title: The Ras signaling inhibitor LOX-PP interacts with Hsp70 and c-Raf to reduce Erk activation and transformed phenotype of breast cancer cells.
abstract: The lysyl oxidase gene (LOX) inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162-amino-acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibits Erk signaling, motility, and tumor formation in a breast cancer xenograft model; however, its mechanism of action is largely unknown. Here, a copurification-mass spectrometry approach was taken using ectopically expressed LOX-PP in HEK293T cells and the heat shock/chaperone protein Hsp70 identified. Hsp70 interaction with LOX-PP was confirmed using coimmunoprecipitation of intracellularly and bacterially expressed and endogenous proteins. The interaction was mapped to the Hsp70 peptide-binding domain and to LOX-PP amino acids 26 to 100. LOX-PP association reduced Hsp70 chaperone activities of protein refolding and survival after heat shock. LOX-PP interacted with the Hsp70 chaperoned protein c-Raf. With the use of ectopic expression of LOX-PP wild-type and deletion proteins, small interfering RNA (siRNA) knockdown, and Lox(-/-) mouse embryo fibroblasts, LOX-PP interaction with c-Raf was shown to decrease downstream activation of MEK and NF-κB, migration, and anchorage-independent growth and reduce its mitochondrial localization. Thus, the interaction of LOX-PP with Hsp70 and c-Raf inhibits a critical intermediate in Ras-induced MEK signaling and plays an important role in the function of this tumor suppressor.
authors.affiliation: Department of Biochemistry, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Sato
authors.forename: Philip C
authors.initials: PC
authors.lastname: Trackman
authors.forename: Joni M
authors.initials: JM
authors.lastname: Mäki
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
authors.forename: Kathrin H
authors.initials: KH
authors.lastname: Kirsch
authors.forename: Gail E
authors.initials: GE
authors.lastname: Sonenshein
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D018631:ras Proteins / Q000037:antagonists & inhibitors*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; D008249:Protein-Lysine 6-Oxidase; D019908:Proto-Oncogene Proteins c-raf; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D018631:ras Proteins