Abstract B122: Sumoylation regulates nuclear signaling of ErbB4 intracellular domain

Abstract

Abstract The members of the ErbB family (EGFR/ErbB1, ErbB2, ErbB3, and ErbB4) of receptor tyrosine kinases (RTK) regulate key cellular processes including proliferation, differentiation, and survival, and play critical roles in several human malignancies. The alternatively spliced ErbB4 JM-a isoform was the first RTK found to signal by a mechanism involving proteolytic release of an intracellular receptor domain (ICD) upon ligand binding. The released ErbB4 ICD can translocate into the nucleus, where it functions as a transcriptional coregulator. ErbB4 ICD has been shown to regulate mammary epithelial development in vivo. Furthermore, nuclear ErbB4 immunoreactivity is frequently observed in clinical breast cancer samples. Although these findings suggest that ErbB4 signaling via ICD is biologically significant, the factors that regulate subcellular distribution and nuclear functions of ErbB4 ICD are largely unknown. We hypothesized that ErbB4 ICD is likely to be regulated by different mechanisms than the ones controlling conventional RTK signaling. One regulatory mechanism that is often employed by nuclear proteins is the small ubiquitin-like modifier (SUMO) system. SUMO proteins modify protein substrates covalently, and alter their function through changes in activity, stability, or subcellular localization. Here, we demonstrate by pull-down and western blotting of cell culture lysates that ErbB4 ICD is modified by SUMO. Full-length ErbB4 was not sumoylated, indicating that SUMO is a specific regulatory modification for the soluble ICD. By using site-directed mutagenesis followed by sumoylation assays, we mapped the major SUMO acceptor site in ErbB4. Wild type and sumoylation deficient mutant of ErbB4 did not differ in their ability to activate canonical RTK signaling pathways downstream of ErbB4. However, fractionation experiments of MCF-7 breast cancer cells showed weaker nuclear targeting of sumoylation deficient mutant compared to wild-type ErbB4 ICD. Protein inhibitor of activated STAT3 (PIAS3), a SUMO E3 ligase that could stimulate the ErbB4 ICD sumoylation, modulated the transcriptional coregulatory activity of ErbB4 ICD in luciferase reporter assays, as well as the function of ErbB4 ICD in HC11 mouse mammary epithelial cell differentiation. Based on these findings we propose that the SUMO system regulates nuclear localization and signaling of ErbB4 ICD. Citation Format: Anna M. Knittle, Maria Sundvall, Katri Vaparanta, Klaus Elenius. Sumoylation regulates nuclear signaling of ErbB4 intracellular domain. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B122.