Sekiya-Kawasaki Mariko, Groen Aaron Chris, Cope M Jamie T V, Kaksonen Marko, Watson Hadiya A, Zhang Chao, Shokat Kevan M, Wendland Beverly, McDonald Kent L, McCaffery J Michael, Drubin David G
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.
J Cell Biol. 2003 Sep 1;162(5):765-72. doi: 10.1083/jcb.200305077.
We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.
我们运用化学遗传学方法来调控出芽酵母Prk1p的活性，Prk1p是一种蛋白激酶，与哺乳动物的GAK和AAK1相关，且作用于几种参与胞吞作用的肌动蛋白调节蛋白。体内Prk1p抑制作用会阻断信息素受体的胞吞作用，并导致皮质肌动蛋白斑迅速聚集成大团块，这些团块包含Abp1p、Sla2p、Pan1p、Sla1p和Ent1p。团块形成依赖于Arp2p，这表明该表型可能是由未受调控的Arp2/3刺激的肌动蛋白组装所致。电子显微镜/免疫电子显微镜分析以及对胞吞膜标记物FM4-64的追踪显示，肌动蛋白团块内存在可能源自胞吞作用的囊泡。去除抑制剂后，肌动蛋白团块迅速解体，且重新出现了正确极化的肌动蛋白斑。我们的结果表明，肌动蛋白团块是由于在一个正常短暂步骤中受阻而形成的，在此步骤中，肌动蛋白组装受到胞吞蛋白的刺激。因此，我们揭示了对一个内在动态的、与肌动蛋白斑相关过程的严格磷酸化调控，并提出Prk1p负向调节胞吞蛋白的肌动蛋白组装刺激活性。
