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.
Highlights
Lysyl oxidase (LOX) is a key extracellular enzyme that controls collagen and elastin crosslinking, which is required for the biosynthesis of functional extracellular matrices
An atypical proline-rich Cblinteracting protein of 85-kDa’’ (CIN85) Src homology 3 (SH3) interacting ligand PxpxxRh was identified in the tumor suppressor protein LOX-PP that functionally inhibits CIN85-mediated invasion by breast cancer cells
Using co-purification/mass spectrometry, CIN85 was identified as a novel interacting partner of the tumor suppressor protein LOX-PP. Their direct interaction was confirmed and the interacting regions mapped to the SH3-B domain of CIN85 and to amino acids 111 to 119 of LOX-PP
Summary
Lysyl oxidase (LOX) (protein-6-oxidase; EC 1.4.3.13) is a key extracellular enzyme that controls collagen and elastin crosslinking, which is required for the biosynthesis of functional extracellular matrices. Ectopic LOX gene expression in gastric cancer cells inhibits tumor formation in nude mice [2] and reduces LOX expression has been reported in many carcinomas (reviewed in [3]). LOX-PP inhibits Ras signaling and the transformed phenotype in Ras-transformed NIH 3T3 fibroblasts [4], and in Her-2/neu-driven NF639 breast cancer cells [5]. Ectopic LOX-PP expression in NF639 or MiaPaCa2 pancreatic cancer cells reduces tumor xenograft formation in nude mice [5,6,7], and prevented growth of pre-existing NF639 tumors [8]. LOX-PP attenuates fibronectin-mediated integrin signaling via the focal adhesion kinase (FAK) - p130Cas pathway, and selectively inhibits integrin-mediated migration of breast cancer cells [9]. To further elucidate the mechanisms of LOX-PP action, co-affinitypurification/mass spectrometry was performed and the ‘‘Cblinteracting protein of 85-kDa’’ (CIN85) [10] identified as an associating protein
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