Phactr3/scapinin, a member of protein phosphatase 1 and actin regulator (phactr) family, interacts with the plasma membrane via basic and hydrophobic residues in the N-terminus.

Akihiro Itoh,Junji Sagara,Atsushi Uchiyama,Shunichiro Taniguchi,Eugene A Permyakov

doi:10.1371/journal.pone.0113289

Abstract

Proteins that belong to the protein phosphatase 1 and actin regulator (phactr) family are involved in cell motility and morphogenesis. However, the mechanisms that regulate the actin cytoskeleton are poorly understood. We have previously shown that phactr3, also known as scapinin, localizes to the plasma membrane, including lamellipodia and membrane ruffles. In the present study, experiments using deletion and point mutants showed that the basic and hydrophobic residues in the N-terminus play crucial roles in the localization to the plasma membrane. A BH analysis (http://helixweb.nih.gov/bhsearch) is a program developed to identify membrane-binding domains that comprise basic and hydrophobic residues in membrane proteins. We applied this program to phactr3. The results of the BH plot analysis agreed with the experimentally determined region that is responsible for the localization of phactr3 to the plasma membrane. In vitro experiments showed that the N-terminal itself binds to liposomes and acidic phospholipids. In addition, we showed that the interaction with the plasma membrane via the N-terminal membrane-binding sequence is required for phactr3-induced morphological changes in Cos7 cells. The membrane-binding sequence in the N-terminus is highly conserved in all members of the phactr family. Our findings may provide a molecular basis for understanding the mechanisms that allow phactr proteins to regulate cell morphogenesis.

Highlights

Protein phosphatase 1 (PP1) and actin regulatory (Phactr) proteins are a family that comprises four members in humans and other vertebrates; phactr1–4 [1]
In HeLa cells, phactr3 was distributed throughout the cells, but it was frequently localized to the plasma membrane including the lamellipodia and membrane ruffles (Fig. 1B)
The phactr protein family is considered to be involved with cell migration and morphogenesis by modulating the actin cytoskeleton, but their regulatory mechanisms are poorly understood [8,9,10,12]

Summary

Introduction

Protein phosphatase 1 (PP1) and actin regulatory (Phactr) proteins are a family that comprises four members in humans and other vertebrates; phactr1–4 [1]. Accumulating evidence indicates the involvement of phactr proteins in human diseases such as myocardial infarction, Parkinson’s disease, and cancers [2,3,4]. Each phactr protein contains four G-actin-binding RPEL motifs, including an N-terminal motif and a C-terminal triple RPEL repeat. RPEL motifs are found in the regulatory domains of myocardin-related transcription factor (MRTF) transcriptional coactivators where they control subcellular localization and activity by sensing signal-induced changes in the G-actin concentration [6,7]. Subcellular localization of phactr that was similar to that of MRTF is controlled by RPEL motifs. Phactr exhibits nuclear accumulation in response to serum-induced G-actin depletion [8]. There is no evidence for the serum-induced nuclear accumulation of phactr proteins other than phactr1 [8,9]

Methods

Results

Conclusion