The E3 ubiquitin ligase HERC1 controls the ERK signaling pathway targeting C-RAF for degradation

// Taiane Schneider 1 , Arturo Martinez-Martinez 1 , Monica Cubillos-Rojas 1 , Ramon Bartrons 1 , Francesc Ventura 1 and Jose Luis Rosa 1 1 Departament de Ciencies Fisiologiques, IDIBELL, Campus Bellvitge, Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain Correspondence to: Jose Luis Rosa, email: joseluisrosa@ub.edu Keywords: ubiquitin; ERK; RAF; proliferation; protein degradation Received: September 08, 2017     Accepted: July 12, 2018     Published: July 31, 2018 ABSTRACT The RAF/MEK/ERK cascade is a conserved intracellular signaling pathway that controls fundamental cellular processes including growth, proliferation, differentiation, survival and migration. Aberrant regulation of this signaling pathway has long been associated with human cancers. A major point of regulation of this pathway occurs at the level of the serine/threonine protein kinase C-RAF. Here, we show how the E3 ubiquitin ligase HERC1 regulates ERK signaling. HERC1 knockdown induced cellular proliferation, which is associated with an increase in ERK phosphorylation and in C-RAF protein levels. We demonstrate that overexpression of wild-type C-RAF is sufficient to increase ERK phosphorylation. Experiments with pharmacological inhibitors of RAF activity, or with interference RNA, show that the regulation of ERK phosphorylation by HERC1 is RAF-dependent. Immunoprecipitation, pull-down and confocal fluorescence microscopy experiments demonstrate an interaction between HERC1 and C-RAF proteins. Mechanistically, HERC1 controls C-RAF stability by regulating its polyubiquitylation in a lysine 48-linked chain. In vitro ubiquitylation assays indicate that C-RAF is a substrate of the E3 ubiquitin ligase HERC1. Altogether, we show how HERC1 can regulate cell proliferation through the activation of ERK signaling by a mechanism that affects C-RAF’s stability.