Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Dev Biol. 2011 Jan 15;349(2):283-95. doi: 10.1016/j.ydbio.2010.10.013. Epub 2010 Oct 20.
O(2) regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O(2)-threshold for culmination implicating it as a direct O(2)-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3(SCF)ubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O(2)-signal. Here we show that genetically induced changes in Skp1 levels also affect the O(2)-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3(SCF)ubiquitin-ligase activity modulate O(2)-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O(2)-sensing.
O(2) 调节着社会性粘菌盘基网柄菌的多细胞发育，表明它可能在其原生土壤环境中作为一个重要的线索。盘基网柄菌表达一种 HIFα 型脯氨酰 4-羟化酶 (P4H1)，其水平影响 culmination 的 O(2)阈值，暗示它作为一个直接的 O(2)传感器，就像在动物中一样。但是盘基网柄菌缺乏 HIFα，动物脯氨酰 4-羟化酶信号的介质，并且 P4H1 可以羟化 Skp1 的脯氨酸 143，Skp1 是 E3(SCF)泛素连接酶的一个亚基。Skp1 羟脯氨酸随后成为五个连续糖基转移酶反应的靶标，这些反应调节 O(2)信号。在这里，我们表明 Skp1 水平的遗传诱导变化也会影响 O(2)阈值，与修饰酶的方向相反，表明后者降低了 Skp1 的活性。与此一致，过表达的 Skp1 羟化程度较差，并且 Skp1 是可检测到的提取物中唯一的 P4H1 底物。Pro143 突变的影响，以及 Skp1 和酶水平扰动的组合的影响，与 Skp1 活性的途径调节一致。然而，一些影响与 Skp1 池修饰的变化不对应，暗示 Skp1 的亚群和可能的其他未知因素。改变的 Skp1 水平也以修饰依赖的方式影响其他发育转变。虽然动物 HIFα 的羟化导致其多泛素化和蛋白酶体降解，但盘基网柄菌 Skp1 水平受其修饰状态的影响很小。这些数据表明 Skp1 和可能的 E3(SCF)泛素连接酶活性调节 O(2)依赖性 culmination 和其他发育过程，并且至少部分介导羟化/糖基化途径在 O(2)感应中的作用。
