Phosphatase activity of small C-terminal domain phosphatase 1 (SCP1) controls the stability of the key neuronal regulator RE1-silencing transcription factor (REST)

Nathaniel Tate Burkholder,Joshua E Mayfield,Xiaohua Yu,Seema Irani,Daniel K Arce,Faqin Jiang,Wendy L Matthews,Yuanchao Xue,Yan Jessie Zhang

doi:10.1074/jbc.ra118.004722

Abstract

The RE1-silencing transcription factor (REST) is the major scaffold protein for assembly of neuronal gene silencing complexes that suppress gene transcription through regulating the surrounding chromatin structure. REST represses neuronal gene expression in stem cells and non-neuronal cells, but it is minimally expressed in neuronal cells to ensure proper neuronal development. Dysregulation of REST function has been implicated in several cancers and neurological diseases. Modulating REST gene silencing is challenging because cellular and developmental differences can affect its activity. We therefore considered the possibility of modulating REST activity through its regulatory proteins. The human small C-terminal domain phosphatase 1 (SCP1) regulates the phosphorylation state of REST at sites that function as REST degradation checkpoints. Using kinetic analysis and direct visualization with X-ray crystallography, we show that SCP1 dephosphorylates two degron phosphosites of REST with a clear preference for phosphoserine 861 (pSer-861). Furthermore, we show that SCP1 stabilizes REST protein levels, which sustains REST's gene silencing function in HEK293 cells. In summary, our findings strongly suggest that REST is a bona fide substrate for SCP1 in vivo and that SCP1 phosphatase activity protects REST against degradation. These observations indicate that targeting REST via its regulatory protein SCP1 can modulate its activity and alter signaling in this essential developmental pathway.

Highlights

The RE1-silencing transcription factor (REST) is the major scaffold protein for assembly of neuronal gene silencing complexes that suppress gene transcription through regulating the surrounding chromatin structure
small C-terminal domain phosphatase 1 (SCP1) had weak activity when only Ser-864 is phosphorylated (Fig. 1A). These results demonstrate that pSer-861 of REST is the preferred dephosphorylation site for SCP1 compared with pSer-864
Our in vitro and cell-based experiments revealed both the physical characteristics and biological implications of the REST/SCP1 interaction. These results strongly suggest that controlling SCP1 phosphatase activity is sufficient to alter REST protein levels and modulate expression of genes targeted by REST

Summary

Results

Overexpression of SCP1 increases REST dephosphorylation [26]. To test if SCP1 can directly dephosphorylate REST, we obtained 12-mer synthetic peptides with the sequence EDLSPPSPPLPK from the C-terminal region of REST(858 – 869), where two degron sites are located [23]. These results demonstrate that pSer-861 of REST is the preferred dephosphorylation site for SCP1 compared with pSer-864 When both Ser-861 and Ser-864 are phosphorylated, SCP1 still shows strong phosphatase activity (kcat/Km of is determined to be 80 Ϯ 40 mMϪ1 sϪ1). Tyr-158 of SCP1 is essential for binding of the C-terminal domain of RNA polymerase II (Fig. S3) as replacing this residue with alanine is sufficient to eliminate SCP1’s substrate specificity without affecting enzymatic activity [27] This pocket is critical for SCP1 recognition of the small molecular inhibitor rabeprazole [30] as its sulfoxide group sits over Tyr-158 (Fig. S3). The structure of SCP1 bound to the doubly phosphorylated REST peptide supports our hypothesis that pSer-861 is the preferred dephosphorylation site and suggests that SCP1 specificity toward REST is dominated by hydrophobic interactions on either side of phosphorylated substrate residue.

PDB code

Discussion

Protein expression and purification

Phosphatase kinetics with peptide substrates

Protein crystallization and substrate complexation

Western blot analysis