Hsp90 Binds Directly to Fibronectin (FN) and Inhibition Reduces the Extracellular Fibronectin Matrix in Breast Cancer Cells

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isola...

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Veröffentlicht in:	PloS one Jg. 9; H. 1; S. e86842
Hauptverfasser:	Hunter, Morgan C., O’Hagan, Kyle L., Kenyon, Amy, Dhanani, Karim C. H., Prinsloo, Earl, Edkins, Adrienne L.
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	United States Public Library of Science 22.01.2014 Public Library of Science (PLoS)
Schlagworte:	Bacterial Proteins Binding Binding sites Biochemistry Biology Biotechnology Breast cancer Breast Neoplasms - metabolism Cancer Cancer cells Cancer metastasis Cell adhesion & migration Cell Line, Tumor Cell migration Chromatography, Affinity Collagen Confocal microscopy Crosslinking Electrophoresis, Polyacrylamide Gel Embryogenesis Embryonic growth stage Endoplasmic reticulum Enzyme-Linked Immunosorbent Assay Epidermal growth factor Extracellular Matrix - metabolism Female Fibronectin Fibronectins Fibronectins - metabolism Fluorescence Heat shock proteins HSP90 Heat-Shock Proteins - metabolism Hsp90 protein Humans Immunoprecipitation Inhibition Internalization Kinases LAMP-1 protein Low density lipoprotein receptors Mass spectrometry Mass spectroscopy Matrix protein MCF-7 Cells Medicine Metastases Metastasis Microscopy Microscopy, Confocal Novobiocin Penicillin Proteins RNA Interference Sepharose - analogs & derivatives Spectroscopy Stability Surface Plasmon Resonance Tandem Mass Spectrometry Tumor cell lines Wound healing South Africa
ISSN:	1932-6203, 1932-6203
Online-Zugang:	Volltext
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Zusammenfassung:	Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.
Bibliographie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: ALE MCH KLOH. Performed the experiments: MCH KLOH AK KCHD. Analyzed the data: MCH KLOH AK KCHD EAP ALE. Wrote the manuscript with contributions from all other authors: ALE MCH.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0086842