Lottersberger Francisca, Panza Andrea, Lucchini Giovanna, Piatti Simonetta, Longhese Maria Pia
Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, 20126 Milan, Italy.
Genetics. 2006 Jun;173(2):661-75. doi: 10.1534/genetics.106.058172. Epub 2006 Apr 28.
14-3-3 proteins are highly conserved polypeptides that participate in many biological processes by binding phosphorylated target proteins. The Saccharomyces cerevisiae BMH1 and BMH2 genes, whose concomitant deletion is lethal, encode two functionally redundant 14-3-3 isoforms. To gain insights into the essential function(s) shared by these proteins, we searched for high-dosage suppressors of the growth defects of temperature-sensitive bmh mutants. Both the protein kinase C1 (Pkc1) and its upstream regulators Wsc2 and Mid2 were found to act as high dosage suppressors of bmh mutants' temperature sensitivity, indicating a functional interaction between 14-3-3 and Pkc1. Consistent with a role of 14-3-3 proteins in Pkc1-dependent cellular processes, shift to the restrictive temperature of bmh mutants severely impaired initiation of DNA replication, polarization of the actin cytoskeleton, and budding, as well as cell wall integrity. Because Pkc1 acts in concert with the Swi4-Swi6 (SBF) transcriptional activator to control all these processes, the defective G(1)/S transition of bmh mutants might be linked to impaired SBF activity. Indeed, the levels of the G(1) cyclin CLN2 transcripts, which are positively regulated by SBF, were dramatically reduced in bmh mutants. Remarkably, budding and DNA replication defects of bmh mutants were suppressed by CLN2 expression from an SBF-independent promoter, suggesting that 14-3-3 proteins might contribute to regulating the late G(1) transcriptional program.
14-3-3蛋白是高度保守的多肽，通过结合磷酸化的靶蛋白参与许多生物学过程。酿酒酵母的BMH1和BMH2基因，其同时缺失是致死性的，编码两种功能冗余的14-3-3亚型。为了深入了解这些蛋白共有的基本功能，我们寻找了温度敏感型bmh突变体生长缺陷的高剂量抑制子。发现蛋白激酶C1（Pkc1）及其上游调节因子Wsc2和Mid2均作为bmh突变体温度敏感性的高剂量抑制子，表明14-3-3与Pkc1之间存在功能相互作用。与14-3-3蛋白在依赖Pkc1的细胞过程中的作用一致，将bmh突变体转移到限制温度会严重损害DNA复制的起始、肌动蛋白细胞骨架的极化和出芽，以及细胞壁完整性。由于Pkc1与Swi4-Swi6（SBF）转录激活因子协同作用来控制所有这些过程，bmh突变体有缺陷的G(1)/S转变可能与SBF活性受损有关。事实上，受SBF正向调控的G(1)周期蛋白CLN2转录本水平在bmh突变体中显著降低。值得注意的是，bmh突变体的出芽和DNA复制缺陷被来自不依赖SBF的启动子的CLN2表达所抑制，这表明14-3-3蛋白可能有助于调节G(1)晚期转录程序。
