Identification of a Specific Inhibitor of Human Scp1 Phosphatase Using the Phosphorylation Mimic Phage Display Method

Protein phosphatases are divided into tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. While substrate trapping mutants are frequently used to identify substrates of Tyr phosphatases, a rapid and simple method to identify Ser/Thr phosphatase substrates is yet to be developed. The TFIIF-associating component of RNA polymerase II C-terminal domain (CTD) phosphatase/small CTD phosphatase (FCP/SCP) phosphatase family is one of the three types of Ser/Thr protein phosphatases. Defects in these phosphatases are correlated with the occurrence of various diseases such as cancer and neuropathy. Recently, we developed phosphorylation mimic phage display (PMPD) method with AlF4-, a methodology to identify substrates for FCP/SCP type Ser/Thr phosphatase Scp1. Here, we report a PMPD method using BeF3- to identify novel substrate peptides bound to Scp1. After screening peptide phages, we identified peptides that bound to Scp1 in a BeF3--dependent manner. Synthetic phosphopeptide BeM12-1, the sequence of which was isolated at the highest frequency, directly bound to Scp1. The binding was inhibited by adding BeF3-, indicating that the peptide binds to the active center of catalytic site in Scp1. The phosphorylated BeM12-1 worked as a competitive inhibitor of Scp1. Thus, PMPD method may be applicable for the identification of novel substrates and inhibitors of the FCP/SCP phosphatase family.