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Acta Physiologica 2013; Volume 207, Supplement 694
92nd Annual Meeting of the German Physiological Society
3/2/2013-3/5/2013
Heidelberg, Germany
ENGINEERED VOLTAGE SENSITIVE PHOSPHATASES REVEAL ALA126 AND THR167/ILE168 AS DETERMINANTS OF THE SUBSTRATE SPECIFICITY OF PTEN
Abstract number: P164
Leitner
1
M.G., Mavrantoni
1
A., Feuer
1
A., Oliver
1
D.,
Halaszovich
1
*C.R.
1
Philipps-University Marburg, Neurophysiology, Marburg, Germany
The tumor suppressor PTEN antagonizes the PI3-kinase/Akt-pathway via its PI(3,4)P2/PIP(3,4,5)P3-3-phosphatase activity. It is a member of the CX5R phosphatase family, named after the conserved active site motif. All members of this family were assumed to share PTEN's phosphatase phenotype, but lately certain CX5R-phosphatases, namely the voltage sensitive phosphatases (VSP) were shown to be PI(4,5)P2/PI(3,4,5)P3-5-phosphatases. Remarkably, the amino acid residues lining the active site pocket of PTEN are highly conserved in the VSPs. Notable differences are an alanine/glycine (position 126 in PTEN) exchange within the CX5R-motif and a TI/ET (167/168 in PTEN) exchange in the so-called TI-loop. We set out to clarify the import of these differences for substrate specificity.
Employing wild-type and engineered voltage sensitive phosphatases, we studied the effects of active site mutations in PTEN and VSPs. The enzymatic activity of the mutants was analyzed in CHO cells using fluorophore tagged phosphoinositide binding domains (PI(3,4,5)P3, Btk-PH; PI(4,5)P2, PLCδ1-PH; PI(3,4)P2, TAPP1-PH). The phosphoinositide concentration dependent membrane association of these domains was quantified using total internal reflection (TIRF) microscopy.
We find the mutants PTEN(A126G) and PTEN(TI167/168ET) to be PI(3,4,5)P3-5-phoshatases, PTEN(A126G,TI167/168ET) is a PI(4,5)P2/PI(3,4,5)P3-5-phosphatase like the VSPs. Curiously, the reciprocal G/A and ET/TI mutations did not alter the specificity of the VSPs.
We conclude that the specificity of PTEN is fully determined by Ala126 and Thr167/Ile168, whereas in the VSPs additional, yet unknown determinants of specificity are present.
Supported by University Medical Center Giessen and Marburg (grant UKGM 32/2011 MR to CRH) and DFG (grant SFB593 TP A12 to DO)
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Acta Physiologica 2013; Volume 207, Supplement 694 :P164