Điều Hòa Sự Chết Tế Bào Của Protease Caspase-9 Qua Phosphorylation
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Tài liệu tham khảo
; R. M. Kluck E. Bossy-Wetzel D. R. Green D. D. Newmeyer ibid p. 1132; E. Bossy-Wetzel
Single-letter abbreviations for the amino acid residues are as follows: A Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K. Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; X any amino acid; and Y Tyr.
M. H. Cardone et al. unpublished data.
Songyang Z., Baltimore D., Cantley L. C., Kaplan D. R., Franke T. F., Proc. Natl. Acad. Sci. U.S.A. 94, 11345 (1997);
NIH 3T3 cells expressing activated oncogenic Akt were generated by viral infection of NIH 3T3 cells with a retrovirus expressing v-akt [
For labeling experiments 4 × 10 5 cells in 35-mm dishes were cultured 1 day after transfection for 3 hours in 1 ml of phosphate-free Dulbecco's modified Eagle's medium containing 1 mCi/ml ortho- 32 P (New England Nuclear) with or without 5% dialyzed serum. Cells were lysed in 20 mM Hepes 1% Triton X-100 0.5% NP-40 150 mM NaCl 20 mM NaF 2 mM Na 3 Va0 4 10 mM β-glycerophosphate and protease inhibitors. Lysates were precleared with protein A– or protein G–Sepharose with preimmune serum. Casp9 was immunoprecipitated with a monoclonal antibody (mAb) to FLAG or a polyclonal antibody to Casp9 washed and analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography or by phosphoimager analysis.
A rabbit antiserum to Casp9 was raised against purified recombinant His 6 -active Casp9 and verified to be specific for Casp9 by immunoblotting experiments using a panel of recombinant caspases including Casp3 Casp6 Casp7 Casp8 and Casp10.
293T cells (10 7 ) were transiently transfected with 25 μg of pCMV6-myrAkt-HA or pcDNA (control) DNA. The activated form of Akt was generated by adding the NH 2 -terminal Src myristoylation sequence to a pre-existing construct expressing Akt-HA in pCMV-6 (19). Cells were lysed 1 day later in 1.5 ml of 20 mM tris-HCl (pH 7.4) 140 mM NaCl 1% NP-40 10 mM NaF 1 mM Na 3 VaO 4 1 mM EDTA and protease inhibitors. After normalizing for protein concentration lysates were precleared with protein G–Sepharose and preimmune serum for 1 hour and incubated at 4°C with 0.5 μg of rat high-affinity mAb to hemagglutinin (HA) (Boehringer-Mannheim) followed by addition of 10 μl of protein G–Sepharose (Pharmacia) for 1 hour. Alternatively endogenous Akt was immunoprecipitated from 267 or 267-Ki-Ras cells with antibody to Akt (Santa Cruz Biotech) producing similar results (4). Immunoprecipitates were washed three times in lysis solution and two times in kinase solution [20 mM Hepes (pH 7.2) 10 mM MgCl 2 10 mM MnCl 2 1 mM DTT and 3 μM ATP].
GST-Akt was expressed from a recombinant baculovirus in Sf9 cells with activated forms of PI3K to achieve kinase activation. GST-Akt was purified from Sf9 lysates by glutathione-Sepharose affinity chromatography.
To determine the effects of Akt-mediated phosphorylation on caspase activity in vitro kinase reactions were performed as described (17) except that 0.1 mM ATP was substituted for [γ- 32 P]ATP. Immobilized Akt was removed by centrifugation and half the sample (20 μl) was incubated with 10 μM Ac-DEVD-pNA (Alexis) and 2 μM purified pro-Casp3 in a final volume of 0.1 ml of caspase buffer (50 mM Hepes 1 mM EDTA 0.1% CHAPS 10% sucrose and 5 mM dithiothreitol). Caspase activity was based on cleavage of the colorimetric substrate Ac-DEVD-pNA (5) and was normalized relative to Akt-untreated (mock) material. For Casp9 measurements the addition of pro-Casp3 created a coupled Casp9 → Casp3 → DEVD-pNA reaction because Casp9 does not efficiently cleave DEVD (16). Activity percent was measured and normalized to mock-treated samples. Anti-HA immune complexes prepared from control-transfected cells and immobilized GST control protein resulted in no significant alterations of caspase activity (4).
Pro-Casp9 and Pro-Casp9(C287A) cDNAs as well as S183A and S196A mutants of these were expressed with NH 2 -terminal His 6 -tags from pET23b in BL21 cells for production of processed Casp9 and unprocessed Casp9 respectively (16). Expression was induced with 0.2 mM isopropyl-β- d -thiogalactopyranoside at OD 600 ≅ 0.6 to 0.8 and ∼25°C for 4 hours for the S183A mutant and for 1 hour for the S196A mutant. Proteins were affinity purified by Ni-chelate Sepharose (Pharmacia).
For MS analysis 1 pmol of a 1.826-kD synthetic peptide corresponding to a V8 fragment containing the Akt phosphorylation site in Casp9 was kinased in vitro or mock treated and spotted onto a SELDI chip (Ciphergen Biosystems Palo Alto CA) and imbedded with cinamininic acid matrix. Alternatively 293T cells were transiently transfected with pCMV6-myrAktHA and pcDNA3-FLAG constructs encoding C287A mutants of either pro-Casp9 or pro-Casp9(S196A). Casp9 (wild type) and Casp9(S196A) were isolated by immunoprecipitation using antibody to FLAG eluted from beads with glycine (pH 3.0) and digested with 0.05 U of V8 protease for 8 hours in 50 mM NH 4 oAc (pH 4.0) at room temperature. The samples were then analyzed by SELDI as described above. An 80-dalton increase in mass indicated that the peptide fragment was phosphorylated.
Casp9 mutants were generated by site-directed polymerase chain reaction (PCR) mutagenesis from a human pro-Casp9 cDNA (V. Dixit) and subcloned into pcDNA3-FLAG pCMV2-FLAG or pET23b plasmids. The primer pairs used to generate the S183A and S196A mutants were 5′-CCGCACCCGCACTGGCGCGAACATCGACTGTGAG-3′ plus 5′-CTCACAGTCGATGTTCGCGCCAGTGCGGGTGCGG-3′; and 5′- CGGCGTCGCTTCTCCGCGCTGCATTTCCTGGTGG-3′ plus 5′-CCACCATGAAATGCAGCGCGGAGAAGCGACGCCG-3′ respectively. PCR was performed for 16 cycles at 95°C for 30 s 55°C for 1 min and 68°C for 12 min. Twenty microliters of the reactions was digested with Dpn I (10 U) for subsequent subcloning into plasmids.
L. del Peso
; X. Yang H. Y. Chang D. Baltimore ibid. p. 319.
Krajewska M., et al., Cancer Res. 57, 1605 (1997).
We thank J. Rhim for 267 Ki-Ras cells; T. G. Sambandan for mass spectroscopy advice; I. Tamm for IAP measurements; S. Kitada for help with cell lines; T. Bobo A. Sinskey and P. Sorger for support; and the members of the Reed lab for helpful discussions. This work was partially supported by a Biomeasure grant to M.C. a Department of Defense Breast Cancer grant to N.R. a Danish Natural Science Foundation grant (9600412) to H.R.S. and grants CA-69381 and CA-69515 from NIH and the National Cancer Institute.