Src family kinases engage differential pathways for encapsulation into extracellular vesicles

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Extracellular vesicles (EVs) are diverse nanoparticles secreted by cells, and understanding their protein cargo is key to deciphering their heterogeneity.
• Src family kinases (Src and Fyn) possess N-terminal modifications like fatty acylation and lysine residues relevant to protein localization and function.

Purpose of the Study:

• To investigate the preferential encapsulation of Src and Fyn kinases into EVs.
• To elucidate the roles of N-terminal fatty acylation and lysine residues in this encapsulation process.
• To explore the impact of different EV biogenesis pathways on protein cargo selection.

Main Methods:

• Genetic manipulation (loss of myristoylation/palmitoylation, lysine mutation) and pharmacological inhibition (filipin III) were used to assess kinase encapsulation.
• TSG101 knockdown was employed to investigate the role of the ESCRT pathway in EV biogenesis and protein sorting.
• Analysis of Src and Fyn kinase levels in EVs under various experimental conditions, including in vivo tumor models.

Main Results:

• Src and Fyn kinases are preferentially encapsulated in EVs.
• Myristoylation and palmitoylation significantly facilitate the encapsulation of Src and Fyn, respectively.
• Lysine residues at specific sites in Src are crucial for its myristoylated form's encapsulation.
• The ESCRT pathway (via TSG101) and lipid raft integrity influence the levels of Src and Fyn in EVs, suggesting distinct biogenesis routes.

Conclusions:

• EV heterogeneity is partly determined by distinct biogenesis pathways that regulate the selective encapsulation of specific proteins like Src and Fyn kinases.
• Fatty acylation and specific protein features (e.g., lysine residues) are critical determinants for protein sorting into EVs.
• This research provides insights into the mechanisms governing protein cargo selection in EVs, relevant for understanding their biological roles and potential as biomarkers.