Phosphorylation of Tristetraprolin (TTP) by the ERK Signaling Pathway in the Early Differentiation of 3T3-L1 Preadipocyte

Previously, our lab has demonstrated that both tristetraprolin (TTP) and MKP-1 are immediate early genes, whose expression were activated and quickly subsided in response to ERK signaling during 3T3-L1 differentiation. Notably, TTP is known as an mRNA-destabilizing protein, which can bind to AU-rich elements (AREs) in the 3’UTR of mRNA and trigger mRNA destabilization. We also found the mRNA target of TTP includes MKP-1 and TTP itself. The function of TTP is tightly regulated by its protein phosphorylation status. The ERK signaling inhibitors U0126 decreased the expression and phosphorylation levels of TTP, indicating that TTP phosphorylation and its function are regulated by ERK signaling. Therefore, this thesis aims to explore the detailed mechanism of how TTP function is regulated by ERK signaling during adipogenesis. Here, we demonstrates that Cnot1, a scaffold protein of Ccr4–Not deadenylase complex, is an essential partner in TTP-mediated mRNA decay by functional luciferase reporter analysis combining with siRNA knockdown and protein-protein interactions analysis. Also, our results reveal that ERK can phosphorylate TTP at serine 220 and increases interaction between TTP with 14-3-3, thus impairing TTP-mediated mRNA decay. Moreover, using mass spectrometry analysis, functional reporter assay and co-immunoprecipitation assay combined with mutants, we demonstrated that TTP was phosphorylated at serine 316 in C-terminal conserved region by ERK signaling, which significantly perturbs Ccr4-Not complex recruitment and TTP mRNA-destabilizing activity. These findings provide a better understanding of how TTP regulate MKP-1 mRNA stability under the control of ERK signaling pathway during 3T3-L1 differentiation.