Examination of serine and threonine phosphorylation of the STAR protein Sam68

The signal transduction and activation of RNA (STAR) proteins are a family of proteins that contain an RNA binding STAR domain. The best characterised member of the STAR family is Sam68, a predominantly nuclear protein that can also be found in the cytoplasm where it acts as an adapter protein in various signaling cascades. One of the main biological functions of Sam68 is its role in alternative splicing of specific target genes such as CD44, cyclin D1 and Bcl-X (Frisone et al., 2015). The function of Sam68 in alternative splicing can be regulated by post-translational modifications (PTMs) such as serine/threonine phosphorylation (Matter et al., 2002) tyrosine phosphorylation (Lukong et al., 2005), arginine methylation (Côté et al., 2003) and lysine acetylation (Babic et al., 2004). The precise mapping of these PTMs on Sam68 and how they affect the function of Sam68 is currently unknown. Here, we aimed to determine site specific mapping of serine and threonine phosphorylation sites in the intrinsically disordered N-terminal domain (NTD) of Sam68 using NMR. Assignment of the N-terminal domain of Sam68 would allow measurements of NMR spectra of the protein in the presence of specific kinases as well to determine the phosphorylation sites at a residue specific level. Furthermore, we aimed to characterise the functional consequences of the phosphorylation in terms of RNA binding and splicing using a series of point mutants where targeted residues could no longer be phosphorylated. We also began preliminary studies of the STAR domain of Sam68 using in-cell NMR in collaboration Professor Yutaka Ito at Tokyo Metropolitan University, Japan.