Wang Y, Dimitrov K, Garrity L K, Sazer S, Beverley S M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Mol Biochem Parasitol. 1998 Oct 30;96(1-2):139-50. doi: 10.1016/s0166-6851(98)00121-2.
Cell cycle control by cdc2-related kinases (CRKs) is essential to the regulation of cell proliferation and developmental processes in many organisms. Alternating phases of growth, arrest, and differentiation are characteristics of the infectious cycle of many trypanosomatid parasites, raising the possibility that members of the trypanosomatid CRK gene family participate in the regulation of these essential processes. Here we describe properties of the CRK3 gene from Leishmania major, which encodes a 36 kDa protein kinase showing 60% amino acid sequence identity with human CDK2, including several conserved sites implicated in regulation of kinase activity. CRK3 mRNA was constitutively expressed throughout the parasite life cycle, but histone H1 kinase activity of an epitope tagged CRK3 protein was greater in log-phase than in stationary-phase promastigotes. When integrated into the genome and expressed at the optimal level, CRK3 was able to rescue the growth defect of a Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)), indicating that CRK3 is a functional homolog of cdc2. Mutants of CRK3 at several key regulatory residues showed the expected dominant negative effects on the S. pombe mutant. This is the first example of functional expression of a trypanosomatid CRK in yeast, opening the way for further genetic studies within this amenable organism.
细胞周期蛋白依赖性激酶（CRKs）对许多生物体中细胞增殖和发育过程的调控至关重要。生长、停滞和分化的交替阶段是许多锥虫寄生虫感染周期的特征，这增加了锥虫CRK基因家族成员参与调控这些重要过程的可能性。在此，我们描述了来自硕大利什曼原虫的CRK3基因的特性，该基因编码一种36 kDa的蛋白激酶，与人类CDK2的氨基酸序列同一性为60%，包括几个与激酶活性调控相关的保守位点。CRK3 mRNA在寄生虫的整个生命周期中持续表达，但表位标记的CRK3蛋白的组蛋白H1激酶活性在对数期前鞭毛体中比在稳定期前鞭毛体中更高。当整合到基因组中并以最佳水平表达时，CRK3能够挽救粟酒裂殖酵母cdc2突变体（cdc2-33(ts)）的生长缺陷，表明CRK3是cdc2的功能同源物。CRK3在几个关键调控残基处的突变体对粟酒裂殖酵母突变体显示出预期的显性负效应。这是锥虫CRK在酵母中功能表达的首个例子，为在这种易于操作的生物体中进行进一步的遗传学研究开辟了道路。
