Article

Sipl1 and Rbck1 are novel Eya1-binding proteins with a role in craniofacial development.

Leibniz Institute for Age Research-Fritz Lipmann Institute, Jena, Germany.
Molecular and Cellular Biology (Impact Factor: 5.04). 10/2010; 30(24):5764-75. DOI: 10.1128/MCB.01645-09
Source: PubMed

ABSTRACT The eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies. For a better understanding of Eya1's function, we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as novel interaction partners of Eya1. We confirmed the interactions by glutathione S-transferase (GST) pulldown analysis and coimmunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Using reverse transcriptase PCR (RT-PCR) and in situ hybridization, we show that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. By morpholino-mediated knockdown, we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 ortholog as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype, with characteristic defects in ear and branchial arch formation.

0 Followers
 · 
101 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identified a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-β-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-α (ERα). Furthermore, FKBPL and RBCK1 associate with ERα at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.Oncogene advance online publication, 5 August 2013; doi:10.1038/onc.2013.306.
    Oncogene 08/2013; DOI:10.1038/onc.2013.306 · 8.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in the human EYA1 gene have been associated with several human diseases including branchio-oto (BO) and branchio-oto-renal (BOR) syndrome, as well as congenital cataracts and ocular anterior segment anomalies. BOR patients suffer from severe malformations of the ears, branchial arches and kidneys. The phenotype of Eya1-heterozygous mice resembles the symptoms of human patients suffering from BOR syndrome. The Eya1 gene encodes a multifunctional protein that acts as a protein tyrosine phosphatase and a transcriptional coactivator. It has been shown that Eya1 interacts with Six transcription factors, which are also required for nuclear translocation of the Eya1 protein. We investigated the effects of seven disease-causing Eya1 missense mutations on Eya1 protein function, in particular cellular localization, ability to interact with Six proteins, and protein stability. We show here that the BOR-associated Eya1 missense mutations S454P, L472R, and L550P lead to enhanced proteasomal degradation of the Eya1 protein in mammalian cells. Moreover, Six proteins lead to a significant stabilization of Eya1, which is caused by Six-mediated protection from proteasomal degradation. In case of the mutant L550P, loss of interaction with Six proteins leads to rapid protein degradation. Our observations suggest that protein destabilization constitutes a novel disease causing mechanism for Eya1.
    PLoS ONE 01/2014; 9(1):e87407. DOI:10.1371/journal.pone.0087407 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-κB (NF-κB) signaling. NF-κB is crucial for inflammatory and immune responses, and aberrant NF-κB signaling is implicated in multiple disorders. We found that LUBAC (linear ubiquitin chain assembly complex), composed of HOIL-1L, HOIP, and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-κB pathway. Moreover, specific deubiquitinases (DUBs), such as CYLD, A20 (TNFAIP3), and OTULIN/gumby, inhibit LUBAC-induced NF-κB activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B cell lymphomas, and Parkinson's disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-κB activation pathway regulation by LUBAC.
    Journal of Biochemistry 08/2013; DOI:10.1093/jb/mvt079 · 3.07 Impact Factor

Full-text (2 Sources)

Download
35 Downloads
Available from
Jun 2, 2014