Ordered Phosphorylation Events in Two Independent Cascades of the PTEN C-tail Revealed by NMR

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• 作者：Florence Cordier ; Alain Chaffotte ; Elouan Terrien ; Christophe Pr茅haud ; Fran莽ois-Xavier Theillet ; Muriel Delepierre ; Monique Lafon ; Henri Buc ; Nicolas Wolff
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：December 19, 2012
• 年：2012
• 卷：134
• 期：50
• 页码：20533-20543
• 全文大小：606K
• 年卷期：v.134,no.50(December 19, 2012)
• ISSN：1520-5126

文摘

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 GSK3尾 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 GSK3尾 occur independently on two clusters of sites (S380鈥揝385 and S361鈥揝370) 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.