Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada.
Histochem Cell Biol. 2011 Aug;136(2):177-89. doi: 10.1007/s00418-011-0839-6. Epub 2011 Jul 16.
The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.
此前已经证明，粘菌（Dictyostelium discoideum）中的哺乳动物细胞周期蛋白依赖性激酶 5（Cdk5）同源物对于该模式生物的最佳生长和分化是必需的，但是该蛋白的亚细胞定位以前尚未研究过。在这项研究中，免疫定位和 GFP 融合构建物将 Cdk5 主要定位于营养细胞的核内。Western blot 显示 Cdk5 存在于核和非核部分，这表明其在这两个细胞区室中具有功能作用。在有丝分裂的早期阶段，Cdk5 逐渐从点状核质分布转变为定位于核内膜附近。在后期和末期，Cdk5 定位于细胞质中，在核质中未检测到。Cdk5 在胞质分裂期间返回核内。蛋白水解活性已被证明是细胞周期的关键调节剂。免疫沉淀结合免疫定位将嘌呤霉素敏感氨肽酶 A（PsaA）鉴定为粘菌中 Cdk5 的潜在结合伴侣。免疫沉淀还鉴定了两种磷酸酪氨酸蛋白（35 和 18 kDa），它们可能在体内与 Cdk5 相互作用。总之，这项工作为 Cdk5 的定位、其在细胞分裂过程中的功能以及其与粘菌中蛋白水解酶的结合提供了新的见解。
