SPARC Fusion Protein Induces Cellular Adhesive Signaling

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Abstract

Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive matricellular protein with a diversity of biological functions associated with morphogenesis, remodeling, cellular migration, and proliferation. We have produced mouse SPARC with a FLAG-tag at the N-terminus of SPARC (Flag-SPARC, FSP) in a Bac-to-Bac baculoviral expression system. After affinity purification, this procedure yields SPARC of high purity, with an electrophoretic mobility of ∼44 kDa under reducing conditions, and ∼38–39 kDa under non-reducing conditions. Unexpectedly, FSP adsorbed to plastic supported cell attachment and spreading, in a calcium-dependent manner. The adhesive activity of native FSP was inhibited by prior incubation with anti-SPARC IgG. Cell adhesion to FSP induced the formation of filopodia and lamellipodia but not focal adhesions that were prominent on cells that were attached to fibronectin. In addition, FSP induced the tyrosine phosphorylation of FAK and paxillin in attached epithelial cells. Erk1/2 and Rac were also activated in cells attached to FSP, but at a lower level in comparison to cells on fibronectin. This study provides new insight into the biological functions of SPARC, a matricellular protein with important roles in cell-extracellualr matrix interactions.

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Most cited references 32

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The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.

A J Ridley, A. Hall (1992)

Actin stress fibers are one of the major cytoskeletal structures in fibroblasts and are linked to the plasma membrane at focal adhesions. rho, a ras-related GTP-binding protein, rapidly stimulated stress fiber and focal adhesion formation when microinjected into serum-starved Swiss 3T3 cells. Readdition of serum produced a similar response, detectable within 2 min. This activity was due to a lysophospholipid, most likely lysophosphatidic acid, bound to serum albumin. Other growth factors including PDGF induced actin reorganization initially to form membrane ruffles, and later, after 5 to 10 min, stress fibers. For all growth factors tested the stimulation of focal adhesion and stress fiber assembly was inhibited when endogenous rho function was blocked, whereas membrane ruffling was unaffected. These data imply that rho is essential specifically for the coordinated assembly of focal adhesions and stress fibers induced by growth factors.

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Focal adhesions, contractility, and signaling.

K. Burridge, M Chrzanowska-Wodnicka (1996)

Focal adhesions are sites of tight adhesion to the underlying extracellular matrix developed by cells in culture. They provided a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction that relate to growth control. The assembly of focal adhesions is regulated by the GTP-binding protein Rho. Rho stimulates contractility which, in cells that are tightly adherent to the substrate, generates isometric tension. In turn, this leads to the bundling of actin filaments and the aggregation of integrins (extracellular matrix receptors) in the plane of the membrane. The aggregation of integrins activates the focal adhesion kinase and leads to the assembly of a multicomponent signaling complex.

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The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.

A Ridley, Alexander H. G. Paterson, Bradley C. Johnston … (1992)

The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.

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Author and article information

Contributors

Hiromi Yanagisawa: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2013

Publication date (Electronic): 21 January 2013

Volume: 8

Issue: 1

Electronic Location Identifier: e53202

Affiliations

[1 ]Benaroya Research Institute, Seattle, Washington, United States of America

[2 ]Department of Biological Structure, School of Medicine, University of Washington, Seattle, Washington, United States of America

[3 ]Institute of Reproduction and Stem Cell Engineering, Central South University, Changsha, China

UT-Southwestern Med Ctr, United States of America

Author notes

* E-mail: qyanresearch@ 123456gmail.com

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: QY. Performed the experiments: LC. Analyzed the data: QY LC EHS. Wrote the paper: QY EHS LC.

Article

Publisher ID: PONE-D-12-20041

DOI: 10.1371/journal.pone.0053202

PMC ID: 3549909

PubMed ID: 23349702

SO-VID: ae67a8fa-27a9-417f-9014-9be0c7f97004

License:

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.

History

Date received : 8 July 2012

Date accepted : 28 November 2012

Page count

Pages: 8

Funding

This work was supported by National Institutes of Health Grant EY014150 and GM40711. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

SPARC Fusion Protein Induces Cellular Adhesive Signaling

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Most cited references 32

The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.

Focal adhesions, contractility, and signaling.

The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.

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