A Novel Protein Associated with Membrane-type 1 Matrix Metalloproteinase Binds p27kip1 and Regulates RhoA Activation, Actin Remodeling, and Matrigel Invasion

Daisuke Hoshino,Taizo Tomari,Makoto Nagano,Naohiko Koshikawa,Motoharu Seiki

doi:10.1074/jbc.m109.041400

Abstract

Pericellular proteolysis by membrane-type 1 matrix metalloproteinase (MT1-MMP) plays a pivotal role in tumor cell invasion. Localization of MT1-MMP at the invasion front of cells, e.g. on lamellipodia and invadopodia, has to be regulated in coordination with reorganization of the actin cytoskeleton. However, little is known about how such invasion-related actin structures are regulated at the sites where MT1-MMP localizes. During analysis of MT1-MMP-associated proteins, we identified a heretofore uncharacterized protein. This protein, which we call p27RF-Rho, enhances activation of RhoA by releasing it from inhibition by p27(kip1) and thereby regulates actin structures. p27(kip1) is a well known cell cycle regulator in the nucleus. In contrast, cytoplasmic p27(kip1) has been demonstrated to bind GDP-RhoA and inhibit GDP-GTP exchange mediated by guanine nucleotide exchange factors. p27RF-Rho binds p27(kip1) and prevents p27(kip1) from binding to RhoA, thereby freeing the latter for activation. Knockdown of p27RF-Rho expression renders cells resistant to RhoA activation stimuli, whereas overexpression of p27RF-Rho sensitizes cells to such stimulation. p27RF-Rho exhibits a punctate distribution in invasive human tumor cell lines. Stimulation of the cells with lysophosphatidic acid induces activation of RhoA and induces the formation of punctate actin structures within foci of p27RF-Rho localization. Some of the punctate actin structures co-localize with MT1-MMP and cortactin. Down-regulation of p27RF-Rho prevents both redistribution of actin into the punctate structures and tumor cell invasion. Thus, p27RF-Rho is a new potential target for cancer therapy development.

Highlights

Malignant tumor cells grow invasively and form distant metastases after moving through multiple tissue barriers
Localization of p27RFRho appears to be important for defining the subcellular region where GDP-RhoA can be activated by GEFs in response to stimuli as summarized in supplemental Fig
MT1-matrix metalloproteinases (MMPs) was detected in the HT1080 cells treated with Lysophosphatidic acid (LPA) (Fig. 7B). p27RF-Rho was idenprecipitate of p27RF-F

Summary

EXPERIMENTAL PROCEDURES

Plasmids—Complementary DNAs (cDNAs) encoding RhoGDI and CA-Lbc were gifts from Y. CDNAs for p27RF-Rho, RhoA, p115RhoGEF, transferrin receptor, and p27kip were obtained from HT1080 cells by reverse transcription-PCR. A p27kip1-binding Protein Regulates RhoA and A375 were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS; Hyclone), 100 units/ml penicillin, and 100 ␮g/ml streptomycin (Invitrogen). The Pulldown Assay for Rho-GTPases—Cells were washed with ice-cold PBS, scraped off the plates in cell-lysis buffer (50 mM Tris-HCl (pH 7.4), 2 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 100 mM NaCl, supplemented with 1 mM dithiothreitol, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 10 ␮g/ml pepstatin-A) on ice. Lysates were centrifuged for 5 min at 20,000 ϫ g at 4 °C. The pellet containing the beads was collected, washed 3 times with ice-cold cell lysis buffer, and subjected to SDS-PAGE followed by Western blot analysis using the indicated antibodies. The unpaired Student’s t test was used for analyzing differences between experimental groups

RESULTS

Findings

DISCUSSION