PtdIns(3,4,5)P3-Dependent and -Independent Roles for PTEN in the Control of Cell Migration

Summary Background Phosphatase and tensin homolog (PTEN) mediates many of its effects on proliferation, growth, survival, and migration through its PtdIns(3,4,5)P3 lipid phosphatase activity, suppressing phosphoinositide 3-kinase (PI3K)-dependent signaling pathways. PTEN also possesses a protein phosphatase activity, the role of which is less well characterized. Results We have investigated the role of PTEN in the control of cell migration of mesoderm cells ingressing through the primitive streak in the chick embryo. Overexpression of PTEN strongly inhibits the epithelial-to-mesenchymal transition (EMT) of mesoderm cells ingressing through the anterior and middle primitive streak, but it does not affect EMT of cells located in the posterior streak. The inhibitory activity on EMT is completely dependent on targeting PTEN through its C-terminal PDZ binding site, but can be achieved by a PTEN mutant (PTEN G129E) with only protein phosphatase activity. Expression either of PTEN lacking the PDZ binding site or of the PTEN C2 domain, or inhibition of PI3K through specific inhibitors, does not inhibit EMT, but results in a loss of both cell polarity and directional migration of mesoderm cells. The PTEN-related protein TPTE, which normally lacks any detectable lipid and protein phosphatase activity, can be reactivated through mutation, and only this reactivated mutant leads to nondirectional migration of these cells in vivo. Conclusions PTEN modulates cell migration of mesoderm cells in the chick embryo through at least two distinct mechanisms: controlling EMT, which involves its protein phosphatase activity; and controlling the directional motility of mesoderm cells, through its lipid phosphatase activity.

similarly generated by PCR with the sense primer PTEN351tail Eco-S. A vector expressing the C2 domain of TPTE was produced similarly by using the TPTE C2 clone S and TPTE C2 clone AS.
Construction of an expression vector for GFP-TPTE based upon EGFP-C1 has been previously described [S3]. All experiments here use expression of TPTEg from the nomenclature of Tapparel et al. [S4]. TPTE-R was produced by using the primer TPTE reactivate S and its reverse complement. Mutagenesis of the potential TPTE active-site cysteine residue was performed, but caused the protein to express poorly in E. coli, in the chick embryo, and in cultured HEK293 cells, presumably through effects on protein stability.

Oligonucleotides
In each case, the indicated oligonucleotide sequence and its reverse complement were used for PCR mutagenesis.

RNA-Interference Experiments
An siRNA ''smartpool'' targeting the chicken PTEN gene was purchased from Dharmacon. siRNA was diluted to a concentration of 1 mM in 20 mM KCl, 6 mM Hepes (pH 7.5), and 200 mM MgCl 2 and electroporated according to the protocol and conditions of the plasmid constructs. Standard whole-mount in situ hybridization used an antisense probe against cPTEN according to the method of Wilkinson and Nieto [S6].

Substrate Preparation and Phosphatase Assays
Poly-Glu:Tyr (4:1) (Sigma) was phosphorylated by using 1 mg of polymer in 0.2 ml of a kinase buffer (50 mM Hepes [pH 7.4], 12 mM MgCl 2 , 1 mM EGTA, 1 mM 2-mercaptoethanol, and 18.5 MBq Figure S2. PTEN Expression in Chick Embryos at Developmental Stages HH1-HH8 Expression of PTEN mRNA was addressed by in situ hybridization in the developing chick embryo. Sections taken as indicated by the dashed line are shown in (C 0 ) and (F 0 ). g-[ 33 P] ATP), with incubation with 1 mg insulin receptor kinase domain (Upstate) for 1 hr at 32 C before addition of 0.5 mg further kinase and incubation for another hour. The reaction was stopped by addition of 0.5 ml 20% TCA at 4 C and centrifuged at 14,000 3 g for 10 min, and the pellet washed once in fresh 10% TCA. The pellet was then resuspended in 500 ml 1M Tris (pH 7.4) and dialysed twice at 4 C against 10 mM Tris (pH 7.4) by using a 3,500 MW cut-off dialysis cassette (Pierce). 3-[ 33 P] PtdIns(3,4,5)P 3 was prepared as previously described [S7].
PtdIns(3,4,5)P 3 assays were conducted with substrate vesicles prepared by sonication of 100 mM phosphatidylcholine, 10 mM unlabeled PtdIns(3,4,5)P 3 , and 100,000 dpm 3-[ 33 P] PtdIns(3,4,5)P 3 . These were incubated in 50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EGTA, and 10 mM DTT with 1 mg enzyme for 30 min at 32 C. PolyGlu-Tyr(P) phosphatase assays were conducted in 50 mM Tris (pH 7.4), 1 mM EGTA, and 10 mM DTT with 1 mg of enzyme and 100,000 dpm (approximately 1 mg) of phosphorylated substrate per assay, also at 32 C for 30 min. Reactions were terminated directly by the addition of 500 ml of ice-cold 1M perchloric acid and 100 mg/ml BSA, left on ice for 30 min, and spun at 15,000 3 g at 4 C for 10 min. The supernatant was removed, and ammonium molybdate was added to a final concentration of 10 mg/ml. After extraction with 2 vol of toluene/isobutanol (1:1 vol/vol), the upper phase was removed and radioactivity was determined by scintillation counting. pNPP assays were performed with 2 mg of enzyme, 20 mM pNPP (Sigma), 50 mM Tris (pH 7.4), 1 mM EGTA, and 10 mM DTT, and activity was measured by absorbance at 405 nm.      Embryos were transfected with expression vectors encoding (A) GFP TPTE C2 or (B) GFP DdPTEN C2 and observed during development as described in the text. Merged bright-field and fluorescence images for the start of the experiment (t = 0) and the end (t = 20 hr) of the experiment are shown. Expression of each C2 domain interferes with the normal directional patterns of migration through the embryo; see, for example, Figure 1 and Figure S1. Figure S8. Overexpression of the PTEN C2 Domain Activates Akt/PKB PTEN null 1321N1 astrocytoma cells were infected with baculoviruses encoding GFP or the indicated GFP-PTEN fusion proteins. Cells were incubated for 24 hr and lysed before the endogenous Akt/PKB was immunoprecipitated and assayed in vitro against the peptide substrate Crosstide. Activity data are presented as the mean + standard deviation. Expression of GFP and PTEN in these lysates was assessed by western blotting with antibodies against GFP and PTEN. The A2B1 monoclonal antibody used to detect PTEN recognizes an epitope that is in the C-terminal tail and is absent in GFP-PTEN C2 only. Figure S9.

The Localization of the Indicated GFP-PTEN Fusion Proteins Is Shown
Embryos were transfected and development was allowed to proceed for 15 hr before cells were photographed. Only the GFP-PTEN C2 + tail construct seems strongly membrane localized, with the C2-only and C2 DPDZ constructs displaying some weak membrane enrichment. Figure S10. TPTE Lacks Detectable Phosphatase Activity (A) An alignment of the active-site P loop of PTEN, TPIP, and TPTE is shown, with the PTEN catalytic-cysteine residue boxed in red. The black boxing shows the threonine and aspartic-acid residues at which TPTE differs from the active phosphatases PTEN and TPIP in this region and the mutation of these residues performed in reactivated TPTE (called TPTE-R).
(B) When expressed as a GFP-fusion protein in HEK293 cells, TPTE is located largely on the plasma membrane.
(C) The protein preparations used in the presented phosphatase assays (here and in Figure 6) are shown with Coomassie total-protein stain and anti-GST western blotting (WB). The GST-TPTE fusion protein contains all of the main intracellular regions of the protein, lacking the N-terminal transmembrane domains.
(D) The activity of the indicated PTEN and TPTE proteins was assayed with either 33 P-radiolabeled PtdIns(3,4,5)P 3 or phosphorylated polyGluTyr as substrate. Data are shown as mean activity and standard deviation from triplicate assays. (E) Activity is shown against the artificial substrate para-nitrophenol phosphate (pNPP) as the mean measured optical density (OD) from duplicate samples.
(F) TPTE lacks activity against other phosphoinositide substrates. Two micrograms of recombinant GST-PTEN or GST-TPTE were incubated with each phosphoinositide lipid for 1 hr at 30 C before released inorganic phosphate was measured with malachite green reagent. Lipids were present as synthetic diC8-soluble compounds at 100 mM. An image of the experiment is shown. No significant increase in measured OD at 620 nM was detected in TPTE samples relative to control (data not shown). WT EMT block 8/9 0/9 9/10 1/6 7/9 Cell migration: directed 1/9 8/9 1/10 0/6 2/9 Cell migration: random 0/9 1/9 0/10 5/6 0/9 PTEN mutant and double-mutant proteins are identified from the combinations of the top row and the left column. Each experimental set shows the three possible experimental outcomes: an EMT block with cells failing to escape the primitive-streak graft; random cell migration, with cells escaping the graft, but migrating randomly through the embryo; or directed cell migration, with cells escaping from the graft and following their normal migratory tracks through the embryo. Numbers represent the frequency of each outcome and the number of experiments performed.