Thayyullathil Faisal, Pallichankandy Siraj, Rahman Anees, Kizhakkayil Jaleel, Chathoth Shahanas, Patel Mahendra, Galadari Sehamuddin
Cell Signaling Laboratory, Department of Biochemistry, College of Medicine and Health Sciences, UAE University, P,O, Box 17666, Al Ain, Abu Dhabi, UAE.
J Mol Signal. 2013 Feb 27;8(1):2. doi: 10.1186/1750-2187-8-2.
Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that selectively activates and induces apoptosis in cancer cells, but not in normal cells. The cancer specific pro-apoptotic function of Par-4 is encoded in its centrally located SAC (Selective for Apoptosis induction in Cancer cells) domain (amino acids 137-195). The SAC domain itself is capable of nuclear entry, caspase activation, inhibition of NF-κB activity, and induction of apoptosis in cancer cells. However, the precise mechanism(s) of how the SAC domain is released from Par-4, in response to apoptotic stimulation, is not well explored.
In this study, we demonstrate for the first time that sphingosine (SPH), a member of the sphingolipid family, induces caspase-dependant cleavage of Par-4, leading to the release of SAC domain containing fragment from it. Par-4 is cleaved at the EEPD131G site on incubation with caspase-3 in vitro, and by treating cells with several anti-cancer agents. The caspase-3 mediated cleavage of Par-4 is blocked by addition of the pan-caspase inhibitor z-VAD-fmk, caspase-3 specific inhibitor Ac-DEVD-CHO, and by introduction of alanine substitution for D131 residue. Moreover, suppression of SPH-induced Akt dephosphorylation also abrogated the caspase dependant cleavage of Par-4.
Evidence provided here shows that Par-4 is cleaved by caspase-3 during SPH-induced apoptosis. Cleavage of Par-4 leads to the generation of SAC domain containing fragment which may possibly be essential and sufficient to induce or augment apoptosis in cancer cells.
前列腺凋亡反应蛋白4（Par-4）是一种肿瘤抑制蛋白，可选择性激活并诱导癌细胞凋亡，而对正常细胞无此作用。Par-4的癌症特异性促凋亡功能由其位于中央的SAC（癌细胞凋亡诱导选择性区域）结构域（氨基酸137 - 195）编码。SAC结构域本身能够进入细胞核、激活半胱天冬酶、抑制NF-κB活性并诱导癌细胞凋亡。然而，关于SAC结构域如何响应凋亡刺激从Par-4中释放的确切机制尚未得到充分研究。
在本研究中，我们首次证明鞘脂家族成员鞘氨醇（SPH）可诱导Par-4的半胱天冬酶依赖性切割，导致含有SAC结构域的片段从其中释放。在体外与半胱天冬酶-3孵育时，以及用几种抗癌药物处理细胞时，Par-4在EEPD131G位点被切割。添加泛半胱天冬酶抑制剂z-VAD-fmk、半胱天冬酶-3特异性抑制剂Ac-DEVD-CHO以及将D131残基替换为丙氨酸可阻断半胱天冬酶-3介导的Par-4切割。此外，抑制SPH诱导的Akt去磷酸化也消除了Par-4的半胱天冬酶依赖性切割。
此处提供的证据表明，在SPH诱导的凋亡过程中，Par-4被半胱天冬酶-3切割。Par-4的切割导致含有SAC结构域的片段生成，该片段可能对诱导或增强癌细胞凋亡至关重要且足够。
