PTEN catalysis of phospholipid dephosphorylation reaction follows a two-step mechanism in which the conserved aspartate-92 does not function as the general acid--mechanistic analysis of a familial Cowden disease-associated PTEN mutation.
PTEN exerts its tumour suppressor function by dephosphorylating the phospholipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP(3)). Herein, we demonstrate that the PTEN-catalysed PIP(3) dephosphorylation reaction involves two-steps: (i) formation of a phosphoenzyme intermediate (PE) in which Cys-124 in the active site is thiophosphorylated, and (ii) hydrolysis of PE. For protein tyrosine- and dual-specificity phosphatases, catalysis requires the participation of a conserved active site aspartate as the general acid in Step 1. Its mutation to alanine severely limits PE formation. However, mutation of the homologous Asp-92 in PTEN does not significantly limit PE formation, indicating that Asp-92 does not act as the general acid. G129E is a common germline PTEN mutations found in Cowden syndrome patients. Mechanistic analysis reveals that this mutation inactivates PTEN by both significantly slowing down Step 1 and abolishing the ability to catalyse Step 2. Taken together, our results highlight the mechanistic similarities and differences between PTEN and the conventional protein phosphatases and reveal how a disease-associated mutation inactivates PTEN.
Xiao
Yi
Y
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Yeong Chit Chia
Joel
J
Gajewski
Joanna E
JE
Sio Seng Lio
Daisy
D
Mulhern
Terrence D
TD
Zhu
Hong-Jian
HJ
Nandurkar
Harshal
H
Cheng
Heung-Chin
HC
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006223:Hamartoma Syndrome, Multiple / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation / Q000235:genetics*; D051059:PTEN Phosphohydrolase / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D013997:Time Factors
D010714:Phosphatidylethanolamines; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D010761:Phosphorus Radioisotopes; C060974:phosphatidylinositol 3,4,5-triphosphate; D001224:Aspartic Acid; C483858:phosphatidylethanolamine; D010749:Phosphoprotein Phosphatases; D051059:PTEN Phosphohydrolase; C487216:Pten protein, rat; D003545:Cysteine; D005998:Glycine