ERK inhibits Capicua repressor function via multisite phosphorylation

Area of Science:

• Molecular Biology
• Cell Signaling
• Genetics

Background:

• The receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway is crucial for cellular functions.
• Capicua (Cic) is a transcriptional repressor targeted by ERK; mutations in human CIC are linked to diseases.
• Understanding Cic phosphorylation by ERK is vital for deciphering its regulatory mechanisms.

Purpose of the Study:

• To identify and characterize specific phosphorylation sites on Drosophila Cic targeted by ERK.
• To validate the in vivo developmental functions of these phosphosites using mutant Cic variants.
• To elucidate the role of multisite phosphorylation in Cic downregulation and degradation.

Main Methods:

• Phosphoproteomic analysis to identify potential ERK target sites on Cic.
• Site-directed mutagenesis to create Cic variants with mutated phosphosites.
• In vivo functional assays in Drosophila to assess the impact of mutations on Cic activity and degradation.

Main Results:

• Identified multiple high-confidence Cic phosphosites directly targeted by ERK.
• Cic mutated at 20 sites showed resistance to proteasomal degradation, acting as a "super-repressor".
• ERK requires simultaneous phosphorylation of multiple Cic sites for full downregulation, suggesting a phosphodegron mechanism.

Conclusions:

• Multisite phosphorylation by ERK is essential for the functional downregulation and degradation of Cic.
• This mechanism involves phosphodegrons recognized by ubiquitin ligases like Ago/FBXW7.
• The findings provide critical insights into signal interpretation downstream of the RTK/ERK pathway.