Methods for Identification of Substrates/Inhibitors of FCP/SCP Type Protein Ser/Thr Phosphatases

Kazuhiro Furukawa,Shunta Imai,Masataka Mizunuma,Atsushi Kaneko,Yoshiro Chuman

doi:10.3390/pr8121598

Abstract

Protein phosphorylation is the most widespread type of post-translational modification and is properly controlled by protein kinases and phosphatases. Regarding the phosphorylation of serine (Ser) and threonine (Thr) residues, relatively few protein Ser/Thr phosphatases control the specific dephosphorylation of numerous substrates, in contrast with Ser/Thr kinases. Recently, protein Ser/Thr phosphatases were reported to have rigid substrate recognition and exert various biological functions. Therefore, identification of targeted proteins by individual protein Ser/Thr phosphatases is crucial to clarify their own biological functions. However, to date, information on the development of methods for identification of the substrates of protein Ser/Thr phosphatases remains scarce. In turn, substrate-trapping mutants are powerful tools to search the individual substrates of protein tyrosine (Tyr) phosphatases. This review focuses on the development of novel methods for the identification of Ser/Thr phosphatases, especially small C-terminal domain phosphatase 1 (Scp1), using peptide-displayed phage library with AlF4−/BeF3−, and discusses the identification of putative inhibitors.

Highlights

Protein dephosphorylation is mainly regulated by protein Ser/Thr phosphatases (PSPs) and protein tyrosine (Tyr) phosphatases (PTPs), and disorder of these enzymes contributes to several illnesses, including cancer and neurological disorders [4,5,6,7,8]
We have focused on the role of AlF4 as a phosphate mimic and mimic and developed the phosphorylation mimic phage display (PMPD) method −using AlF4 − and the phage-displayed peptide library to developed the PMPD method using AlF4 and the phage-displayed peptide library to potentially potentially identify substrate candidate peptides for small C-terminal domain phosphatase 1 (Scp1) [43]
Isolation of Putative Peptides Derived from Substrates/Inhibitors of Scp1 by PMPD Methods

Summary

Introduction

Protein phosphorylation is strictly regulated by protein kinases and phosphatases, with more than 95% of phosphorylation affecting serine (Ser) and threonine (Thr) residues [1,2,3]. The small C-terminal domain (CTD) phosphatase 1 (Scp1), which is known as Nuclear LIM interactor-interacting factor 3 (NLI-IF) or CTDSP1, belongs to the FCP/SCP type phosphatase and was originally identified as a protein Ser/Thr phosphatase targeting the CTD of the largest subunit of the RNA polymerase II (RNAPII), which includes tandem heptapeptide repeats of the sequence Y1 S2 P3 T4 S5 P6 S7. Recent investigations on Scp revealed that it dephosphorylates various substrates associated with several signaling pathways, including cell cycle regulation, neuronal gene silencing, and osteoblast differentiation Most of these substrates were identified using an expression screening strategy for protein phosphatases (Table 1)

Scp1 Substrate Identification by PMPD Method with AlF4

Methods

PMPD Methods

Findings

Conclusions