title: Inhibition of CIN85-mediated invasion by a novel SH3 domain binding motif in the lysyl oxidase propeptide.
abstract: The lysyl oxidase gene 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 inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the "Cbl-interacting protein of 85-kDa" (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Sato
authors.forename: Yingshe
authors.initials: Y
authors.lastname: Zhao
authors.forename: Misa
authors.initials: M
authors.lastname: Imai
authors.forename: Philip C
authors.initials: PC
authors.lastname: Simister
authors.forename: Stephan M
authors.initials: SM
authors.lastname: Feller
authors.forename: Philip C
authors.initials: PC
authors.lastname: Trackman
authors.forename: Kathrin H
authors.initials: KH
authors.lastname: Kirsch
authors.forename: Gail E
authors.initials: GE
authors.lastname: Sonenshein
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D064593:Heterografts; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009361:Neoplasm Invasiveness; D009368:Neoplasm Transplantation; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C433810:SH3KBP1 protein, human; D008249:Protein-Lysine 6-Oxidase; D050721:Proto-Oncogene Proteins c-cbl; C067419:CBL protein, human