Expression, generation, and purification of unphosphorylated and phospho-Ser-380/Thr-382/Thr-383 form of recombinant PTEN phosphatase.
The dual specificity phosphatase PTEN exerts its tumour suppressor and cell-migration regulatory functions by dephosphorylating the phospholipid substrate, phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P(3)), and phosphotyrosine protein substrates. PTEN functions are regulated by phospholipid binding, interactions with other cellular proteins and phosphorylation at multiple sites. Precisely, how the phosphorylation and binding events modulate PTEN activity and structure remains mostly unclear. Detailed studies of this issue require the availability of significant quantity of both the unphosphorylated and phosphorylated forms of purified recombinant PTEN. Here, we describe the successful expression and purification of recombinant rat PTEN using a baculovirus-infected Spodoptera frugiperda (Sf9) cell expression system. The recombinant PTEN was purified to near homogeneity using four sequential column chromatographic steps. The specific enzymatic activity of the purified preparation in dephosphorylating PI(3,4,5,)P(3) and the artificial phosphotyrosine substrate poly(Glu/Tyr) are 6.7 nmol/min/microg and 0.006 pmol/min/microg, respectively. Intriguingly, similar to PTEN expressed in mammalian cells, the recombinant PTEN was phosphorylated in the infected insect cells at Ser-380, Thr-382, and Thr-383 at the C-terminal tail. Treatment with alkaline phosphatase fully dephosphorylated these sites. After the treatment, the unphosphorylated PTEN and alkaline phosphatase could be separated by ion exchange column chromatography. The availability of the phosphorylated and unphosphorylated forms of recombinant PTEN permits future investigations into the three-dimensional structures of the phosphorylated and unphosphorylated forms of PTEN, and the role of phosphorylation in regulating PTEN activity, phospholipid- and protein-binding affinities.
Gajewski
Joanna E
JE
Department of Biochemistry & Molecular Biology, The University of Melbourne, Parkville, Vic. 3010, Australia.
Bird
Megan J
MJ
Crowhurst
Meredith O
MO
Sio-Seng Lio
Daisy
D
Liu
Junjun
J
Wettenhall
Richard E H
RE
Zhu
Hong-Jian
HJ
Cheng
Heung-Chin
HC
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D051059:PTEN Phosphohydrolase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D018411:Spodoptera; D013912:Threonine / Q000737:chemistry*
D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D051059:PTEN Phosphohydrolase