Phosphorylation-linked complex profiling identifies assemblies required for Hippo signal integration

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Predicting functional relevance of phosphorylation sites computationally is established.
• Experimental analysis of the interplay between protein phosphorylation and protein-protein interactions (PPIs) is challenging.
• Understanding these interdependencies is crucial for deciphering cellular signaling networks.

Purpose of the Study:

• To develop and validate an experimental strategy for investigating the interdependencies between protein phosphorylation and protein complex formation.
• To apply this strategy to YAP1, a key regulator of organ size and tissue homeostasis.
• To elucidate how Hippo pathway signaling controls YAP1 phosphorylation and its association with different protein complexes.

Main Methods:

• Systematic charting of a target protein's phosphorylation landscape.
• Native complex separation (AP-BNPAGE) and protein correlation profiling to assign proteoforms to specific complexes.
• Analysis of proteoforms and complexes in cells with altered expression of key regulatory proteins.

Main Results:

• Identified multiple YAP1 phosphosites linked to distinct protein complexes.
• Inferred the regulatory roles of Hippo pathway members on YAP1 phosphorylation and complex formation.
• Detected a PTPN14/LATS1/YAP1 complex and proposed a model for PTPN14-mediated inhibition of YAP1.

Conclusions:

• The developed strategy effectively establishes interdependencies between protein phosphorylation and complex formation.
• Hippo pathway signaling dynamically regulates YAP1 phosphorylation and its interaction partners.
• PTPN14 may inhibit YAP1 activity through enhanced complex formation and LATS1/2-mediated phosphorylation.