Actopaxin (α-Parvin) Phosphorylation Is Required for Matrix Degradation and Cancer Cell Invasion

Jeanine Pignatelli,Dominic Clarke,David P Lalonde,Sara E Lalonde,Christopher E Turner

doi:10.1074/jbc.m112.385229

Abstract

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.

Highlights

The process of matrix degradation is an integral component of cell invasion
Quantitation of both the percent of cells associated with matrix degradation (Fig. 1C) and the area of matrix degradation/cell area (Fig. 1D) demonstrated that the wild type (WT) and S4D/ S8D actopaxin cells had a significant enhancement in matrix degradation whereas the Quint actopaxin cells significantly inhibited matrix degradation compared with parental cells
To examine the role p38 MAPK activity in actopaxin-induced matrix degradation, WT, Quint, and S4D/S8D actopaxin-expressing cells were plated in the presence of the p38 MAPK inhibitor SB203580 (Fig. 6, B and C). p38 MAPK inhibition abolished matrix degradation in WT and S4D/S8D actopaxin cells. p38 MAPK has been shown to be regulated in a Rac1-dependent manner [34]; we evaluated whether treatment with the Rac1 inhibitor reduced p38 MAPK phosphorylation

Summary

Introduction

The process of matrix degradation is an integral component of cell invasion. Results: Actopaxin phosphorylation is required for matrix degradation. Conclusion: Actopaxin can act upstream of Rho GTPase signaling via an interaction with ␤-PIX and PAK to control matrix degradation. Phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Rac was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Actopaxin forms a complex with the Rac1/Cdc GEF ␤-PIX and Rac1/Cdc effector PAK1, to regulate actopaxindependent matrix degradation. This study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling

Methods

Results

Conclusion