Cloning and Characterization of a Novel Adaptor Protein, CIN85, That Interacts with c-Cbl

Hematology Branch, National Institutes of Health, Bethesda, Maryland, 20892, USA.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 03/2000; 268(2):321-8. DOI: 10.1006/bbrc.2000.2147
Source: PubMed


The c-Cbl protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel c-Cbl-interacting protein termed CIN85 with a molecular mass of 85 kDa which shows similarity to adaptor proteins, CMS and CD2AP. CIN85 mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. CIN85 was basally associated with c-Cbl. For interaction of CIN85 with c-Cbl, the second SH3 domain of CIN85 was shown to serve as a central player. The CIN85-c-Cbl association was enhanced shortly after stimulation of 293 cells with epidermal growth factor (EGF) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of c-Cbl. Our results suggest that CIN85 may play a specific role in the EGF receptor-mediated signaling cascade via its interaction with c-Cbl.

2 Reads
  • Source
    • "CIN85 is identified as a Cbl-interacting protein of 85 kDa and belongs to adaptor/scaffold proteins [1,2]. It consists of three Src homology (SH3) domains, a proline-rich region and a putative α-helical coiled-coil domain at the extreme C-terminal end [1]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Results Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. Conclusions As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.
    BMC Biochemistry 07/2014; 15(1):13. DOI:10.1186/1471-2091-15-13 · 1.44 Impact Factor
  • Source
    • "Notably, 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-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) [10] identified as an associating protein. "
    [Show abstract] [Hide abstract]
    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.
    PLoS ONE 10/2013; 8(10):e77288. DOI:10.1371/journal.pone.0077288 · 3.23 Impact Factor
  • Source
    • "The ubiquitin ligase that mediates nephrin ubiquitination has not been determined. c-Cbl is the E3 enzyme originally identified as a binding partner of CIN85 and mediates ubiquitination of many receptors [66]. It will be interesting to determine whether c-Cbl cooperates with CIN85 and ubiquitinates nephrin in podocytes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Podocytes are highly specialized epithelial cells localized in the kidney glomerulus. The distinct cell signaling events and unique cytoskeletal architecture tailor podocytes to withstand changes in hydrostatic pressure during glomerular filtration. Alteration of glomerular filtration leads to kidney disease and frequently manifests with proteinuria. It has been increasingly recognized that cell signaling and cytoskeletal dynamics are coupled more tightly to membrane trafficking than previously thought. Membrane trafficking coordinates the cross-talk between protein networks and signaling cascades in a spatially and temporally organized fashion and may be viewed as a communication highway between the cell exterior and interior. Membrane trafficking involves transport of cargo from the plasma membrane to the cell interior (i.e., endocytosis) followed by cargo trafficking to lysosomes for degradation or to the plasma membrane for recycling. Yet, recent studies indicate that the conventional classification does not fully reflect the complex and versatile nature of membrane trafficking. While the increasing complexity of elaborate protein scaffolds and signaling cascades is being recognized in podocytes, the role of membrane trafficking is less well understood. This review will focus on the role of membrane trafficking in podocyte health and disease.
    Pediatric Nephrology 08/2012; 28(9). DOI:10.1007/s00467-012-2281-y · 2.86 Impact Factor
Show more