Microorganisms, culture conditions and genetic methods
Escherichia coli DH5α strain was used as the host strain for plasmid constructions and protein production. It was grown in LB (1 % peptone, 0.5 % yeast extract, 1 % NaCl, pH 7.5) medium supplemented with 50 mg/l ampicillin. Yeast strain used in this work was THY-AP4 (MATa, ura3, leu2, lexA::lacZ::trp1, lexA::HIS3, lexA::ADE2). Yeast transformation was carried out using the lithium acetate protocol . Yeast cultures were grown in synthetic complete (SC) medium lacking the corresponding supplements to maintain selection for plasmids .
pCMV-HA-laforin, pFLAG-laforin, pFLAG-laforin C266S and pcDNA3-HA-malin plasmids were described previously . Plasmids pBridge-laforin and pBridge-laforin C266S were obtained by subcloningBamHI/SalI fragments containing either laforin WT or C266S in pBridgeLexA/v-src . Plasmids pCMV-myc-PKM1 and pCMV-myc-PKM2 were obtained by subcloning an EcoRI/XhoI fragment containing the corresponding ORF in pCMV-myc vector. Other plasmids used in this study were pCMV-6xHisUbiq (from Dr. M. Rodriguez, Proteomics Unit, CIC-BioGUNE, Vizcaya, Spain); pCMV-6xHisUbiq-K48R and pCMV-6xHisUbiq-K63R (from Dr. Ch. Blattner, Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany); pCMV-Mdm2 (from Dr. M. Gentry, University of Kentucky, Lexington, KY; ).
Two-hybrid screening and analysis
A modified form of yeast two-hybrid screening named yeast substrate-trapping system  was used to identify proteins that interacted with LexA-laforin C266S (plasmid pBridge-laforin C266S). Plasmid pBridge-laforin C266S also contains the ORF corresponding to the mammalian protein kinase v-src under the control of the MET25 promoter. Saccharomyces cerevisiae THY-AP4 strain containing pBridge-laforin C266S plasmid was transformed with a commercial human brain cDNA library in pACT2 vector (Clontech, Madrid, Spain). Transformants were selected in SC + 2 % glucose plates lacking tryptophan, leucine, methionine and histidine and were subsequently screened for β-galactosidase activity using a filter lift assay . pACT2-plasmids were recovered from positive clones and used to transform again S. cerevisiae THY-AP4 strain containing pBridge-laforin C266S or laforin WT, to confirm the interaction. The strength of the interaction was determined by measuring β-galactosidase activity in permeabilized yeast cells and expressed in Miller units as described by Ludin and collaborators .
Cell culture, transfection and preparation of crude extracts
Human embryonic kidney (HEK293) and human osteosarcoma (U2OS) cells were grown in Dulbecco’s modified Eagle’s medium (Lonza, Barcelona, Spain), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 10 % of inactivated fetal bovine serum (Lonza, Barcelona, Spain) in a humidified atmosphere at 37 °C with 5 % CO2. When indicated, cells were transfected with 1 μg of each plasmid using X-treme GENE HP transfection reagent (Roche Diagnostics, Barcelona, Spain) according to the manufacturer’s instructions. Cell extracts were prepared using lysis buffer A [25 mMTrisHCl at pH 7.4, 15 mM EDTA at pH 8, 50 mMNaF, 0.6 M sucrose, 15 mM Na4P2O7, 1 % nonidet P40, 10 mMNaCl, 1 mM PMSF, and a complete protease inhibitor mixture (Roche Diagnostics, Barcelona, Spain)]. Cells were lysed by repeated passage through 24G × 5/8″ needle and whole lysates were centrifuged at 10,000 × g for 15 min. The supernatants were collected and 25 μg of total protein subjected to SDS-PAGE, transferred into PVDF membrane and revealed with the appropriated antibodies.
Co-immunoprecipitation and western blotting
Mammalian HEK293 cells were transfected with the corresponding plasmids. Twenty-four hours after transfection, cells were harvested with lysis buffer B [50 mMTrisHCl pH 7.5, 10 mMNaCl, 2 mM EDTA, 15 % glycerol, 1 % nonidet P40, complete protease inhibitor cocktail (Roche Diagnostics, Barcelona, Spain) and 1 mM PMSF]. Cells were lysed by repeated passage through 24G×5/8″ needle, whole lysates were centrifuged at 10,000 × g for 15 min and the soluble fraction was collected for immunoprecipitation. Co-immunoprecipitation experiments were carried out with 500 μg of soluble protein extracts, in a final volume of 500 μl of lysis buffer, adding 1 μl of anti-FLAG monoclonal antibodies (Sigma, Madrid, Spain) as in . For western blot analysis, proteins (25 μg) were denatured using sample buffer (125 mMTrisHCl pH 6.8, 20 % Glicerol, 0.01 % bromophenol blue, 4 % SDS) and heating to95 °C for 5 min. The samples were subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Madrid, Spain). Membranes were blocked with 5 % milk in TBS-tween for 1 h and incubated with the following specific antibodies: anti-myc, anti-FLAG, anti-HA and anti-actin from Sigma (Madrid, Spain); anti-Mdm2 from Santa Cruz Biotechnology (Madrid, Spain). Thereafter, blots were washed with TBS-tween and further incubated for 1 h with the corresponding secondary antibody conjugated with horseradish peroxidase. Finally, membranes were washed (3× 15 min) with TBS-tween and analyzed by chemiluminiscence (ECL Western Blotting Detection Reagents, GE Healthcare, UK) using an image reader LAS-4000 (GE Healthcare, UK).
Analysis of ubiquitination
For ubiquitination assays, HEK293 cells were co-transfected with pCMV-myc-PKM1 or pCMV-myc-PKM2, 6xHis-tagged ubiquitin (WT, K48R or K63R) plasmids and, when indicated, with pcDNA3-HA-malin and pCMV-HA-laforin, or pCMV-Mdm2 plasmids, using X-treme GENE transfection reagent, according to the manufacturer’s instructions (Roche Diagnostics, Barcelona, Spain). After 18 h of transfection, cells were lysed in guanidinium hydrochloride to inhibit the action of deubiquitinases and ubiquitinated proteins purified by metal affinity chromatography . Bound proteins and clarified extracts were analyzed by immunoblotting with the appropriated antibodies.
Determination of pyruvate kinase enzymatic activity
HEK293 cells transfected with the indicated plasmids were resuspended in lysis buffer C [50 mMTrisHClpH 7.5, 1 mM EDTA, 150 mMNaCl, 1%NP-40, 1 mM DTT, 10 μM fructose 1,6 bisphosphateand complete protease inhibitor mixture (Roche Diagnostics, Barcelona, Spain)]. Cells were lysed by repeated passage through 24G × 5/8″ needle and whole lysates were centrifuged at 10,000 × g for 15 min. Clarified extracts were used to determine the pyruvate kinase activity by standard methods (reaction buffer: 40 mM K2HPO4 pH 7.6, 0.58 mM phosphoenolpyruvate, 0.11 mM NADH, 6.8 mM MgSO4, 1.5 mM ADP, 10 units lactate dehydrogenase, 10 μM fructose 1,6 bisphosphate). The disappearance of NADH absorbance was measured spectrophotometrically at 340 nm. One unit of enzymatic activity is defined as the amount of enzyme that is able to oxidize 1 μmol of NADH per 1 min at 25 °C. Enzymatic activity was normalized by the total amount of protein in the sample.
Human osteosarcoma U2OS cells transfected with the indicated plasmids were grown on 12-well plates containing coverslips. Cells were fixed with 4 % paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. Then, cells were permeabilized with 0.2 % Triton X-100 in PBS for 15 min, blocked 1 h with 10 % fetal bovine serum, 5 % nonfat dried milk, 0.5 % BSA and 0.1 % Triton X-100 in PBS, and incubated overnight at 4 °C in the same buffer containing anti-HA and anti-PKM2 antibodies. Samples were washed three timeswith PBS and incubated with a 1/500 dilution of anti-mouse Texas Red and anti-rabbit Alexa-Fluor 488 (Invitrogen, Madrid, Spain). Then, cellswere washed three times with PBS and mounted on slices using Aqua-Poly/Mount coverslipping medium (Polysciences, Inc. Eppelheim, Germany). Images were acquired with an uprightLeica DM RXA2microscope using an PL APO 63× oil 1.4 N.A. immersion objective, and the Leica IM50 software. At least 100 cells were counted in each condition.
Statistical data analysis
Data are expressed as means with standard deviation. Statistical significance of differences between the groups was evaluated by a paired Student’s t-test with two-tailed distribution. The significance has been considered at ** p < 0.01, as indicated.