Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (UBA), C1428EGA Buenos Aires, Argentina; CONICET-UBA, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Buenos Aires C1428EHA, Argentina.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Mol Cell. 2018 Mar 15;69(6):938-952.e6. doi: 10.1016/j.molcel.2018.02.018.
We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Such collaborative phosphorylation can broaden regulatory inputs and diversify output dynamics of signaling pathways.
我们报告了一个意想不到的由细胞周期蛋白依赖性激酶 (CDK) 和丝裂原活化蛋白激酶 (MAPK) 共同调节的系统，涉及到单个底物的协同多部位磷酸化。在芽殖酵母中，蛋白质 Ste5 通过 G1 阻滞途径控制信号转导。进入细胞周期后，CDK 通过多个磷酸化位点抑制 Ste5，破坏其膜结合。我们使用定量延时显微镜，在不同的细胞周期阶段检查了 Ste5 膜募集的动力学。令人惊讶的是，在 S 期，Ste5 的募集应该被阻断，但我们观察到一个初始的募集，然后是一个陡峭的下降。这种延迟的抑制揭示了 CDK 活性和途径 MAPK Fus3 的负反馈的共同需求。突变、质谱和电泳分析表明，CDK 和 MAPK 修饰共享的位点，当两个激酶都活跃并能够结合它们在 Ste5 上的 docking 位点时，这些位点被最广泛地磷酸化。这种协同磷酸化可以拓宽信号通路的调节输入，并使输出动力学多样化。
