The full-length cDNAs encoding PKB and PDK1 were amplified by PCR and subcloned 5′ or 3′ of the F (amino acids [aa]1 to 158) and F (aa 159 to 239) fragments of GFP (pCMS-EGFP; Clontech, Palo Alto, Calif.) into the eukaryotic expression vector pMT3 (24
). The PKB-F fusion was also inserted in a pMT3 vector where the ampicillin resistance gene had been replaced by a chloramphenicol resistance gene (pMT3-chloramphenicol) for the purpose of the cDNA library screen. In all cases, a 10-amino-acid flexible linker consisting of (Gly-Gly-Gly-Gly-Ser)2
was inserted between the cDNA and the GFP fragments to ensure that the orientation and arrangement of the fusions in space were optimal to bring the GFP fragments into close proximity. The F-GCN4 and GCN4-F constructs consisted of fusions with GCN4 leucine zipper-forming sequences used as controls. For the GFP PCA-based cDNA library screen, a human brain cDNA library was excised from the vector pEXP1 (ClonCapture cDNA library; Clontech) using Sfi
I restriction sites and inserted into the pMT3 vector, 3′ of the F fragment of GFP and a 10-amino-acid flexible linker. The PCA-cDNA library fusion expression vectors were divided into several pools (according to the size of the inserted cDNAs, from 0.5 to 4.6 kb) and amplified at 30°C in liquid medium. After the human Ft1 (hFt1) gene was isolated, a Myc tag-hFt1 fusion was also constructed. The GSK3β (Ser9Ala) point mutant was generated using the QuikChange site-directed mutagenesis kit (Stratagene).
COS-1, HEK293, and HEK293T cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, Utah). The human Tag-Jurkat cell line was grown in RPMI 1640 (Invitrogen) supplemented with 10% FBS, 1 mM sodium pyruvate, and 10 mM HEPES. The Tag-Jurkat cells express the simian virus 40 large T antigen and harbor an integrated β-galactosidase reporter plasmid where three tandem copies of the NF-AT binding site direct transcription of the lacZ
PCA-based cDNA library screen.
COS-1 cells were plated in 150-mm dishes 24 h before transfection. Cells were transfected (10 μg of total DNA/dish) using Lipofectamine reagent (Invitrogen), at around a 60% confluence, with pMT3 vector harboring the human brain cDNA library fused to the F fragment of GFP (F-cDNA library) and the pMT3-chloramphenicol vector containing the full-length PKB fused to the F fragment of GFP (PKB-F). The F-cDNA library fusions were transfected in several pools, according to their size. At 48 h after transfection, positive clones (folding and reconstitution of GFP from its fragments) were collected on a fluorescence-activated cell sorter (FACS) analyzer (FACScalibur; Becton Dickinson, Franklin Lakes, N.J.). The total DNA from each pool of positive cells was extracted (DNeasy tissue kit; Qiagen, Chatsworth, Calif.), transformed in DH5α bacterial cells, and plated on Luria-Bertani (LB) agar containing 100 μg of ampicillin/ml (no propagation of the chloramphenicol-resistant vector harboring the PKB-F fusion). DNA plasmids containing the F-cDNA fusions were extracted from individual clones and retransfected separately with PKB-F or the F fragment alone (negative control) to discard negative clones that entered the pool during the cell sorting. After this second round of selection, the DNA plasmids corresponding to the positive clones were submitted to sequence analysis.
Cell transfection and fluorometric analysis.
COS-1 and HEK293T cells were split in 12-well plates 24 h before transfection. Cells were transfected, at around 60% confluence, with different pairs of the GFP PCA fusion expression vectors (1 μg of total DNA/well), using Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. For the microplate measurements, 48 h after transfection cells were washed one time with phosphate-buffered saline (PBS), gently trypsinized, and resuspended in 200 μl of PBS. The total cell suspensions were transferred to 96-well black microtiter plates (Dynex Technologies, Chantilly, Va.) and subjected to fluorometric analysis (Spectra MAX GEMINI XS; Molecular Devices, Sunnyvale, Calif.). Afterwards, the data were normalized to total protein concentration in cell lysates (Bio-Rad protein assay; Bio-Rad, Hercules, Calif.). The background fluorescence intensity corresponding to nontransfected cells was subtracted from the fluorescence intensities of all of the samples. For the fluorescence microscopy, 48 h after transfection cells were washed two times with PBS, incubated for 5 h in serum-free medium, and untreated or treated with 300 nM wortmannin (Calbiochem, San Diego, Calif.) for the last hour. Afterwards, cells were stimulated for 30 min with 10% serum (FBS; HyClone). Cells were washed two times with PBS and mounted on glass slides. Fluorescence microscopy was performed on live cells (Zeiss Axiophot microscope; 100× objective lens).
Preparation of cell lysates and immunoblot analysis.
HEK293T and Tag-Jurkat cells were cotransfected with PDK1 and PKB expression vectors, or with PKB and GSK3β expression vectors, in the presence or absence of hFt1, in 12-well plates as described above. The amounts of DNA transfected in each experiment were kept constant by adding empty vector. HEK293T cells were serum starved for 16 h and stimulated for 15 min with 10% serum (FBS; HyClone) prior to lysis. Tag-Jurkat cells were stimulated for 30 min with 5 μg of phytohemagglutinin (PHA)/ml plus 500 nM phorbol-12-myristate-13-acetate (PMA; Calbiochem) prior to lysis. Cells were lysed 48 h posttransfection (lysis buffer, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 150 mM NaCl, 20 mM Tris [pH 8.0], 20 μg of aprotinin/ml, 5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 mM NaF, and 5 mM sodium vanadate). Equal amounts of total proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes, and immunoblotted with antibodies according to the manufacturer's instructions. Anti-PKB, anti-PKB-Thr308-p, anti-PKB-Ser473-p, and anti-GSK3β-Ser9-p were from Cell Signaling (Beverly, Mass.). Anti-GSK3β was from BD Biosciences (San Diego, Calif.). Anti-hFt1 is a custom-made antibody (Washington Biotechnology, Baltimore, Md.). Bound antibodies were detected using horseradish peroxidase-conjugated anti-immunoglobulin G (anti-IgG; Cell Signaling) and a chemiluminescence detection system (NEN Life Science Products, Boston, Mass.).
In vitro kinase assay.
HEK293T cells were cotransfected with PDK1 and PKB expression vectors, in the presence or absence of hFt1, in 12-well plates as described above. Cells were serum starved for 16 h and stimulated for 15 min with 10% serum (FBS) prior to lysis. PKB was immunoprecipitated with immobilized anti-PKB antibodies (Cell Signaling). Immunoprecipitates were washed four times, and the pellets were incubated in kinase buffer (25 mM Tris [pH 7.5], 5 mM β-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM sodium vanadate, 10 mM MgCl2) containing 200 μM ATP and 1 μg of a pure protein substrate (paramyosin fused to GSK-3α/β cross-tide, corresponding to 20 residues surrounding GSK-3α/β-Ser21/9) (Akt kinase assay kit; Cell Signaling) in each reaction mixture. Phosphorylation of the substrate was detected by immunoblotting using anti-GSK3α/β-Ser21/9-p antibodies (Cell Signaling). Expression levels of PKB and hFt1 were determined by immunoblotting total cell lysates with their corresponding antibodies.
In vitro binding assay.
Recombinant PKB and PDK1 were purified from Escherichia coli as His tag fusion proteins (pQE vector; Qiagen). Approximately 5 μg of glutathione S-transferase (GST) or the GST-hFt1 fusions (GST-hFt1-N [aa 1 to 116], GST-hFt1-Ub [aa 117 to 217] and GST-hFt1-C [aa 218 to 292]) was incubated with 3 μg of recombinant PKB or PDK1 for 2 h at 4°C in binding buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 1% Nonidet P-40). Proteins were eluted from glutathione Sepharose using 10 mM glutathione in 50 mM Tris, pH 8.0 (Amersham Pharmacia Biotech, Piscataway, N.J.). PKB or PDK1 that associated with the GST-hFt1 fragment fusions was detected by immunoblotting with anti-PKB (Cell Signaling) and anti-PDK1 (Biosource International, Camarillo, Calif.) antibodies, respectively. Expression levels of the GST-hFt1 fragment fusions were detected by immunoblotting with anti-GST antibodies (Cell Signaling). The same strategy was used to study the association of hFt1 with the GST-PKB fusions (GST-PKB-N [aa 1 to 149], GST-PKB-CAT [aa 150 to 408], and GST-PKB-C [aa 409 to 480]) and the GST-PDK1 fusions (GST-PDK1-N [aa 1 to 81], GST-PDK1-CAT [aa 82 to 342], GST-PDK1-C [aa 343 to 456], and GST-PDK1-PH [aa 457 to 556]).
Endogenous PKB and PDK1 were immunoprecipitated from HEK293 cell lysates with anti-PKB (Cell Signaling) and anti-PDK1 (Biosource International) antibodies, respectively, and a protein G Plus-protein A-agarose suspension (Calbiochem). Immunoprecipitates were washed four times with lysis buffer (described above), separated on SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The membrane was immunoblotted with an anti-hFt1 antibody (custom-made antibody; Washington Biotechnology). Expression levels of the endogenous proteins were detected by immunoblotting total lysates (cell extracts before immunoprecipitations) with their corresponding antibodies. For the PKB-PDK1 coimmunoprecipitation experiments, HEK293T cells were cotransfected with PKB and PDK1 expression vectors, in the presence of hFt1 or a truncated version of hFt1 lacking its C-terminal domain (aa 218 to 292) (hFt1ΔCT). PKB was immunoprecipitated, and the amount of PDK1 in the immune complexes was determined by immunoblotting with anti-PDK1 antibodies.
Tag-Jurkat cells were transfected at 106 cells/well in 12-well plates (2 μg of total DNA/well) using DMRIE-C reagent (Invitrogen) with different combinations of expression vectors (as indicated). The next day, 1 μg of PHA/ml and 50 ng of PMA/ml were added to the growth medium to enhance promoter activity and gene expression. At 48 h after transfection, cells were harvested and incubated for 7 to 8 h in serum-free medium containing 1 μM dexamethasone (Calbiochem). Another set of experiments was also performed by simultaneously adding 5 μg of neutralizing anti-human Fas ligand NOK-1 mouse antibody (BD Biosciences)/ml or control mouse anti-IgG antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). Cells were washed one time with PBS and kept overnight at 4°C in the same buffer. The next day, cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) according to the manufacturer's instructions (BD Biosciences) and analyzed by flow cytometry (FACScalibur; Becton Dickinson). The transition between early and late apoptosis was very sharp in these experiments. Thus, simultaneous detection of annexin V (early apoptosis) and propidium iodide (late apoptosis) staining was performed.
NF-AT transcription reporter assay.
Tag-Jurkat cells were transfected and treated with dexamethasone as described for the apoptosis detection experiment except that the cells were incubated with dexamethasone for only 4 h, with or without the addition of 200 nM FK506 (Calbiochem). After this incubation, cells were spun down and resuspended in 400 μl of deionized water. One quarter of this cell suspension was mixed with 100 μl of 1 mM fluorescein digalactoside (FDG; Molecular Probes, Eugene, Oreg.) prepared in deionized water and transferred to a 96-well black microtiter plate (Dynex Technologies). The resulting hypotonic solution permeabilizes the cells, allowing the FDG to enter. The plate was incubated at 37°C for 30 min, and the conversion of FDG to fluorescein was detected by fluorometry (Spectra MAX GEMINI XS; Molecular Devices). The background fluorescence intensity corresponding to cells not treated with FDG was subtracted from the fluorescence intensities of all of the samples.