Tay Bridge Is a Negative Regulator of EGFR Signalling and Interacts with Erk and Mkp3 in the Drosophila melanogaster Wing

Abstract

The regulation of Extracellular regulated kinase (Erk) activity is a key aspect of signalling by pathways activated by extracellular ligands acting through tyrosine kinase transmembrane receptors. In this process, participate proteins with kinase activity that phosphorylate and activate Erk, as well as different phosphatases that inactivate Erk by de-phosphorylation. The state of Erk phosphorylation affects not only its activity, but also its subcellular localization, defining the repertoire of Erk target proteins, and consequently, the cellular response to Erk. In this work, we characterise Tay bridge as a novel component of the EGFR/Erk signalling pathway. Tay bridge is a large nuclear protein with a domain of homology with human AUTS2, and was previously identified due to the neuronal phenotypes displayed by loss-of-function mutations. We show that Tay bridge antagonizes EGFR signalling in the Drosophila melanogaster wing disc and other tissues, and that the protein interacts with both Erk and Mkp3. We suggest that Tay bridge constitutes a novel element involved in the regulation of Erk activity, acting as a nuclear docking for Erk that retains this protein in an inactive form in the nucleus.

Tay bridge and extracellular‐regulated kinase activity are required for motoneuron function in the Drosophila neural system

“…tay is expressed in a generalized manner in all epithelial cells of the imaginal discs, and in the wing disc is required to antagonize Ras/ERK signaling . Considering the link between vertebrate AUTS2 and neural development, we first analyzed the expression of Tay in neuronal cells developing from the eye imaginal disc.…”

“…OK6‐GAL4 is a PGal4 insertion into the Rapgap1 locus, and its expression is restricted to motoneurons . We also used the UAS (Upstream Activating Sequence) lines UAS‐Erk sem , UAS‐tay and UAS‐mCD8GFP , the RNA interference lines UAS‐Mkp3‐i (23911), UAS‐tay‐i (29021) and UAS‐rolled‐i (35641) UAS‐raf‐i (20909) and UAS‐cic‐i (40867) from the VDRC Stock Center (Dr. Bohr‐Gasse 3, A‐1030 Vienna Austria), and UAS‐EGFR‐i (10079R‐2) and UAS‐ras‐i (9375R‐1) from NIG‐Fly (Kyoto; Japan). Lines not described in the text can be found in flybase …”

“…We used the rabbit antibody anti‐diphosphorylated ERK 1&2 Cell Signaling Technology (Schuttersveld 2 2316 ZA Leiden; The Netherlands) mouse monoclonal antibodies anti‐Arm, anti‐FasII, anti‐Dlg and anti‐Elav (Hybridoma Bank at University of Iowa; Iowa City, Iowa), and the rabbit antibody anti‐Mira . We also used guinea pig anti‐Tay . Alexa Fluor secondary antibodies (used at 1:200 dilution) were from Invotrogen (Thermo Fisher Scientific 168 Third Avenue Waltham, MA USA).…”

“…Drosophila Tay bridge ( tay ) encodes a nuclear protein of 2486 amino acids expressed in the embryonic central nervous system (CNS) and in other ectodermal derivatives such as the imaginal discs . The analysis of tay loss‐ and gain‐of‐function conditions in the wing disc uncovered a functional relationship between Tay and the epidermal growth factor receptor (EGFR) signaling pathway, and it was suggested that Tay facilitates the inactivation of extracellular‐regulated kinase (ERK), acting as a nuclear anchor that promotes ERK nuclear retention in an inactive state and/or its inactivation by nuclear phosphatases . The analysis of tay viable alleles also showed that mutant tay flies display reduced walking speed, reduced sensitivity to the effects of alcohol and defective compensation of rotatory stimuli during walking .…”

“…Despite the structural and sequence differences between Tay and AUTS2, it is intriguing that both genes play a role in neural development and that tay mutant flies display behavioral phenotypes reminiscent of alterations in AUTS2. Furthermore, overexpression of human AUTS2 in Drosophila wing discs cause a phenotype indicative of altered ERK signaling, suggesting that both proteins retain to some extent similar interaction domains . The Ras‐ERK signaling pathway plays a variety of roles during the development of the nervous system, ranging from the patterning of neural progenitors along the Dorso‐Ventral (D/V) axes in the neuroectoderm to the regulation of synaptic plasticity at the neuromuscular junction .…”