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: 4.78). 10/2010; 30(24):5764-75. DOI: 10.1128/MCB.01645-09
Source: PubMed


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.

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    • "Furthermore, differences in the expression profiles and tissue distribution could account for some of the relatively mild phenotypes, such as for example in Icap1-knockout mice, in which it is mostly bone formation that appears to be affected (Bouvard et al., 2007), or in the kindlin-3 (Fermt3)-knockout mouse, in which only leukocytes are affected (Moser et al., 2008). Another complication is that several integrin inhibitors also have integrin-independent functions, such as SHARPIN (Gerlach et al., 2011; He et al., 2010; Ikeda et al., 2011; Landgraf et al., 2010; Tokunaga et al., 2011) and filamins (for a review, see Zhou et al., 2010), which will also contribute to the overall phenotype observed. Finally, mice lacking one integrin inactivator might compensate by increasing the expression or activity of other inactivators, albeit examples of this remain to be identified. "
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    ABSTRACT: Integrins are heterodimeric transmembrane adhesion receptors composed of α- and β-subunits. They are ubiquitously expressed and have key roles in a number of important biological processes, such as development, maintenance of tissue homeostasis and immunological responses. The activity of integrins, which indicates their affinity towards their ligands, is tightly regulated such that signals inside the cell cruicially regulate the switching between active and inactive states. An impaired ability to activate integrins is associated with many human diseases, including bleeding disorders and immune deficiencies, whereas inappropriate integrin activation has been linked to inflammatory disorders and cancer. In recent years, the molecular details of integrin 'inside-out' activation have been actively investigated. Binding of cytoplasmic proteins, such as talins and kindlins, to the cytoplasmic tail of β-integrins is widely accepted as being the crucial step in integrin activation. By contrast, much less is known with regard to the counteracting mechanism involved in switching integrins into an inactive conformation. In this Commentary, we aim to discuss the known mechanisms of integrin inactivation and the molecules involved.
    Full-text · Article · Jul 2012 · Journal of Cell Science
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    • "The reasons for the presumed lack of interaction between Eya1 and Six1a in the neuronal lineage are unknown. However, three possible reasons are: (1) a co-factor for which Eya1 has more affinity than Six1a is present in the neuronal lineage, preventing Eya1 binding to Six1a and allowing Gro1 to bind Six1a in this lineage; (2) either Six1a, Eya1, or both, are modified at the post-translational level (for example, by phosphorylation or methylation) in the neuronal lineage and such modifications prevent their mutual interactions, leaving Six1a available to interact with Gro1; or (3) the Eya1-Six1a complex is more labile in vivo than it is in vitro and such a complex needs to be stabilized by a third partner only expressed in the sensory lineage but absent in the neuronal lineage, such Eya1 co-factors have been recently characterized in mice and zebrafish (Landgraf et al. 2010). "
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    ABSTRACT: While genes involved in the differentiation of the mechanosensory hair cells and the neurons innervating them have been identified, genes involved in balancing their relative numbers remain unknown. Six1a plays a dual role by promoting hair cell fate while inhibiting neuronal fate in these two lineages. Genes homologous to six1a act as either transcriptional activators or repressors, depending on the partners with which they interact. By assaying the in vivo and in vitro effects of mutations in presumptive protein-protein interacting and DNA-binding domains of Six1a, we show that, in the developing zebrafish inner ear, Six1a promotes hair cell fate by acting as a transcriptional activator and inhibits neuronal fate by acting as a transcriptional repressor. We also identify several potential partners for Six1a that differ between these two lineages. The dual role of Six1a in the developing otocyst provides a mechanism for balancing the relative number of hair cells and neurons during organogenesis of the inner ear.
    Full-text · Article · Sep 2011 · Developmental Biology
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    ABSTRACT: Precise control of the innate immune response is essential to ensure host defense against infection while avoiding inflammatory disease. Systems-level analyses of Toll-like receptor (TLR)-stimulated macrophages suggested that SHANK-associated RH domain-interacting protein (SHARPIN) might play a role in the TLR pathway. This hypothesis was supported by the observation that macrophages derived from chronic proliferative dermatitis mutation (cpdm) mice, which harbor a spontaneous null mutation in the Sharpin gene, exhibited impaired IL-12 production in response to TLR activation. Systems biology approaches were used to define the SHARPIN-regulated networks. Promoter analysis identified NF-κB and AP-1 as candidate transcription factors downstream of SHARPIN, and network analysis suggested selective attenuation of these pathways. We found that the effects of SHARPIN deficiency on the TLR2-induced transcriptome were strikingly correlated with the effects of the recently described hypomorphic L153P/panr2 point mutation in Ikbkg [NF-κB Essential Modulator (NEMO)], suggesting that SHARPIN and NEMO interact. We confirmed this interaction by co-immunoprecipitation analysis and furthermore found it to be abrogated by panr2. NEMO-dependent signaling was affected by SHARPIN deficiency in a manner similar to the panr2 mutation, including impaired p105 and ERK phosphorylation and p65 nuclear localization. Interestingly, SHARPIN deficiency had no effect on IκBα degradation and on p38 and JNK phosphorylation. Taken together, these results demonstrate that SHARPIN is an essential adaptor downstream of the branch point defined by the panr2 mutation in NEMO.
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