additional serinethreonine phosphorylation reduces binding affinity but preserves interface topography of substrate proteins to the c-cbl tkb domain额外serinethreonine磷酸化减少亲和力但保存界面基质蛋白质的地形c-cbl tkb域.pdfVIP

Additional Serine/Threonine Phosphorylation Reduces Binding Affinity but Preserves Interface Topography of Substrate Proteins to the c-Cbl TKB Domain 1 2 2 2 1 Qingxiang Sun , Rebecca A. Jackson , Cherlyn Ng , Graeme R. Guy *, J. Sivaraman * 1 Department of Biological Sciences, National University of Singapore, Singapore, Singapore, 2 Institute of Molecular and Cell Biology, Biopolis, Singapore, Singapore Abstract The E3-ubiquitin ligase, c-Cbl, is a multi-functional scaffolding protein that plays a pivotal role in controlling cell phenotype. As part of the ubiquitination and downregulation process, c-Cbl recognizes targets, such as tyrosine kinases and the Sprouty proteins, by binding to a conserved (NX/R)pY(S/T)XXP motif via its uniquely embedded SH2 domain (TKB domain). We previously outlined the mode of binding between the TKB domain and various substrate peptide motifs, including epidermal growth factor receptor (EGFR) and Sprouty2 (Spry2), and demonstrated that an intrapetidyl hydrogen bond forms between the (pY-1) arginine or (pY-2) asparagine and the phosphorylated tyrosine, which is crucial for binding. Recent reports demonstrated that, under certain types of stimulation, the serine/threonine residues at the pY+ 1 and/or pY+2 positions within this recognition motif of EGFR and Sprouty2 may be endogenously phosphorylated. Using structural and binding studies, we sought to determine whether this additional phosphorylation could affect the binding of the TKB domain to these peptides and consequently, whether the type of stimulation can dictate the degree to which su