14-3-3蛋白对神经元存活及其缺血损伤的保护作用和机制
摘要
14-3-3蛋白存在于所有真核生物中,在脑组织中含量非常丰富,约占脑组织中可溶性蛋白总含量的1%。已知哺乳动物细胞中14-3-3蛋白共有7种亚型:γ、β、ζ、ε、σ、τ、η,在细胞增殖分裂、生长分化、凋亡、代谢等生命活动过程中发挥重要作用。14-3-3蛋白在脑内的本质功能还不清楚。前期研究表明在缺血缺氧的星形胶质细胞中14-3-3γ蛋白的表达增加。本项目主要研究14-3-3蛋白对神经元存活及缺血损伤的保护作用。首先为了探讨14-3-3蛋白对神经细胞存活的作用,在小鼠成神经瘤细胞(N2a)及原代皮层神经元细胞中过表达14-3-3蛋白抑制肽Difopein以抑制14-3-3与其配体蛋白的结合,Hoechst染核细胞计数,MTT及荧光微孔板细胞内荧光蛋白动态检测结果都显示抑制内源性14-3-3的功能后神经元死亡明显增加,证明在正常情况下神经元的存活依赖于14-3-3蛋白功能的正常发挥。其次为了研究14-3-3蛋白对缺血缺氧诱导的神经元损伤是否也有保护作用,我们在N2a细胞中分别过表达Difopein和14-3-3不同亚型后予缺糖缺氧(OGD)处理,Hoechst染核及MTT检测结果证实抑制14-3-3蛋白加剧了OGD导致的细胞损伤,虽然过表达14-3-3不同亚型对细胞有保护作用,但14-3-3γ对OGD诱导的细胞损伤的保护作用最明显。我们进一步探讨了14-3-3γ在神经元中抗凋亡的保护机制。在N2a细胞中抑制14-3-3功能后caspase3明显激活,siRNA抑制内源性的14-3-3γ并予缺糖缺氧处理后发现Bax表达增加,caspase3激活,表明14-3-3γ抗凋亡作用主要是通过抑制Bax-caspase3途径来实现的。在大鼠脑缺血再灌注(MCAO)模型中免疫荧光双标结果也证实14-3-3γ阳性的细胞中P53、Bax表达量低,表明14-3-3γ是通过下调P53、Bax水平从而抑制线粒体凋亡途径。同时在缺血半阴影区神经元内还发现14-3-3γ有明显的核转位,提示14-3-3γ可能通过与转录因子相结合直接调控凋亡基因表达来发挥其细胞保护作用。最后我们在动物水平研究了过表达14-3-3γ对脑缺血损伤的保护作用。综上所述,14-3-3蛋白为神经元存活所必需;14-3-3蛋白,其中主要是γ亚型对缺血损伤的神经细胞有重要的保护作用;14-3-3γ通过P53-Bax-caspase3通路发挥其抗凋亡作用。研究结果将为脑中风的防治提供新的思路。
The 14-3-3 proteins are a group highly conserved regulatory molecules found in alleukaryotes. 14-3-3 proteins are highly expressed in the brain and seven mammalianisoforms (β,γ,ε,η,ζ,σ,τ) are identified. 14-3-3 proteins are involved in manybiologically important processes, including cell differentiation, proliferation,metabolism control, apoptosis. However the exact physiological function of the 14-3-3proteins in neurons remains unclear. We have previously reported that 14-3-3γprotectedischemic astrocytes from apoptosis. Here, we studied the role of 14-3-3 on neuronalsurvival and the protective effects of 14-3-3γon ischemic neurons in vitro and in vivo.First, we studied the role of endogenous 14-3-3 proteins in neuronal survival by usingdifopein (dimeric fourteen-three-three peptide inhibitor), which specifically blocks theinteraction of 14-3-3 and its ligands. Expression of difopein in N2a cells and primaryculture of cerebral cortical neurons caused severe cell death as measured by Hoechststaining, MTT and Fluorescence microplate assays, suggesting that 14-3-3 proteins wereessential for neuronal survival in normal condition. Then, the protective effects of 14-3-3 proteins on ischemic neuronal cells were studied by over-expressing difopein and 14-3-3 isoforms (β,γ,ζ,η,ε,τ,σ) in N2a cells, Hoechst staining and MTT assaydemonstrated that blocking 14-3-3 function exacerbated cell death evidently underOGD-treatment and the gamma isoform of 14-3-3 was the most effective in protectingN2a cell from OGD-induced death. We further investigated the protective mechanism of14-3-3 proteins on neuronal apoptosis under ischemia. Caspase 3 was activated afterblocking 14-3-3 function and inhibiting endogenous 14-3-3γby siRNA techniquedemonstrated that caspase 3 was activated and the expression of Bax was increased in N2a cells under OGD condition, suggesting that 14-3-3 protects ischemic neurons fromapoptosis through Bax-caspase pathway. In MACO rats, immunohistofluorescenceanalysis showed that the expression of 14-3-3γwas separated from those of P53 and Baxin cortical neurons, suggesting that 14-3-3 suppresses mitochondrial-mediated apoptoticpathway by down-regulating P53 and Bax expression. Moreover, we found thatcytoplasmic 14-3-3γtranslocated into nucleus in cortical neurons at the penumbra area,indicating that 14-3-3 works as a transcription co-factor. Finally, we studied theprotective role of 14-3-3γin ischemic neuron in vivo by viral-transfection. Takentogether, our data demonstrated that 14-3-3 proteins were required for neuronal survivaland were protective in ischemic neurons. Theγisoform was the most effective inproviding protection to ischemic neurons. 14-3-3γexerts its anti-apoptotic effectthrough P53-Bax-caspase 3 pathway.
The 14-3-3 proteins are a group highly conserved regulatory molecules found in alleukaryotes. 14-3-3 proteins are highly expressed in the brain and seven mammalianisoforms (β,γ,ε,η,ζ,σ,τ) are identified. 14-3-3 proteins are involved in manybiologically important processes, including cell differentiation, proliferation,metabolism control, apoptosis. However the exact physiological function of the 14-3-3proteins in neurons remains unclear. We have previously reported that 14-3-3γprotectedischemic astrocytes from apoptosis. Here, we studied the role of 14-3-3 on neuronalsurvival and the protective effects of 14-3-3γon ischemic neurons in vitro and in vivo.First, we studied the role of endogenous 14-3-3 proteins in neuronal survival by usingdifopein (dimeric fourteen-three-three peptide inhibitor), which specifically blocks theinteraction of 14-3-3 and its ligands. Expression of difopein in N2a cells and primaryculture of cerebral cortical neurons caused severe cell death as measured by Hoechststaining, MTT and Fluorescence microplate assays, suggesting that 14-3-3 proteins wereessential for neuronal survival in normal condition. Then, the protective effects of 14-3-3 proteins on ischemic neuronal cells were studied by over-expressing difopein and 14-3-3 isoforms (β,γ,ζ,η,ε,τ,σ) in N2a cells, Hoechst staining and MTT assaydemonstrated that blocking 14-3-3 function exacerbated cell death evidently underOGD-treatment and the gamma isoform of 14-3-3 was the most effective in protectingN2a cell from OGD-induced death. We further investigated the protective mechanism of14-3-3 proteins on neuronal apoptosis under ischemia. Caspase 3 was activated afterblocking 14-3-3 function and inhibiting endogenous 14-3-3γby siRNA techniquedemonstrated that caspase 3 was activated and the expression of Bax was increased in N2a cells under OGD condition, suggesting that 14-3-3 protects ischemic neurons fromapoptosis through Bax-caspase pathway. In MACO rats, immunohistofluorescenceanalysis showed that the expression of 14-3-3γwas separated from those of P53 and Baxin cortical neurons, suggesting that 14-3-3 suppresses mitochondrial-mediated apoptoticpathway by down-regulating P53 and Bax expression. Moreover, we found thatcytoplasmic 14-3-3γtranslocated into nucleus in cortical neurons at the penumbra area,indicating that 14-3-3 works as a transcription co-factor. Finally, we studied theprotective role of 14-3-3γin ischemic neuron in vivo by viral-transfection. Takentogether, our data demonstrated that 14-3-3 proteins were required for neuronal survivaland were protective in ischemic neurons. Theγisoform was the most effective inproviding protection to ischemic neurons. 14-3-3γexerts its anti-apoptotic effectthrough P53-Bax-caspase 3 pathway.
引文
【1】MOORE BW，et al Specific acid proteins in the nervous system Prentice Hall． New-Jersey,Englewood Clitt~，343-359(1967)
【2】一Boston PF一，et al Human 14-3-3 protein：radioimmunoassay,tissue distribution，mad cerebrospinal fluid levels in patients with neurological disorders—J Neurochem…May；38(5)：1475-82(1982)
【3】一Fu H一，et al 14-3-3 proteins：structure，function．and regulation…Annu Rev Pharmacol Toxicol一；40：617-47(2000)
【4】一Masters SC一．et al 14-3-3 proteins mediate an essential anti-apoptotic signal—J Biol Chem—Nov 30；276(48)：45 193-200(2001)
【5】一Shikano S一，et al 14-3-3 proteins：regulation of endoplasmic reticulum localization and surface expression of membrane proteins—Trends Cell Biol—Jul；16(7)：370-5(2006)
【6】一Chen XQ一．et al 14-3-3gamma is upregulated by in vitro ischemia and binds to protein kinase Raf in primary cultures of astrocytes—．Glia— Jun；42(4)：3 15-24(2003)
【7】一Rapp UR一，et al BAD association with membranes is regulatedna by Raf kinases and association with 14-3-3 proteins—Adv Enzyme Regul一47：281- 5(2007)
【8】一Meller R_，et al Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro—．Cell Death Differ一 May；10(5)：539-47(2003)
【9】一Masters SC一，14-3-3 inhibits Bad-induced cell death through interaction with serine-136一Mol Pharmac01．Dec；60(6)：1325-31(2001)
【1 0】一Chen XQ一，et al The association of 14-3-3gamma and actin plays a role in cell division and apoptosis in astrocytes—．Biochem Biophys Res Commun Aug 23；296(3)：657-63(2002)
【11】FU H，et al 14-3-3 proteins：structure，function，and regulation Annu RevPharmacol Toxicol 40：617-647(2000)
【12】Masters SC一，et al 14-3-3 proteins mediate an essential anti-apoptotic signal—JBiolChem—Nov 30；276(48)：45193-200(2001、
【13】_Nomura M一，et al 14-3-3 Interacts directly with and negatively regulates pro- apoptotic Bax—J Biol Chem—Jan 17；278(3)：2058-65(2003)
【14】一Iijima_r_Mitochondrial membrane potential and ischemic neuronal death—Neurosci Res—Jul；55(3)：234-43(2006)
【1 5】Tsuruta F，et al JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins EMBO J 23(8)：1 889 99(2004)
【16】PowellDW,et al Proteomicidentification ofl4-3-3zetaas am~ogen-activated protein kinase-activated protein kinase 2 substrate：role in dimer formation and ligand binding Mol Cell Biol 23(15)：5376 87(2003)
【17】HamaguchiA．et al Sphingosine-dependent proteinkinase-1．directedto 14-3- 3，is identified as the kinase domain of protein kinase C delta J Biol Chem 278(42)：41557——65(2003)
【18】Ma Y，et al Sphingosine activates protein kinase A type II by a novel cAMP- independent mechanism J Biol Chem 280(28)：260 1 1 7(2005)
【19】DeGregori J、et al Distinct roles for E2F proteins in cell growth control and apoptosis Proc Natl Acad Sci USA 94(14)：7245 50(1997)
[1 ] Van Hemert, et al. 14-3-3 proteins: key regulators of cell division, signallingandapoptosis. Bioessays 23:936-946 (2001).
[2] MOORE BW, et al. Specific acid proteins in the nervous system. PrenticeHall, New Jersey, Englewood Cliffs, 343-359 (1967).
[3] Xiao, B. et al. Structure of a 14-3-3 protein and implications for coordinationof multiple signalling pathways. Nature 376: 188-191 (1995).
[4] FU H, et al. 14-3-3 proteins: structure, function, and regulation. Annu RevPharmacol Toxicol 40: 617-647 (2000).
[5] Zhang L, et al. Suppression of apoptosis signal-regulating kinase 1-inducedcell death by 14-3-3 proteins. Proc Natl Acad Sci USA 96(15):8511-5 (1999).
[6] Xing H, et al. 14-3-3 proteins block apoptosis and differentially regulateMAPK cascades. EMBO J 19(3):349-58 (2000).
[7] Masters SC, et al. 14-3-3 proteins mediate an essential anti-apoptotic signal. JBiol Chem 276(48);45193-200 (2001).
[8] Won J, et al. Cleavage of 14-3-3 protein by caspase-3 facilitates Badinteraction with Bcl-x(L) during apoptosis. J Biol Chem 278(21): 19347-51(2003).
[9] Nomura M, et al. 14-3-3 interacts directly with and negatively regulates pro-apoptotic Bax. J Biol Chem 278(3):2058-65(2003).
[10] Lei K, et al. The Bax subfamily of Bcl2-related proteins is essential forapoptotic signal transduction by c-Jun NH(2)-terminal kinase. Mol Cell Biol22(13):4929-42(2002). [11l] Tsuruta F, et al. JNK promotes Bax translocation to mitochondria throughphosphorylation of 14-3-3 proteins. EMBO J 23(8): 1889-99 (2004).
[12] Aitken A, et al. 14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta In vivo stoichiometric phosphorylation in brain at aSer-Pro-Glu-Lys motif. J Biol Chem 270(11): 5706-9 (1995).
[13] Liu D, et al. Crystal structure of the zeta isoform of the 14-3-3 protein. Nature376(6536):191-4(1995).
[14] Yoshida K, et al. JNK phosphorylation of 14-3-3 proteins regulates nucleartargeting of c-Abl in the apoptotic response to DNA damage. Nat CellBiol7(3):278-85(2005).
[15] DeGregori J, et al. Distinct roles for E2F proteins in cell growth control andapoptosis. ProcNatlAcadSciUSA94(14):7245-50 (1997).
[16] Sunayama J, et al. JNK antagonizes Akt-mediated survival signals byphosphorylating 14-3-3. J Cell Biol 170(2):295-304 (2005).
[17] Pendergast AM, etal. Stress and death: breaking up the c-Abl/14-3-3 complexin apoptosis. Nat Cell Biol 7(3):213^ (2005)
[18] Tzivion G, et al. A dimeric 14-3-3 protein is an essential cofactor for Rafkinase activity. Nature 394(6688):88-92 (1998).
[19] Shen YH, et al. Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding. MolBiol Cell 14(11): 4721-33 (2003).
[20] Megidish T, et al. The signal modulator protein 14-3-3 is a target ofsphingosine- or N,N-dimethyl sphingosine-dependent kinase in 3T3(A31) cells.Biochem Biophys Res Commun 216(3):739-47 (1995).
[21] Megidish T, et al. A novel sphingosine-dependent protein kinase (SDKl)specifically phosphorylates certain isoforms of 14-3-3 protein. J Biol Chem273(34):21834-45(1998).
[22] Woodcock JM, et al. The dimeric versus monomeric status of 14-3-3zeta iscontrolled by phosphorylation of Ser58 at the dimer interface. J Biol Chem278(38):36323-7(2003). [23 ] Suzuki E, et al. Sphingosine-dependent apoptosis: a unified concept based onmultiple mechanisms operating in concert. Proc Natl Acad Sci USA101(41): 14788-93 (2004).
[24] ObsiTet al. Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. A role for scaffolding in enzyme regulation. Cell105(2):257-67(2001).
[25] Powell DW, et al. Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimerformation and ligand binding. Mol Cell Biol 23(15):5376-87 (2003).
[26] Hamaguchi A, etal. Sphingosine-dependent protein kinase-1, directed to 14-3-3, is identified as the kinase domain of protein kinase C delta. J Biol Chem278(42):41557-65(2003).
[27] Ma Y, et al. Sphingosine activates protein kinase A type II by a novel cAMP-independent mechanism. J Biol Chem 280(28): 26011-7 (2005).
[28] Danial NN, et al. Cell death: critical control points. Cell 116(2):205-19(2004).
[29] Bakhshi A, et al. Cloning the chromosomal breakpoint of t(14;18) humanlymphomas: clustering around JH on chromosome 14 and near a trans criptionalunit on 18. Cell 41(3):899-906 (1985).
[30] Li, Z. et al. Down-regulation of 14-3-3 suppresses anchorage-independentgrowth of lung cancer cells through anoikis activation. Proc. Natl. Acad. Sci. U. S.A. 105, 162-167(2008)
【2】一Boston PF一，et al Human 14-3-3 protein：radioimmunoassay,tissue distribution，mad cerebrospinal fluid levels in patients with neurological disorders—J Neurochem…May；38(5)：1475-82(1982)
【3】一Fu H一，et al 14-3-3 proteins：structure，function．and regulation…Annu Rev Pharmacol Toxicol一；40：617-47(2000)
【4】一Masters SC一．et al 14-3-3 proteins mediate an essential anti-apoptotic signal—J Biol Chem—Nov 30；276(48)：45 193-200(2001)
【5】一Shikano S一，et al 14-3-3 proteins：regulation of endoplasmic reticulum localization and surface expression of membrane proteins—Trends Cell Biol—Jul；16(7)：370-5(2006)
【6】一Chen XQ一．et al 14-3-3gamma is upregulated by in vitro ischemia and binds to protein kinase Raf in primary cultures of astrocytes—．Glia— Jun；42(4)：3 15-24(2003)
【7】一Rapp UR一，et al BAD association with membranes is regulatedna by Raf kinases and association with 14-3-3 proteins—Adv Enzyme Regul一47：281- 5(2007)
【8】一Meller R_，et al Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro—．Cell Death Differ一 May；10(5)：539-47(2003)
【9】一Masters SC一，14-3-3 inhibits Bad-induced cell death through interaction with serine-136一Mol Pharmac01．Dec；60(6)：1325-31(2001)
【1 0】一Chen XQ一，et al The association of 14-3-3gamma and actin plays a role in cell division and apoptosis in astrocytes—．Biochem Biophys Res Commun Aug 23；296(3)：657-63(2002)
【11】FU H，et al 14-3-3 proteins：structure，function，and regulation Annu RevPharmacol Toxicol 40：617-647(2000)
【12】Masters SC一，et al 14-3-3 proteins mediate an essential anti-apoptotic signal—JBiolChem—Nov 30；276(48)：45193-200(2001、
【13】_Nomura M一，et al 14-3-3 Interacts directly with and negatively regulates pro- apoptotic Bax—J Biol Chem—Jan 17；278(3)：2058-65(2003)
【14】一Iijima_r_Mitochondrial membrane potential and ischemic neuronal death—Neurosci Res—Jul；55(3)：234-43(2006)
【1 5】Tsuruta F，et al JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins EMBO J 23(8)：1 889 99(2004)
【16】PowellDW,et al Proteomicidentification ofl4-3-3zetaas am~ogen-activated protein kinase-activated protein kinase 2 substrate：role in dimer formation and ligand binding Mol Cell Biol 23(15)：5376 87(2003)
【17】HamaguchiA．et al Sphingosine-dependent proteinkinase-1．directedto 14-3- 3，is identified as the kinase domain of protein kinase C delta J Biol Chem 278(42)：41557——65(2003)
【18】Ma Y，et al Sphingosine activates protein kinase A type II by a novel cAMP- independent mechanism J Biol Chem 280(28)：260 1 1 7(2005)
【19】DeGregori J、et al Distinct roles for E2F proteins in cell growth control and apoptosis Proc Natl Acad Sci USA 94(14)：7245 50(1997)
[1 ] Van Hemert, et al. 14-3-3 proteins: key regulators of cell division, signallingandapoptosis. Bioessays 23:936-946 (2001).
[2] MOORE BW, et al. Specific acid proteins in the nervous system. PrenticeHall, New Jersey, Englewood Cliffs, 343-359 (1967).
[3] Xiao, B. et al. Structure of a 14-3-3 protein and implications for coordinationof multiple signalling pathways. Nature 376: 188-191 (1995).
[4] FU H, et al. 14-3-3 proteins: structure, function, and regulation. Annu RevPharmacol Toxicol 40: 617-647 (2000).
[5] Zhang L, et al. Suppression of apoptosis signal-regulating kinase 1-inducedcell death by 14-3-3 proteins. Proc Natl Acad Sci USA 96(15):8511-5 (1999).
[6] Xing H, et al. 14-3-3 proteins block apoptosis and differentially regulateMAPK cascades. EMBO J 19(3):349-58 (2000).
[7] Masters SC, et al. 14-3-3 proteins mediate an essential anti-apoptotic signal. JBiol Chem 276(48);45193-200 (2001).
[8] Won J, et al. Cleavage of 14-3-3 protein by caspase-3 facilitates Badinteraction with Bcl-x(L) during apoptosis. J Biol Chem 278(21): 19347-51(2003).
[9] Nomura M, et al. 14-3-3 interacts directly with and negatively regulates pro-apoptotic Bax. J Biol Chem 278(3):2058-65(2003).
[10] Lei K, et al. The Bax subfamily of Bcl2-related proteins is essential forapoptotic signal transduction by c-Jun NH(2)-terminal kinase. Mol Cell Biol22(13):4929-42(2002). [11l] Tsuruta F, et al. JNK promotes Bax translocation to mitochondria throughphosphorylation of 14-3-3 proteins. EMBO J 23(8): 1889-99 (2004).
[12] Aitken A, et al. 14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta In vivo stoichiometric phosphorylation in brain at aSer-Pro-Glu-Lys motif. J Biol Chem 270(11): 5706-9 (1995).
[13] Liu D, et al. Crystal structure of the zeta isoform of the 14-3-3 protein. Nature376(6536):191-4(1995).
[14] Yoshida K, et al. JNK phosphorylation of 14-3-3 proteins regulates nucleartargeting of c-Abl in the apoptotic response to DNA damage. Nat CellBiol7(3):278-85(2005).
[15] DeGregori J, et al. Distinct roles for E2F proteins in cell growth control andapoptosis. ProcNatlAcadSciUSA94(14):7245-50 (1997).
[16] Sunayama J, et al. JNK antagonizes Akt-mediated survival signals byphosphorylating 14-3-3. J Cell Biol 170(2):295-304 (2005).
[17] Pendergast AM, etal. Stress and death: breaking up the c-Abl/14-3-3 complexin apoptosis. Nat Cell Biol 7(3):213^ (2005)
[18] Tzivion G, et al. A dimeric 14-3-3 protein is an essential cofactor for Rafkinase activity. Nature 394(6688):88-92 (1998).
[19] Shen YH, et al. Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding. MolBiol Cell 14(11): 4721-33 (2003).
[20] Megidish T, et al. The signal modulator protein 14-3-3 is a target ofsphingosine- or N,N-dimethyl sphingosine-dependent kinase in 3T3(A31) cells.Biochem Biophys Res Commun 216(3):739-47 (1995).
[21] Megidish T, et al. A novel sphingosine-dependent protein kinase (SDKl)specifically phosphorylates certain isoforms of 14-3-3 protein. J Biol Chem273(34):21834-45(1998).
[22] Woodcock JM, et al. The dimeric versus monomeric status of 14-3-3zeta iscontrolled by phosphorylation of Ser58 at the dimer interface. J Biol Chem278(38):36323-7(2003). [23 ] Suzuki E, et al. Sphingosine-dependent apoptosis: a unified concept based onmultiple mechanisms operating in concert. Proc Natl Acad Sci USA101(41): 14788-93 (2004).
[24] ObsiTet al. Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. A role for scaffolding in enzyme regulation. Cell105(2):257-67(2001).
[25] Powell DW, et al. Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimerformation and ligand binding. Mol Cell Biol 23(15):5376-87 (2003).
[26] Hamaguchi A, etal. Sphingosine-dependent protein kinase-1, directed to 14-3-3, is identified as the kinase domain of protein kinase C delta. J Biol Chem278(42):41557-65(2003).
[27] Ma Y, et al. Sphingosine activates protein kinase A type II by a novel cAMP-independent mechanism. J Biol Chem 280(28): 26011-7 (2005).
[28] Danial NN, et al. Cell death: critical control points. Cell 116(2):205-19(2004).
[29] Bakhshi A, et al. Cloning the chromosomal breakpoint of t(14;18) humanlymphomas: clustering around JH on chromosome 14 and near a trans criptionalunit on 18. Cell 41(3):899-906 (1985).
[30] Li, Z. et al. Down-regulation of 14-3-3 suppresses anchorage-independentgrowth of lung cancer cells through anoikis activation. Proc. Natl. Acad. Sci. U. S.A. 105, 162-167(2008)