Hassan P, Fergusson D, Grant K M, Mottram J C
Wellcome Centre for Molecular Parasitology, University of Glasgow, Anderson College, 56 Dumbarton Road, G11 6NU, Scotland, Glasgow, UK.
Mol Biochem Parasitol. 2001 Apr 6;113(2):189-98. doi: 10.1016/s0166-6851(01)00220-1.
The Leishmania mexicana CRK3 gene encodes a cdc2-related protein kinase with activity towards histone H1. Attempts to disrupt both alleles of CRK3 in the promastigote life-cycle stage resulted in changes in cell ploidy, which were avoided only when an extra copy of CRK3 was expressed from an episome. This provides strong evidence that CRK3 is essential to L. mexicana. The cyclin-dependent kinase specific inhibitor flavopiridol inhibited affinity purified histidine tagged CRK3 (CRK3his) with an IC(50) value of 100 nM and inhibited in vitro growth of L. mexicana promastigotes. Incubation of promastigotes with 2.5 microM flavopiridol for 24 h led to cell cycle arrest with an accumulation of 95% of cells in G2 or early mitosis (G2/M). Release from cell cycle arrest resulted in a semi-synchronous re-entry into the cell cycle; samples taken at 2, 4, and 6 h after release from the block were enriched for cells in G1 (68%), S-phase (70%), and G2/M phase (61%), respectively. This method of synchronisation was used to show that the majority of CRK3his activity towards the substrate histone H1 was present at G2/M. These data suggest that CRK3 has an essential role in controlling cell cycle progression at the G2/M-phase transition in L. mexicana promastigotes.
墨西哥利什曼原虫CRK3基因编码一种与cdc2相关的蛋白激酶，对组蛋白H1具有活性。在前鞭毛体生命周期阶段尝试破坏CRK3的两个等位基因导致细胞倍性发生变化，只有当CRK3的一个额外拷贝从附加体表达时，这种变化才得以避免。这提供了强有力的证据，证明CRK3对墨西哥利什曼原虫至关重要。细胞周期蛋白依赖性激酶特异性抑制剂黄酮哌啶醇以100 nM的IC(50)值抑制亲和纯化的组氨酸标签CRK3（CRK3his），并抑制墨西哥利什曼原虫前鞭毛体的体外生长。用2.5 microM黄酮哌啶醇孵育前鞭毛体24小时导致细胞周期停滞，95%的细胞积累在G2期或早期有丝分裂期（G2/M）。从细胞周期停滞中释放导致半同步重新进入细胞周期；从阻滞中释放后2、4和6小时采集的样本分别富含G1期（68%）、S期（70%）和G2/M期（61%）的细胞。这种同步化方法用于表明CRK3his对底物组蛋白H1的大部分活性存在于G2/M期。这些数据表明，CRK3在控制墨西哥利什曼原虫前鞭毛体G2/M期转换的细胞周期进程中具有重要作用。
