Ca 2+ -Induced Apoptosis Through Calcineurin Dephosphorylation of BAD
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Single-letter amino acid code: A alanine; C cysteine; D aspartic acid; E glutamic acid; F phenylalanine; G glycine; H histidine; I isoleucine; K lysine; L leucine; M methionine; N asparagine; P proline; Q glutamine; R arginine; S serine; T threonine; V valine; W tryptophan; and Y tyrosine.
H.-G. Wang et al. data not shown.
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N. Pathan et al. data not shown.
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Though BAD(S75E S99E) fails to bind 14-3-3 protein it nevertheless is incapable of binding Bcl-x L . Similarly after phosphorylation by Raf-1 BAD does not bind 14-3-3 but is impaired in binding to Bcl-x L . Thus phosphorylation-mediated suppression of BAD dimerization to Bcl-x L does not necessarily correlate with BAD–14-3-3 complex formation.
Furuya Y., Lundmo P., Short A. D., Gill D. L., Isaacs J. T., Cancer Res. 54, 6167 (1994).
Mitochondrial targeting of BAD in l -glutamate–treated neurons was confirmed by two-color confocal microscopy with polyclonal antiserum to BAD in conjunction with monoclonal antibody to mitochondrial Hsp60.
In Fig. 1A a total of 8.5 μg of DNA was transfected in 100-mm dishes including 0.5 μg of pFLAG-CMV2-BAD and 2.5 μg each of pMEP encoding HA epitope–tagged ΔCnA [a Ca 2+ - and calmodulin-independent form of the A subunit of calcineurin (2)] and the B subunit (CnB) and either 3 μg of M-Raf– (9) or HA-Akt(E40K)–producing (6) plasmids or parental plasmid DNA as a control (designated by “−” in the figure). In Fig. 1B 293 cells were transiently transfected with a total of 5.5 μg of DNA including 0.5 μg of pFLAG-CMV2 encoding wild-type BAD or BAD (S75E S99E) mutant 1 μg of pcDNA3-M-Raf-1 or pCMV6-HA-Akt(E40K) and 2 μg each of pMEP encoding HA-tagged ΔCnA and CnB or parental plasmid DNA (−). The mutants BAD(S75E S99E) and Akt(E40K) were generated by polymerase chain reaction mutagenesis with pFLAG-CMV2-BAD as the template to substitute Ser 75 (TCC)/Ser 99 (TCG) for Glu 75 (GAA)/Glu 99 (GAG) or pCMV6-HA-AKT respectively. Plasmids encoding GFP-BAD and GFP-BAD(S75E S99E) were generated by cloning these BAD cDNAs into pEGFP-C2 (Clontech) with Eco RI and Xho I sites.
For in vitro dephosphorylation reactions recombinant baculovirus GST-Akt was purified from NP-40 Sf9 cell lysates with GSH-agarose (Sigma). A 1-μg sample of GST-huBad or GST-huBad(S75E S99E) (hu designates human protein) was phosphorylated in vitro at 30°C for 1 hour. The kinase reaction was stopped by NP-40 lysis buffer and GST-containing proteins were recovered with GSH-agarose. The precipitates were washed in NP-40 lysis buffer phosphate-buffered saline (PBS) and calcineurin phosphatase buffer [50 mM tris (pH 7) 0.1 mM CaCl 2 2 mM NiCl 2 bovine serum albumin (0.25 mg/ml) calmodulin (0.02 mg/ml; Boehringer-Mannheim). The precipitates were resuspended in 100 μl of phosphatase buffer and the reaction was started by addition of 2 μg of calcineurin (Upstate Biotechnology Lake Placid NY). Proteins were separated by 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by autoradiography. Alternatively GST-BAD immobilized on glutathione-Sepharose was 32 P-labeled with baculovirus-produced Raf-1 (9) then treated with various amounts of immunoprecipitates which were prepared from 293T cells transiently transfected with HA-CnA/B (20 to 200 μg of total protein lysate) or pMEP control (“C”) plasmid (200 μg of total protein lysate) respectively with anti-HA. Release of 32 P label from GST-BAD was measured after 0.5 hour at 30°C. Anti-HA immunoblot analysis was performed to quantify input HA-ΔCnA/B used for the in vitro dephosphorylation reactions. For PKA recombinant GST-huBAD or GST-huBAD(S75E S99E) (1 μg) was incubated with 1 mU of PKA (Boehringer-Mannheim) in 40 μl of PKA kinase buffer (40 mM tris-HCl 20 mM MgCl 2 pH 7.4) containing 1 mM dithiothreitol and 1 mM adenosine triphosphate (ATP) at 30°C for 1 hour. Dephosphorylation reactions were carried out at 37°C with 2 μg of calcineurin. Samples were analyzed by SDS-PAGE and immunoblotting with anti-phospho-mBAD(S112) [New England Bio Labs (NEBL)] (serine residue S112 in mouse BAD corresponds to S75 in human BAD) and anti-phospho-mBAD(S136) (corresponding to S99 in human BAD). Total BAD protein was assessed using an anti-BAD monoclonal antibody (15) and ECL detection.
Cell lysates (9) were subjected to immunoprecipitation with monoclonal anti-BAD (15) immobilized on protein G–Sepharose. Proteins were separated by SDS-PAGE and analyzed by immunoblotting with antiserum to BAD anti-S112 mBAD (NEBL) anti–14-3-3 (Santa Cruz Biotechnologies) or anti-CnA (Chemicon).
Two days after transfection 293 cells were lysed in 0.5 ml of NP-40 lysis buffer (137 mM NaCl 20 mM tris-HCI pH 8.0 1.5 mM MgCl 2 1 mM EDTA 0.2% NP-40) containing protease and phosphatase inhibitors (9). Lysates were mixed with 15 μl of anti-FLAG M2 affinity gel for 3 hours at 4°C. The beads were washed three times with 1 ml of NP-40 lysis buffer supplemented with protease and phosphatase inhibitors boiled in Laemmli buffer and the eluted proteins were subjected to 12% SDS-PAGE and immunoblot analysis with rabbit antisera specific for Bcl-x L or BAD (15).
We transiently transfected 293 cells with normalized total amounts of plasmid DNA including 1 μg of pEGFP-BAD or pEGFP-BAD(S75E S99E) 1 μg each of HA-ΔCnA– and CnB-expressing plasmids 1 μg of PPase 2A– or PPase 2C–producing plasmids 3 μg of M-Raf or HA-Akt (E40K) or various combinations of these as indicated. The location of GFP was determined by confocal microscopy (9). In some experiments mitochondria labeling was performed by addition of 50 nM Mitotracker (Molecular Probes) to culture medium for 0.5 hour.
For immunofluorescence cells were fixed in 4% paraformaldehyde/PBS (pH 7.4) for 0.5 hour and blocked in 5% (v/v) goat serum. After permeabilization in 0.2% Triton X-100 for 10 min cells were incubated with polyclonal rabbit anti-HA or anti-Hsp60 (1:500; Berkeley Antibody) which were detected with Cy3- or rhodamine-conjugated goat anti–rabbit IgG (1:100; Chemicon) or with anti-BAD (15) followed by fluorescein isothiocyanate (FITC)–conjugated rabbit anti-mouse IgG (1:100; DAKO) and analyzed with a Bio-Rad MRC 1024 confocal laser scanning microscope.
Rat E17 hippocampal neuronal cultures free of glia were prepared as described (26). The hippocampal neurons were transiently transfected by a calcium phosphate precipitation method (MBS transfection kit Stratagene) (26) and exposed to 1 mM l -glutamate 3 days later for 0.5 hour followed by incubation in DMEM for an additional 4 to 24 hours in 5% CO 2 /10% O 2 at 37°C.
Comparisons were also performed of neurons that were cultured in insulin-containing medium for 3 days then either exposed for 0.5 hour to 1 mM l -glutamate before they were returned to usual culture conditions or exposed continuously to 10 μM staurosporine (13). Replicate cultures of cells were fixed at either 6 hours for analysis of GFP-BAD (direct visualization) and myc–NF-AT (anti-myc indirect immunofluorescence) location or at 24 hours for assessment of apoptosis by staining with 4′6′-diamidino-2-phenylindole (DAPI) (10 μg/ml).
We thank T. Brown for manuscript preparation E. Smith for graphics D. Bredesen for CSM14.1 cells and the NIH (AG-1593 CA-69381 HD25938 NS-36821) AACR (H.G.W.) and Leukemia Society of America (N.P.) for support.