Functional analysis of the protein phosphatase activity of PTEN.

EXPERIMENTAL Plasmids All GFP–PTEN constructs were generated using the GFP–C2 vector (Clontech) to produce N-terminally GFP-tagged proteins. GFP–PTEN(C124S)-4A and GFP–PTEN(C124S)-4D were generated from GFP–PTEN-4A or GFP–PTEN-4D by site-directed mutagenesis using the QuikChange® II site-directed mutagenesis kit (Stratagene).

Hippocampal slice culture and transfection

Hippocampal slices were prepared from P7 rats as described previously [ 15 ]. Animal experimentation was carried out according to the Animal Care and Use Committee of Cold Spring Harbor Laboratory guidelines (protocol number 08-04-02). Genes were delivered at 6 DIV (days in vitro ) using biolistic gene transfer [180 psi (1 psi=6.9 kPa), Helios Gene Gun, Bio-Rad Laboratories] [ 16 ]. Plasmid DNAs were coated on to 1.6 μm gold beads; co-expression was achieved by coating the beads with DsRed ( Discosoma red fluorescent protein) and GFP–PTEN or one of the GFP–PTEN mutant constructs. For a standard preparation, we used 14 μg of DsRed DNA and 36 μg of GFP–PTEN wild-type or mutant DNA with 12.5 mg of gold beads.

Two-photon laser-scanning microscopy and image analysis

The procedures were based on those described in [ 17 – 19 ]. At 48 h after transfection, slices were perfused with ACSF [artificial cerebrospinal fluid; 127 mM NaCl, 25 mM NaHCO 3 , 1.25 mM NaH 2 PO 4, 25 mM D -glucose, 2.5 mM KCl, 4 mM MgCl 2 , 2 mM CaCl 2 and 1 mM MgSO 4 (pH 7.28 at 23°C) aerated with 95% O 2 /5% CO 2 ] at room temperature (23°C). Transfected CA1 pyramidal neurons were identified by epifluorescence illumination. For each cell, a standard region encompassing the initial bifurcation of the CA1 neuron was chosen for data acquisition and two to three segments of apical dendrites were sampled. High-resolution three-dimensional image stacks, consisting of sections of 512 pixels×512 pixels were collected in 0.5 μm steps on a custom-built instrument based on a Fluoview laser-scanning microscope (Olympus America) with an excitation wavelength of 910 nm. All clear protrusions emanating laterally from the dendritic shaft, irrespective of shape, were counted in the DsRed images and the spine density was measured manually using custom MATLAB software [ 18 ] by an observer who was blinded to genotype. All images in the Figures are projections of three-dimensional stacks. Values are means±S.E.M., and statistical significance was determined using Student's t test. Expression levels of GFP-tagged proteins were compared by measuring the fluorescence intensity of single spines at a fixed region of interest, standardized according to the co-transfected cytoplasmic marker DsRed. No significant differences in expression were detected. PTEN purification, PTEN C-tail preparation and PTEN phosphatase assays GST–PTEN, GST–PTEN G129E, GST–PTEN C124S and GST–PTEN Y138L were expressed and purified as described previously [ 12 ]. PTEN C-tail peptide (DHYRYSDTTDSDPENE, 0.5 mM) was phosphorylated with recombinant casein kinase II (a gift from Dr D. Litchfield, Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada) in a reaction mixture consisting of 50 mM Tris/HCl (pH 7.6), 2 mM DTT (dithiothreitol), 150 mM NaCl, 10 mM MgCl 2 , 0.2 mg/ml BSA and 0.1 mM [γ- 32 P]ATP (specific activity 100 c.p.m./pmol) and incubated at 30°C for 10 min. Phosphorylated peptide then was purified with minitrap G-10 columns (GE Healthcare, catalogue number 28-9180-10). Purified peptide substrates were freeze-dried to dryness and resuspended in phosphatase assay buffer, consisting of 50 mM Hepes (pH 7.0), 10 mM MgCl 2 and 10 mM DTT. The standard phosphatase assay contained 5 μM substrate, 50 mM Hepes (pH 7.0), 10 mM MgCl 2 and 10 mM DTT. The reaction was initiated by the addition of 0.2 μg of enzyme to pre-warmed (30°C) substrate mixture, resulting in a final volume of 30 μl. The reactions were allowed to proceed at 30°C for various times up to 15 min and stopped by the addition of a suspension of activated charcoal in 900 mM HCl, 90 mM sodium pyrophosphate and 2 mM sodium phosphate. Protein extraction, immunoprecipitation and immunoblotting GFP or GFP-tagged PTEN constructs were transfected into HEK (human embryonic kidney)-293 cells. At 2 days after transfection, cells were harvested in ice-cold lysis buffer [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM 2-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin and 1 mM PMSF]. Protein extracts (1 mg) were incubated with a mouse anti-GFP antibody (Invitrogen) bound to magnet beads (Invitrogen) overnight at 4°C. Antibodies used for immunoblotting [anti-phospho-PTEN antibody and anti-(total PTEN) antibody] were from Cell Signaling Technology.