2 transport to the nucleus. Indeed, EHD2 has been described as a transcriptional repressor [ 27 ].…”

Section: Resultsmentioning

confidence: 93%

“…On the other hand, despite normal dimerization and binding, EHD2 NPF-to-APA displayed a dramatic relocation to the nucleus ( Fig 3D ). Although EHD2 has a bipartite nuclear localization sequence and many cells display a portion of their EHD2 in the nucleus ( Fig 3C ) [ 27 ], the NPF-to-APA mutant displayed a primarily nuclear localization ( Fig 3D ). Accordingly, despite the yeast two-hybrid binding, we were unable to detect EHD2 NPF-to-APA interactions with Syndapin2 by co-immunoprecipitation, likely due to the mutant’s mislocalization exclusively to the nucleus ( S2 Fig and S1 Table ).…”

Section: Resultsmentioning

confidence: 99%

Role of the EHD2 Unstructured Loop in Dimerization, Protein Binding and Subcellular Localization

2015

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The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2’s homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.

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EHD2 shuttles to the nucleus and represses transcription

Cited by 26 publications

References 57 publications

C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation

C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation

SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment

Role of the EHD2 Unstructured Loop in Dimerization, Protein Binding and Subcellular Localization

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