Proximity proteomics identifies PAK4 as a component of Afadin-Nectin junctions

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• p21-activated kinase 4 (PAK4) is a ubiquitously expressed kinase downstream of Cdc42.
• PAK4 is enriched at cell-cell junctions, suggesting a role in junctional regulation.

Purpose of the Study:

• To investigate the local protein environment and substrates of PAK4 at cell-cell junctions.
• To understand the mechanism of PAK4 localization and identify its binding partners and phosphorylation targets.

Main Methods:

• Biotin-ligasecherichia coli (BioID) proximity labeling using PAK4-BirA-GFP in U2OS cells.
• Reciprocal BioID experiments with Afadin.
• Co-precipitation assays.
• Phosphoproteome analysis following PAK inhibitor treatment.

Main Results:

• BioID identified 27 PAK4-proximal proteins, predominantly cell-cell junction components, with Afadin/AF6 showing the highest labeling.
• Afadin BioID reciprocally identified over 50% of PAK4-proximal proteins, highlighting their interaction.
• PAK4 localization to cell junctions was dependent on Afadin.
• Seventeen PAK4 phosphorylation sites were identified on proximal junctional proteins.

Conclusions:

• Mammalian group II PAKs, including PAK4, are localized to the Afadin/nectin sub-compartment of cell junctions.
• PAK4 exhibits distinct spatial segregation from conventional cadherin and tight junctions.
• The study provides novel insights into PAK4 function and substrate specificity at specific cell-cell junctional domains.