Điều Hòa Sự Chết Tế Bào Của Protease Caspase-9 Qua Phosphorylation
Tóm tắt
Caspase là các protease nội bào hoạt động như các chất khởi xướng và tác nhân thực hiện của quá trình apoptosis. Chất kinase Akt và p21-Ras, một chất kích hoạt Akt, đã kích thích phosphorylation của pro-caspase-9 (pro-Casp9) trong tế bào. Quá trình xử lý proteolytic của pro-Casp9 do cytochrome c kích thích bị khiếm khuyết trong các chế phẩm từ tế bào có biểu hiện Ras hoạt động hoặc Akt. Akt phosphoryl hóa recombinant Casp9 trong ống nghiệm tại serine-196 và ức chế hoạt động protease của nó. Pro-Casp9 đột biến (Ser196Ala) kháng lại phosphorylation và ức chế do Akt ở trong ống nghiệm và trong tế bào, dẫn đến sự khởi động apoptosis kháng Akt. Do đó, caspase có thể được điều hòa trực tiếp bởi phosphorylation của protein.
Từ khóa
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 unpublished data.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.