Characterization of the initiation translation factor eIF4G1 as A PKA substrate in Saccharomyces cerevisiae

ADRIANA C. CAMARANO; PAULA PORTELA; CLARA A. SOLARI; SILVIA M. MORENO; PÍA VALACCO

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Celular (SAIB); 2017

SAIB

PKA has a central role in the control of the metabolism, stress resistance and proliferation in Saccharomyces cerevisiae. In response to nutrient consumption, S. cerevisiae arrests cell cycle, decreases global protein synthesis and initiates translation through internal ribosome entry sites (IRES). We have recently demonstrated that PKA connects the nutritional state of the cell with translation at different levels: through interaction with translation factors, by localization in mRNA granules and affecting the protein abundance of translation initiation factor eIF4G1 by a post-translational mechanism. Phosphoproteomic assays have reported that eIF4G1 is a phosphoprotein. The aim of this work was to characterize eIF4G1 as a PKA substrate and to identify the phosphorylation target residues. For this purpose, the full length and different fragments of recombinant proteins derived from GST-eIF4G1 were expressed and purified using a pGEX system. Using these proteins, we assayed in vitro phosphorylation with Tpk1 and Cas the catalytic subunit from S. cerevisiae and mammals respectively. The GST-eIF4G1 phosphorylation was studied by western blot using an anti-P-Ser antibody and autoradiography for the incorporation of γ-32P [ATP]. The phosphorylated residues were identified by nano-LC MS/MS applying an iron trap chromatography. Our results demonstrate that eIF4G1 is a PKA substrate in vitro and identify Thr934 as a novel phosphorylation target residue in eIF4G1. Since, Thr934 residue localizes on the RNA-binding domain RNA3 at the extreme C-terminus, we will focus on studying the role of this phosphorylation site over eIF4G1 protein abundance and its recruitment to the 43S preinitiation complex on the mRNA.