Interrogating endothelial barrier regulation by temporally resolved kinase network generation

Area of Science:

• Cell Biology
• Biochemistry
• Systems Biology

Background:

• Kinases regulate endothelial barrier function, but their dynamic roles and signaling networks are not fully understood.
• Endothelial barrier integrity is critical for tissue homeostasis and preventing inflammation.

Purpose of the Study:

• To develop a novel methodology, Temporally REsolved KInase Network Generation (TREKING), for reconstructing time-resolved phosphosignaling networks.
• To identify kinases and pathways regulating endothelial barrier integrity under different inflammatory stimuli.

Main Methods:

• TREKING integrates a 28-kinase inhibitor screen with machine learning and network reconstruction.
• Utilized brain endothelial cells stimulated with thrombin, with or without TNF preconditioning.
• Employed temporal Western blotting and genetic/pharmacological interventions (lentivirus-mediated knockdown, drug targeting).

Main Results:

• TREKING predicted over 100 kinases involved in endothelial barrier regulation, revealing condition-specific pathways.
• MAPK-activated protein kinase 2 (MAPKAPK2/MK2) showed dynamic roles, weakening the barrier early but strengthening it late under thrombin-only stimulation.
• Identified a MAP3K20/ZAK-MAPK14/p38α axis controlling late MAPKAPK2/MK2 activation in the thrombin-alone condition.

Conclusions:

• TREKING is a powerful tool for dissecting dynamic kinase signaling networks in biological processes.
• Kinase networks exhibit complex, condition-specific temporal dynamics in regulating endothelial barrier function.
• Understanding these networks offers insights into therapeutic strategies for inflammatory diseases affecting endothelial barriers.