Human HEK293, 293T, and U2OS cell lines were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) penicillin/streptomycin (Invitrogen, Carlsbad, CA). Caco2 cells were maintained in MEM Alpha supplemented with 20% FBS and 1% (vol/vol) penicillin/streptomycin. Human mammary epithelial MCF10A cells were cultured in DMEM/F12 complemented with 5% horse serum (Invitrogen), 10 mg/ml insulin (Sigma-Aldrich, St. Louis, MO), 100 ng/ml cholera toxin (Sigma-Aldrich), 0.5 mg/ml hydrocortisone (Sigma-Aldrich), 20 ng/ml epidermal growth factor (PeproTech, Rocky Hill, NJ), and 1% of penicillin and streptomycin (Invitrogen). All cell lines were cultured in a humidified incubator at 37°C with 5% CO2.
pcDNA3-LATS2-FLAG (wild type and kinase dead) plasmids were a kind gift of Tadashi Yamamoto (University of Tokyo, Tokyo, Japan). The LATS2 deletion constructs were cloned into the pC113-GFP-LAP plasmid (GFP, green fluorescent protein; a gift from Iain Cheeseman, Broad Institute, Cambridge, MA). pcDNA4-AMOTL2-Myc and pcDNA4-AMOTL2-ΔPDZ-Myc (lacking the C-terminal PDZ motif) were kindly provided by Anming Meng (Tsinghua University, Beijing, China), and AMOTL2 deletion constructs were PCR amplified using Herculase Tag polymerase (Stratagene, Santa Clara, CA) and cloned in pcDNA4/myc plasmid using standard procedures. All clones were verified by DNA sequencing. The pCS-GFP-AMOTL1 plasmid came from the Fernandes lab stock, and the pBABE-AMOT80 and pBABE-AMOT130 plasmids were provided by Lars Holmgren (Karolinska Institute) and were subcloned into the pcDNA4/myc plasmid. pcDNA4-YAP2, pcDNA4-YAP2WW, pcDNA4-YAP2-ΔPDZ, p2xFLAG-YAP2, and EGFP-MST2 (plasmids 19060, 19000, 21125, 19045, and 19050 [Addgene, Cambridge, MA], submitted by Marius Sudol, Weis Center for Research, Danville, PA), 3x-FLAG-pCMV5-TOPO TAZ and 3x-FLAG-pCMV5-TOPO TAZ (S89A) (plasmids 24809 and 24815 [Addgene], submitted by Jeff Warner) were obtained from Addgene. HA-MOB1 plasmid DNA was a kind gift of Thanos Halazonetis (University of Geneva, Geneva, Switzerland). FLAG-Trip6 and FLAG-Wtip plasmids were kindly provided by Mary Beckerle (University of Utah, Salt Lake City, Utah) and Sigmar Stricker (Max-Planck-Institute, Berlin, Germany), respectively. The cDNA clones for JUP, Frmd4a, PEZ, and AJUBA were obtained from Open Biosystems (Thermo Biosystems, Huntsville, AL) and cloned in pcDNA3.1 with a Myc epitope tag at the N-terminus of the proteins or in the pC113-GFP vector. YAP2 was PCR amplified and cloned into the GST vector pGEX-5×2. The lentiviral shRNA plasmid pLKO.1 and the packaging plasmids psPAX.2 and pMD2.G were obtained from Addgene. The point mutations in LATS2 (S872A, S872E, T1041A, T1041E) were made using the Quick-Change Site Mutagenesis Kit (Stratagene). All PCR-generated clones were verified by DNA sequencing.
The rabbit anti-LATS2 antibody used for immunofluorescence (Abe et al., 2006
) was kindly provided by Tadashi Yamamoto (University of Tokyo). The rabbit anti-LATS2 antibody (A300-479A) used for immunoprecipitation and Western blot analysis was obtained from Bethyl Laboratories (Montgomery, TX). Mouse anti-tubulin and mouse anti-FLAG (M2) were purchased from Sigma-Aldrich. Mouse anti-HA (16B12) was obtained from Covance (Berkeley, CA). The rabbit anti-YAP (sc15407), mouse anti-YAP (sc10199), mouse anti-GFP (9996), rabbit anti-Myc (sc789), mouse anti-Myc 9E10 (sc46), and goat anti-AMOTL2 (82501) were obtained from Santa Cruz Biotechnology. The rabbit anti-pYAP-S127 (4911S), anti–rabbit pLATS2-S872 (9157S), and rabbit pLATS2-T1041 (9159S) were purchased from Cell Signaling Technology (Beverly, MA). The rabbit anti-AMOT antibody was generated by the Fernandes lab (Université Laval, CHUQ-CHUL Research Centre).
LAP purification and mass spectrometry
U2OS cells were transiently transfected with LAP-tagged pC113-GFP or pC113-LATS2 plasmids using Lipofectamine 2000 (Invitrogen). Stable clones were selected with 800 μg/ml G418 (Invitrogen) in the medium, and pooled cells were subjected to flow sorting to obtain a single positive line that stably expressed the full-length recombinant protein. A large-scale purification of GFP (as control) or the LATS2 protein complex was carried out as described (Cheeseman and Desai, 2005
), and resultant protein complexes were subjected to mass spectrometry analysis.
In vivo YAP phosphorylation assay and Western blotting
For detection of LATS2-mediated phosphorylation of YAP (Ser127), HEK293 cells were transfected in 12-well plates with empty vector, YAP2, and LATS2 with different upstream regulators as indicated, using Lipofectamine 2000. Forty hours after transfection, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.0% Nonidet P-40, 2% glycerol) supplemented with 1× protease inhibitor cocktail (Sigma-Aldrich), 100 nM sodium vanadate (Sigma-Aldrich), and 50 mM sodium fluoride (Sigma-Aldrich), and lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4°C. After determining the protein concentration by bicinchoninic acid assay (Pierce, Thermo Fisher Scientific, Rockford, IL), 25 μg of protein lysate was mixed with 2× SDS sample buffer, boiled for 5 min at 100°C, and subjected to electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA). Membranes were blocked using 5% dry milk (Bio-Rad, Hercules, CA) or bovine serum albumin in phosphate-buffered saline (PBS) containing 0.1% Tween 20. Subsequently, the membranes were probed with pYAP-S127 (1:3000) or appropriate antibodies for 1 h to overnight, followed by incubation with horseradish peroxidase– or alkaline phosphatase (AP)–conjugated secondary antibodies and detected with enhanced chemiluminescence or AP substrates, respectively. Blots were stripped using Western blot strip buffer (Pierce) and then reprobed as needed.
For immunoprecipitation, plasmids were transfected into HEK293 cells using Lipofectamine 2000. Forty hours later, cells were collected and lysates were processed in immunoprecipitation buffer as mentioned previously. An amount of 500 μg of total protein lysate was precleared with Protein G agarose beads (Pierce) for 1 h at 4°C. Then, the lysates were incubated with appropriate primary antibodies for 2 h at 4°C on a rotating platform. Subsequently, 20 μl of Protein G agarose was added to each mixture, and the samples were incubated for an additional hour. The beads were washed five times with immunoprecipitation buffer. After the final wash, residual buffer was removed with a 1-cc syringe. Beads were then mixed with 2× SDS sample buffer, boiled for 5 min at 100°C, and subjected to SDS–PAGE and Western blot analysis as described previously. To pull down endogenous proteins from MCF10A cells, 600–800 μg of protein lysate was mixed with 1 μg of goat anti-AMOTL2 or rabbit anti-LATS2 antibodies.
The siRNA sequence corresponding to firefly luciferase 5′ CGUACGCGGAAUACUUCGA 3′ (used as a control and referred to as GL2) and human-LATS2 5′ TCAACGTGGACCTGTATGA 3′ (Aylon et al., 2006
) were synthesized at Dharmacon (Lafayette, CO). SMARTpool siRNA, consisting of four siRNAs targeting multiple sites on AMOTL2 (AMOTL2-siRNA), and AMOT (targeting both AMOT80 and AMOT130) were obtained from Dharmacon. The sequences for AMOTL2 siRNAs are 5′ GAAAGCAGGUUAAAGGUGC 3′, 5′ CAAGGGCUCUCUUCUAGUG 3′, 5′ GAACGGCUCCUUCAGUUGU 3′, and 5′ CAGAACAACUGCGAGAGAA 3′; and the sequences for AMOT are 5′ CCACAUCGUUUGUCUAUAC 3′, 5′ GACCUGCAAUCCAGACAAA 3′, 5′ CCAAAGACGACACAUCGAA 3′, and 5′ CAAGCUAGAGGGCGAGAUU 3′. For the production of lentiviral shRNA particles the sequences specific to AMOTL2 (Wang et al., 2011
) and GL2 were cloned in pLKO.1 vector
For knockdown experiments in HEK293 cells, 100–200 nM of each siRNA were mixed with LATS2-FLAG, pcDNA4-YAP2, and HA-MOB1 plasmids and transfected using Lipofectamine 2000. Cells were incubated for 48–72 h and analyzed for protein expression using immunoblotting with specific antibodies. For lentiviral shRNA production, 1 d before transfection 5 × 105 293T cells were plated on a 60-mm plate and incubated at 37°C overnight. Next morning, 4 μg of pLKO.1-GL2 or pLKO1-AMOTL2 shRNA transfer vector were mixed with 3 μg of PAX (packing), 1 μg of MD2G (envelope) plasmids, and 16 μl of Lipofectamine 2000. After incubating for 20 min at room temperature, the mixture was added dropwise into each plate. Twenty hours later, the media was replaced with 3 ml of OPTI-MEM. The media containing Lentivirus particles were collected 48 h later and passed through a 0.45-μm filter and directly added to MCF10A cells with 6 μg/ml Polybrene. Subsequently, the stable pools of cells were selected for 5 d in growth medium containing 2 μg/ml puromycin.
For in vitro kinase assays, HEK293 cells were transfected with either wild-type LATS2-FLAG or active-site mutants of LATS2-FLAG as indicated. Forty hours after transfection, cells were lysed in immunoprecipitation buffer, and 300 μg of protein lysate was processed for immunoprecipitation of LATS2 as described earlier. Samples were processed for kinase assays as described earlier (Yang et al., 2004
), with some modification. The immunoprecipitates were washed three times in immunoprecipitation buffer; once with immunoprecipitation buffer containing 500 mM NaCl and 1 mM dithiothreitol (DTT; rotation for 2 min at 4°C); twice with kinase assay buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM MgCl2
, 1 mM DTT, and 1× phosphatase inhibitor. Beads were then mixed with 20 μl of kinase assay mix containing 2 μg of GST-YAP2, 10 μM ATP, and 5 μCi of [γ-32
P]ATP and incubated at 30°C for 30 min. The reaction was stopped by adding 5× SDS sample dye, boiled at 100°C for 5 min, and subjected to SDS–PAGE. After electrophoresis, gels were dried, and exposed to a PhosphorImager screen for 1–5 h, scanned on a Storm PhosphorImager (GE Healthcare, Piscataway, NJ), and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
Caco2, U2OS, and MCF01A cells cultured in coverslips were fixed with 4% paraformaldehyde for 10 min and permeabilized/blocked with 0.5% Triton X-100 and 5% normal goat serum for 20 min. Cells were subsequently incubated with appropriate primary antibodies overnight at 4°C. They were washed three times in PBS and incubated with Alexa Fluor–conjugated secondary antibodies (Molecular Probes, Invitrogen) for 1 h at room temperature. After three washes with PBS, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The coverslips were mounted on slides using ProLong Gold antifade reagent (Invitrogen) and viewed using fluorescence microscopy (DMR; Leica Microsystems, Wetzlar, Germany). Images were acquired and processed using Slidebook software 5.0 (Intelligent Imaging Innovations, Denver, CO) and Photoshop (Adobe, San Jose, CA).