Dissecting the functional behavior of the differentially phosphorylated prolyl isomerase, Pin1

Protein post‐translational modifications (PTMs) play an intricate role in a diverse range of cellular processes creating a complex PTM code that governs cell homeostasis. Understanding the molecular build‐up and the critical factors regulating this PTM code is essential for targeted therapeutic design whereby PTM mis‐regulation is prevalent. Here, we focus on Pin1, a peptidyl‐prolyl cis‐trans isomerase whose regulatory function is altered by a diverse range of PTMs. Through employing advanced mass spectrometry techniques in combination with fluorescence polarization and enzyme activity assays, we elucidate the impact of combinatorial phosphorylation on Pin1 function. Moreover, two phosphorylation sites were identified whereby Ser71 phosphorylation preceded Ser16 phosphorylation, leading to the deactivation of Pin1's prolyl isomerase activity before affecting substrate binding. Together, these findings shed light on the regulatory mechanisms underlying Pin1 function and emphasize the importance of understanding PTM landscapes in health and disease.