Functional studies with Ki‐1/57 protein and structural analyses of human protein RACK1

Abstract

The Ki‐1/57 protein has been discovered through the cross reactivity of the monoclonal antibody Ki‐1 in Hodgkin lymphoma cells. Recent studies have identified sumoylation as important post‐translational modification that regulates the biological functions of protein. In sumoylation, SUMO (small ubiquitin‐like modifier) is covalently attached to a lysine residue in a ΨKXE sequence (where Ψ is a large hydrophobic residue and X represents any amino acid). Furthermore, we found that Ki‐1/57 is a target for sumoylation in vitro and in vivo. The study objective was to generate mutants of the protein Ki‐1/57 to try identify which lysine residue are covalently attached to Sumo. RACK1(receptor of activated kinase 1) was isolated in a yeast two‐hybrid screen to interact with the protein Ki‐1/57. We performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X‐ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution. Fluorescence spectroscopy experiments after titration of Ki‐1/57(122–413) in solution with RACK1 and sedimentation equilibrium experiments showed that this association is strong.Supported by: FAPESP, CNPq