Caracterização parcial do complexo SCF1 contendo a proteína FBXO25 fosforilada

Abstract

Partial characterization of SCF1 complex containing the protein phosphorylated FBXO25. Medeiros, A. C., 2015. Dissertação de mestrado 73p. Programa de Pós-graduação em Bioquímica da FMRP/USP. The FBXO25 is an E3 ligase RING type (Really Interesting New Gene), SCF oligomeric type, responsible for the specific recognition of the substrate to be degraded via the ubiquitinproteasome system (SUP). SUP is the main intracellular proteolytic mechanism responsible for 80-90% degradation of cytosolic and nuclear proteins. The FBXO25 is capable of forming a complex SCF1 (formed by the interaction of proteins Skp1, Cul1, Roc1 protein and a type Fbox), resulting in an active SCF complex which is able to ubiquitinate their substrates. This protein accumulates in the nucleus forming a subnuclear structure called FANDs (FBXO25 Associated Nuclear Domains) that are involved in nuclear ubiquitination. In this work, we purify complex SCF1 (WT or ΔF-box, which is not able to interact with Skp1), treated or not with PMA, by immunoprecipitation technique. We identified by mass spectrometry, an essential phosphorylation site for FBXO25, when it is phosphorylated under the action of the mitogenic reagent PMA. We also search for differentially ubiquitinated substrates for these complexes, by testing in ProtoArrays® identifying substrates involved in the signaling pathway ERK1 / 2.