Pin1 Catalyzes Conformational Changes of Thr-187 in p27Kip1 and Mediates Its Stability through a Polyubiquitination Process

Wei Zhou,Qiaoyun Yang,Choon Bing Low,Balakrishna Chandrababu Karthik,Yu Wang,Akihide Ryo,Shao Q Yao,Daiwen Yang,Yih-Cherng Liou

doi:10.1074/jbc.m109.022814

Abstract

The cis-trans peptidylprolyl isomerase Pin1 plays a critical role in regulating a subset of phosphoproteins by catalyzing conformational changes on the phosphorylated Ser/Thr-Pro motifs. The phosphorylation-directed ubiquitination is one of the major mechanisms to regulate the abundance of p27(Kip1). In this study, we demonstrate that Pin1 catalyzes the cis-trans conformational changes of p27(Kip1) and further mediates its stability through the polyubiquitination mechanism. Our results show that the phosphorylated Thr-187-Pro motif in p27(Kip1) is a key Pin1-binding site. In addition, NMR analyses show that this phosphorylated Thr-187-Pro site undergoes conformational change catalyzed by Pin1. Moreover, in Pin1 knock-out mouse embryonic fibroblasts, p27(Kip1) has a shorter lifetime and displays a higher degree of polyubiquitination than in Pin1 wild-type mouse embryonic fibroblasts, suggesting that Pin1 plays a critical role in regulating p27(Kip1) degradation. Additionally, Pin1 dramatically reduces the interaction between p27(Kip1) and Cks1, possibly via isomerizing the cis-trans conformation of p27(Kip1). Our study thus reveals a novel regulatory mechanism for p27(Kip1) stability and sheds new light on the biological function of Pin1 as a general regulator of protein stability.

Highlights

With Ser-178, which has not yet been well studied, the phosphorylation of Ser-10 and Thr-187 has been well characterized to be important for the regulation of p27Kip1 function
We show that Pin1 binds to p27Kip1, mainly through the phosphorylated Thr-187-Pro motif, and causes subsequent prolyl isomerization of this cell cycle protein
To confirm that Pin1 could interact with p27Kip1, we performed glutathione S-transferase (GST) pulldown assays using recombinant GST-Pin1, GST-Pin1W34A, and GST-Pin1R68A/R69A mutants or GST proteins to pull down FLAG-p27Kip1 protein overex

Summary

EXPERIMENTAL PROCEDURES

Constructs, Reagents, and Antibodies—Full-length cDNAs for p27Kip, Cks, and Skp were cloned from a HeLa cDNA library and inserted into the pXJ-40-FLAG vector and/or pXJ40-GFP vector Antibodies used for Western blotting and pulldown assays were as follows: polyclonal antibody against p27Kip (C-19), monoclonal antibody against p27Kip (F-8), polyclonal antibodies against Ser(P)-10-p27Kip, Skp (H-435), Cks (FL-79), and ubiquitin (P4D1) (Santa Cruz Biotechnology); polyclonal antibody against Thr(P)-187-p27Kip (Zymed Laboratories Inc.); monoclonal antibodies against ␣-tubulin and FLAG (Sigma). Beads were rocked with lysates from cells overexpressing WT or mutant p27Kip constructs for 3 h at 4 °C, followed by five washes with mammalian lysis buffer. Protein Stability Assay—Pin1-WT and -KO MEF cells were transfected with FLAG-p27Kip WT or mutant constructs. The supernatants of cell lysates were incubated with FLAG-M2 beads at 4 °C for 3 h, followed by five washes with mammalian lysis buffer and Western blotting using anti-ubiquitin antibody. Reciprocal experiments were carried out by co-overexpressing FLAG-ubiquitin and GFPp27Kip in Pin1-WT and KO MEF cells. TOSCY experiments [35] were carried out at a mixing time of 75 ms and 8 scans

RESULTS

The crystallographic structure of

DISCUSSION