DYRK protein kinases in Chlamydomonas reinhardtii: Biochemical characterization and physiological study of a DYRK mutant strain

双特异性酪氨酸磷酸化调节激酶（DYRKs）是目前研究的所有真核生物中发现的具有共同结构和生化特性的激酶。DYRK成员参与几种信号传导途径，这对细胞稳态和发育很重要。然而，DYRK蛋白在植物中的作用仍然未知。最近，一个DYRK突变体的衣原体（std 1）分离在一个正向遗传方法筛选淀粉降解突变体。基于系统发育分析，我们发现了一个新的亚群（DYRKP），它只由绿色谱系的DYRK同源物组成。最近的数据表明，DYRKP-1（Std 1）不仅在淀粉降解中发挥作用，导致初始产氢量下降，而且在自养条件下，导致更高的淀粉积累，光合活性和生物量生产的氮剥夺的细胞应激反应中发挥作用。在这个项目中，我们希望进一步表征DYRK突变体的生理和相应的激酶生化。是否只有氮剥夺触发观察到的表型？在不同的光照强度下会发生什么？我们也有兴趣找出是否有较高的淀粉积累和较高的光合活性之间的联系，使用无淀粉菌株。在生物化学方法中，我们希望产生HA标记的菌株，该菌株将用于定位蛋白质，并且对于未来的分析也非常有用，例如寻找激酶底物和相互作用伴侣。最后，我们计划通过定点突变检测衣原体DYRKP-1保守的自磷酸化基序的功能。

Dual-specificity tyrosine-phosphorylation regulated kinases (DYRKs) are found in all eukaryotes studied so far and share common structural and biochemical properties. DYRK members participate in several signalling pathways, which are important for cell homeostasis and development. However, the role of DYRK proteins in plants is still unknown. Recently, a DYRK mutant of Chlamydomonas (std1) was isolated in a forward genetic approach to screen for starch degradation mutants. Based on phylogenetic analysis, we discovered a new subgroup (DYRKP) that is only composed of DYRK homologs of the green lineage. Recent data indicate that DYRKP-1 (Std1) does not only play a role in starch degradation leading to a decreased initial hydrogen production, but also in the cellular stress response to nitrogen deprivation during autotrophic condition causing higher starch accumulation, photosynthetic activity and biomass production in the mutant. In this project we want to further characterize the DYRK mutant physiologically and the corresponding kinase biochemically. Does only nitrogen deprivation trigger the observed phenotype? What happens in different light intensities? We are also interested to find out if there is a link between higher starch accumulation and higher photosynthetic activity using a starchless strain. In a biochemical approach we want to generate an HA-tagged strain that will be used to localize the protein and will also be very useful for future analysis like the search for kinase substrates and interaction partners. Finally, we plan to test the functionality of the conserved autophosphorylation motif of Chlamydomonas DYRKP-1 via site-directed mutagenesis.