title: Ordered phosphorylation events in two independent cascades of the PTEN C-tail revealed by NMR.
abstract: PTEN phosphatase is a tumor suppressor controlling notably cell growth, proliferation and survival. The multisite phosphorylation of the PTEN C-terminal tail regulates PTEN activity and intracellular trafficking. The dynamical nature of such regulatory events represents a crucial dimension for timing cellular decisions. Here we show that NMR spectroscopy allows reporting on the order and kinetics of clustered multisite phosphorylation events. We first unambiguously identify in vitro seven bona fide sites modified by CK2 and GSK3β kinases and two new sites on the PTEN C-terminal tail. Then, monitoring the formation of transient intermediate phosphorylated states, we determine the sequence of these reactions and calculate their apparent rate constants. Finally, we assess the dynamic formation of these phosphorylation events induced by endogenous kinases directly in extracts of human neuroblastoma cells. Taken together, our data indicate that two cascades of events controlled by CK2 and GSK3β occur independently on two clusters of sites (S380-S385 and S361-S370) and that in each cluster the reactions follow an ordered model with a distributive kinetic mechanism. Besides emphasizing the ability of NMR to quantitatively and dynamically follow post-translational modifications, these results bring a temporal dimension on the establishment of PTEN phosphorylation cascades.
authors.affiliation: Département de Biologie Structurale et Chimie, Institut Pasteur, F-75015 Paris, France. florence.cordier@pasteur.fr
authors.forename: Florence
authors.initials: F
authors.lastname: Cordier
authors.forename: Alain
authors.initials: A
authors.lastname: Chaffotte
authors.forename: Elouan
authors.initials: E
authors.lastname: Terrien
authors.forename: Christophe
authors.initials: C
authors.lastname: Préhaud
authors.forename: François-Xavier
authors.initials: FX
authors.lastname: Theillet
authors.forename: Muriel
authors.initials: M
authors.lastname: Delepierre
authors.forename: Monique
authors.initials: M
authors.lastname: Lafon
authors.forename: Henri
authors.initials: H
authors.lastname: Buc
authors.forename: Nicolas
authors.initials: N
authors.lastname: Wolff
mesh_terms: D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D051059:PTEN Phosphohydrolase / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D051059:PTEN Phosphohydrolase