Phosphorylation-mediated PTEN conformational closure and deactivation revealed with protein semisynthesis
David Bolduc,
Meghdad Rahdar,
Becky Tu-Sekine,
Sindhu Carmen Sivakumaren,
Daniel Raben,
L Mario Amzel,
Peter Devreotes,
Sandra B Gabelli,
Philip Cole
Affiliations
David Bolduc
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, United States
Meghdad Rahdar
Department of Pharmacology, University of California, San Diego, San Diego, United States
Becky Tu-Sekine
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States
Sindhu Carmen Sivakumaren
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, United States
Daniel Raben
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States
L Mario Amzel
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States
Peter Devreotes
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, United States
Sandra B Gabelli
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States; Departments of Medicine and Oncology, Johns Hopkins University School of Medicine, Baltimore, United States
Philip Cole
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, United States
The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380–385) and these phosphorylations are proposed to induce a reduction in PTEN’s plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN’s regulation and suggest pharmacologic approaches for direct PTEN activation.
