Rohlfing Anne-Katrin, Schill Tillmann, Müller Christian, Hildebrandt Petra, Prowald Alexandra, Hildebrandt Jan-Peter
Animal Physiology and Biochemistry, Ernst Moritz Arndt-University Greifswald, Biotechnikum, Walther Rathenau-Strasse 49 a, 17489 Greifswald, Germany.
J Comp Physiol B. 2005 Oct;175(7):511-22. doi: 10.1007/s00360-005-0019-x. Epub 2005 Oct 26.
Cell cycle arrest in potentially dividing cells is often mediated by inhibitors of G1/S-phase cyclin-dependent kinases. The cyclin E/CDK2-inhibitor p27(Kip1) has been implicated in this context in epithelial cells. We cloned and sequenced p27(Kip1) of ducklings (Anas platyrhynchos) and used an in vitro assay system to study the mechanism of p27(Kip1) downregulation in the nasal gland which precedes an increase in proliferation rate upon initial exposure of the animals to osmotic stress. Western blot studies revealed that p27(Kip1) is downregulated during 24 h of osmotic stress in ducklings with the steepest decline in protein levels between 5 and 8 h. As indicated by the results of Northern blot and semi-quantitative PCR studies, protein downregulation is not accompanied by similar changes in mRNA levels indicating that Kip1 is regulated mainly at the translational (synthesis) or posttranslational level (degradation). Using recombinant duck Kip1 protein expressed in E. coli, we showed that Kip1 is subject to polyubiquitinylation by cytosolic enzymes from nasal gland cells indicating that loss of Kip1 may be regulated, at least in part, by acceleration of protein degradation. In cultured nasal gland tissue, attenuation of Kip1 expression could be induced by activation of the muscarinic acetylcholine receptor indicating that mAChR-receptor signalling may play a role in the re-entry of quiescent gland cells into the cell cycle.
潜在分裂细胞中的细胞周期停滞通常由G1/S期细胞周期蛋白依赖性激酶的抑制剂介导。细胞周期蛋白E/细胞周期蛋白依赖性激酶2抑制剂p27（Kip1）在此背景下与上皮细胞有关。我们克隆并测序了雏鸭（绿头鸭）的p27（Kip1），并使用体外检测系统研究了在动物初次暴露于渗透应激后增殖率增加之前鼻腺中p27（Kip1）下调的机制。蛋白质印迹研究表明，在雏鸭渗透应激24小时期间，p27（Kip1）下调，蛋白质水平在5至8小时之间下降最为显著。如Northern印迹和半定量PCR研究结果所示，蛋白质下调并未伴随着mRNA水平的类似变化，这表明Kip1主要在翻译（合成）或翻译后水平（降解）受到调控。使用在大肠杆菌中表达的重组鸭Kip1蛋白，我们表明Kip1可被鼻腺细胞的胞质酶多聚泛素化，这表明Kip1的缺失可能至少部分受蛋白质降解加速的调控。在培养的鼻腺组织中，毒蕈碱型乙酰胆碱受体的激活可诱导Kip1表达的减弱，这表明毒蕈碱型乙酰胆碱受体信号传导可能在静止腺细胞重新进入细胞周期中发挥作用。
