Modular interaction domains in phosphorylation and epigenetic signaling – specificity, affinity and interactome analysis
Professor Shawn Li
Department of Biochemistry
Tuesday, November 20, 2012
C2-361 (Reading Room)
Abstract: Modular interaction domains play an important role in cellular signal transduction by mediating the formation of specific protein-protein complexes in the cytosol as well as in the nucleus. Thousands of such domains are found in humans, making them a significant feature of the human genome. Our lab has been focused on characterizing the specificity and affinity of and networking by two classes of modular domains: the Src homology 2 or SH2 domain that reads out phosphotyrosine in kinase signaling and chromatin-binding modules that bind to methyllysine sites. Using peptide arrays, we defined the specificity for the majority of the human SH2 complement; and by comprehensive structural bioinformatic analysis, we uncovered a new paradigm for encoding SH2 domain specificity by surface loops. In addition, we used phage display library screening to evolve the SH2 domain in vitro and identified variants, which we term “superbinders”, that bind to phosphotyrosine with greatly enhanced affinities. These superbinders are capable of blocking cancer cell growth in vitro and causing precocious differentiation of mouse ES cells.
In contrast to the SH2 domains, protein modules such as the chromodomain (CD) recognize methylated Lys residues found on both histone and non-histone proteins. However, our knowledge on the prevalence and function of non-histone protein methylation is poor. We have developed a strategy that combines peptide array, bioinformatics and mass spectrometry to systematically identify lysine methylation sites in proteins and methyllysine-mediated protein-protein interactions. We demonstrate the utility of this approach by identifying the first methyllysine-driven interactome of heterochromatin protein (HP) 1β and uncovering numerous methylated proteins and the corresponding methyllysine sites. We show, by heavy methyl SILAC and MRM-MS, that lysine methylation is a highly dynamic post-translational modification that undergoes widespread and large changes under cellular stress. Furthermore, we provide evidence that lysine methylation plays a pivotal role in the function of DNA-dependent protein kinase (DNA-PK) and its interaction with HP1β during DNA damage response. Our approach may be used to uncover the protein methylome and the methyl interactome when it is extended systematically to methyllysine-binding modules.