Nocodazole induces apoptosis in human Jurkat T cells through G2/M arrest-dependent phosphorylation of Bcl-2 and Bim by p34cdc2 kinase and subsequent provoking mitochondrial death signaling pathway (85.16)

Abstract

Abstract Treatment of human Jurkat T cells with nocodazole, a microtubule-depolymerizing agent, provoked apoptosis along with G2/M-arrest, Bcl-2 phosphorylation at Thr-56 and Ser-70 residues, Bim phosphorylation, Δψm loss, and caspase cascade activation. Whereas these nocodazole-induced apoptotic events were abrogated by Bcl-2 overexpression, the G2/M-arrest and phosphorylation of Bcl-2 and Bim were sustained. Although Bcl-2 and Bim were mainly located in mitochondrial fraction regardless of their phosphorylation status, their physical interaction became reduced upon phosphorylation. FADD-positive Jurkat clone A3 and FADD-deficient Jurkat clone I2.1 exhibited similar susceptibilities to the apoptogenic activity of nocodazole. Further analysis using selective caspase inhibitors elucidated that the activation of caspase-9 and -3, leading to activation of caspase-7 and -8, was crucial for nocodazole-induced apoptosis. In the presence of the G1/S blocking agent (hydroxyurea), nocodazole failed to induce G2/M-arrest, phosphorylation of Bcl-2 and Bim, and mitochondria-dependent apoptotic events. Nocodazole-induced phosphorylation of Bcl-2 and Bim, Δψm loss, and apoptotic events were significantly reduced by pretreatment with p34cdc2 kinase inhibitor (roscovitine). Consequently, these results indicated that G2/M-arrest and resultant phosphorylation of Bcl-2 and Bim dictated by p34cdc2 kinase, contributed to mitochondria-dependent apoptosis in Jurkat T cells exposed to nocodazole.