Talk abstracts

Talk on Friday 02:50-03:10pm submitted by Rupak Mukhopadhyay

A Kinase-Dependent, Negative Feedback Mechanism in GAIT-Mediated Translational Silencing

Rupak Mukhopadhyay (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195), Paul L. Fox (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195)

Abstract:
Recent evidence indicates translational control of inflammatory protein expression is important for resolution of chronic inflammation. In U937 cells, after 16 h of IFN-gamma (IFN) treatment, human ribosomal protein L13a is phosphorylated and released from the 60S ribosomal subunit. Phosphorylated L13a then interacts with 3 other proteins to form the IFN-Gamma Activated Inhibitor of Translation (GAIT) complex that binds a defined, 29-nt GAIT element in the 3’-UTR of ceruloplasmin (Cp) mRNA, and blocks its translation. L13a phosphorylation and GAIT complex activation occur almost simultaneously suggesting that L13a phosphorylation is the rate-limiting step in GAIT-mediated translational control. Kinase activity assays using lysates from IFN-treated U937 cells show delayed appearance of L13a-kinase activity after about 12-16 h, thereby explaining the delayed phosphorylation and recruitment of L13a to the GAIT complex, and consequent translational silencing of Cp mRNA. Immunoprecipitation of IFN-treated lysates with phosphoamino acid-specific antibodies followed by immunoblot using anti-L13a antibody, shows serine phosphorylation of L13a. Mass spectrometric analysis of recombinant, phosphorylated L13a from baculovirus-infected insect cells reveals Ser77 as a candidate phosphorylation site. This site was confirmed by the absence of phosphorylation of exogenous L13a in U937 cells transfected with L13a containing a Ser77-to-Ala mutation. Bioinformatic analysis suggests R-X-X-S77 as a possible kinase-recognition motif. In vitro phosphorylation of mutant peptides with cell lysates confirms the requirement for the Arg residue. Immunocomplex-kinase assay using IFN-treated 16 h cell lysate indicates death-associated protein kinase 3 (DAPK3) is responsible for L13a phosphorylation. SiRNA treatment confirmed both DAPK3, and its family member DAPK1, are involved in this phosphorylation cascade. Both kinases have putative GAIT element in their 3’-UTR and their expressions are regulated at the translational level after IFN-treatment. Therefore, we propose a negative feedback mechanism in which kinases responsible for L13a phosphorylation, and consequent translational silencing, are targets of the same regulatory pathway.

Keywords: GAIT, Ribosomal protein L13a, Phosphorylation