The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain

Area of Science:

• Molecular Biology
• Gene Regulation
• Protein Phosphorylation

Background:

• Tristetraprolin (TTP) proteins bind AU-rich elements and regulate mRNA destabilization.
• TTP's activity is modulated by extensive cellular phosphorylation.
• The C-terminal NOT1-binding domain is crucial for TTP function.

Purpose of the Study:

• To investigate the role of TTP phosphorylation at Serine316 in regulating mRNA stability.
• To identify the kinases and phosphatases involved in TTP Ser316 phosphorylation.
• To explore the functional consequences of TTP phosphorylation on the CCR4-NOT deadenylase complex interaction.

Main Methods:

• Generated an antibody against phospho-Serine316 TTP.
• Examined TTP phosphorylation in LPS-stimulated RAW264.7 cells.
• Utilized CRISPR/Cas9 to create TTP knockout RAW264.7 cells.
• Created phosphorylation-mimic and non-phosphorylated TTP mutants (S316D, S316A).
• Performed immunoprecipitation, GST pull-down, and RNA pull-down assays.

Main Results:

• Serine316 phosphorylation is mediated by RSK1 and MK2 and reversed by PP2A.
• Phosphorylation at Ser316 weakens TTP interaction with CNOT1 and dissociates it from the CCR4-NOT complex.
• TTP knockout cells showed increased TNFα mRNA stability.
• Overexpression of non-phosphorylated S316A TTP restored TTP activity, decreasing TNFα mRNA.
• Phosphorylation at Ser316, Ser52, and Ser178 independently contribute to CCR4-NOT complex recruitment.

Conclusions:

• Serine316 phosphorylation modulates TTP-mediated mRNA stability by regulating TTP interaction with the CCR4-NOT deadenylase complex.
• This phosphorylation event serves as a critical switch for controlling gene expression through mRNA decay pathways.