Scapinin, the Protein Phosphatase 1 Binding Protein, Enhances Cell Spreading and Motility by Interacting with the Actin Cytoskeleton

Copyright (c) 2009 Sagara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.ArticlePLOS ONE. 4(1):e4247 (2009)journal articl

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.

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

Induction of scapinin expression in Hela cells and its effect on cell proliferation.

<p>(<i>A</i>) Tetracycline-induced expression of scapinin in Hela cells. Hela cells were cultured with tetracycline at the indicated concentrations for 24 hours. Cell lysates were separated by SDD-polyacrylamide gel electrophoresis, and scapinin expression levels were measured with Western blotting using anti-scapinin monoclonal antibody. Human whole brain lysate (Clontech Laboratories Inc.) was also loaded. Twenty microgram proteins were applied to each lane. Scapinin expression levels of Hela cells were expressed as folds against the whole brain lysate. (<i>C</i>) The effect of scapinin on the proliferation of Hela cells. Hela cells were plated at 1×10⁴ cells/ well in 96-well plates and cultured with (open bars) or without (closed bars) 0.1 µg/ml tetracycline. At each time point, the number of viable cells was assessed by an MTT assay, and the absorbance was measured at a wavelength of 570 nm with a 96-well plate reader. Data are expressed as mean±SEM of four separate experiments.</p

GFP-scapinin-induced morphological changes in Cos7 cells.

<p>pEGFP empty vectors (<i>A</i>) and pEGFP-scapinin (<i>B</i>) were transfected into Cos7 cells, and the cells were cultured for 16 hours. The cell morphology and the distribution of GFP-scapinin were monitored with fluorescent microscopy and photographed. (<i>C</i>) The distribution of GFP-scapinin in ruffles. (<i>D</i>) Colocalization of scapinin and actin in ruffles. Cos7 cells expressing GFP-scapinin were fixed at 16 hours and then stained with rhodamine-phalloidin (red) to visualize actin. Localization of GFP-scapinin (green) and actin (red) was observed with a confocal microscopy. Ruffles were shown by arrows. Bars: 20 µm.</p

Distribution of scapinin and actin in Hela cells.

<p>(<i>A</i>) Hela cells grown on glass coverslips were cultured in the presence (Tet+) or absence (Tet−) of 0.1 µg/ml tetracycline and were then fixed at 20 hours. After permeabilization, the distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively. Scapinin and actin are colocalized (arrows). (<i>B</i>) Confocal microscopic observation. Hela cells were cultured in the presence of 0.1 µg/ml tetracycline for 20 hours and were then stained with anti-scapinin antibody (green) and rhodamine-phalloidin (red) as in (<i>A</i>). Scapinin and actin are colocalized (arrows). Bar: 20 µm. (<i>C</i>) Absence of scapinin in actin stress fibers. Hela cells grown on a glass coverslip were cultured in the presence of 0.1 µg/ml tetracycline and were then fixed at 8 hours. The distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively as (<i>A</i>). There are four cells; two cells express low levels of scapinin (asterisks), and the other two cells express high levels of scapinin (arrow heads). Bar: 20 µm.</p

The RPEL repeats of scapinin interact with purified skeletal muscle actin and inhibit actin polymerization <i>in vitro</i>.

<p>(<i>A</i>) GST or GST-RPEL repeats (350–422 aa) were covalently conjugated to CNBr-agarose beads (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004247#pone-0004247-g001" target="_blank">Figure 1C</a>) and were incubated with purified skeletal muscle actin. After washing with RIPA buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mM Tris-HCl pH 8.0, 150 mM NaCl), bound proteins were eluted with SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and stained with coomassie brilliant blue (CBB). GST and GST-RPEL proteins (shown by a asterisk) were partly released from the beads by elution with SDS sample buffer. (<i>B</i>) Inhibition of actin polymerization by the RPEL repeats. Skeletal muscle actin (18 µM) was incubated with GST- RPEL repeats (350–422 aa) or GST at the indicated concentrations to polymerize at room temperature for 30 minutes, and then filamentous actin (P) and monomeric actin (S) were separated by ultracentrifugation. Aliquots were analyzed by SDS polyacrylamide gel electrophoresis and stained with coomassie brilliant blue (CBB). Since the contamination of supernatants in the pellet fraction was technically inevitable, a small portion of GST-RPEL construct was seen in the pellet. (<i>C</i>) The density of each actin band was measured by a densitometer and plotted.</p

Enhancement of cell spreading and motility by scapinin.

<p>(<i>A</i>) Enhancement of cell spreading by scapinin. Inducible Hela cells were seeded onto an 8-chambered glass-slide and then cultured with (Tet+) or without (Tet−) 0.1 µg/ml tetracycline. Cell morphology was monitored with a light microscope and photographed at 12 hours. (<i>B</i>) Inducible Hela cells were treated as in (<i>A</i>), and cell morphology was monitored at specified times. Adherent and non-adherent cells were counted under a microscope. Data are expressed as mean±SED from four independent experiments. Student's t test: *: P<0.05; **: P<0.01. The parental Hela cells were also cultured with (Tet+) or without (Tet−) of tetracycline, and cell morphology was monitored. (<i>C</i>) Enhancement of cell motility by scapinin. To measure cell motility, we used a wound healing assay. Hela cells were cultured on 6-well plates until confluence. The confluent monolayer cultures were treated with (Tet+) or without (Tet−) 0.1 µg/ml tetracycline for 4 hours and were then wounded with a straight scratch using a yellow pipette tip. After washing them three times with serum-free DMEM, the wounded monolayer cultures were further incubated (Tet+) with or without (Tet−) 0.1 µg/ml tetracycline in DMEM containing 1% fetal bovine serum. To reduce cell growth, the serum concentration was reduced to 1%. At 1, 24, and 48 hours after the wounding, the cell monolayer was photographed. The front of cell migration at 24 hours was shown in (<i>D</i>).</p

Time course of morphological changes of Cos7 cells induced by a PP1-binding deficient mutant of GFP-scapinin (F515A).

<p>After transfection of pEGFP-scapinin F515A mutant, morphological changes of Cos7 cells were monitored and photographed at indicated times under a fluorescence microscopy. Bar: 20 µm.</p