Identification and characterization of phosphatases, master regulators of cellular stress signalling pathways

蛋白质的可逆磷酸化是一种翻译后修饰，控制着几乎所有细胞过程中涉及的广泛的信号事件。几乎三分之一的细胞蛋白质被磷酸化，强调了这种严格调控的信号网络的巨大重要性。为了控制这么多靶标的修饰，细胞编码大约400种具有不同特异性的激酶。相反，逆转蛋白质磷酸化的蛋白磷酸酶长期以来被认为是非特异性酶，因为编码这些酶的基因数量相当少。此外，纯化的催化实体在体外测定中显示出对它们的底物的相当非歧视性的活性。然而，近年来的研究表明，许多磷酸酶是专性寡聚酶，形成了大量的全酶具有不同的底物特异性。蛋白磷酸酶1（PP 1）在真核细胞中占丝氨酸/苏氨酸去磷酸化事件的大部分。迄今为止，已鉴定出约200个PP 1调节亚基，这些亚基决定了PP 1的特异性和亚细胞定位。尽管它们在控制细胞信号传导方面具有巨大的重要性，但对特定PP 1全酶的生物学知之甚少。在Bertolotti实验室期间，我计划鉴定和表征新的磷酸酶全酶，在细胞应激信号的调节和终止中发挥重要作用。为此，我的目的是研究PP 1相互作用组在内质网（ER）和胞质应激条件下，以确定监管亚基，这是参与这些途径的调节和信号终止。此外，我的目的是阐明参与泛素结合酶UBE 2 J1，这是一个关键的球员在ER蛋白质稳态的去磷酸化的信号事件。UBE 2 J1在不同的压力下被磷酸化，这种修饰似乎对细胞存活至关重要。在第三个项目中，我将专注于一个特定的PP 1调节亚基的特性可能参与胞质应激信号。总之，新的PP 1全酶的鉴定和表征将加深我们对应激反应途径和相关疾病的理解。

The reversible phosphorylation of proteins is a post-translational modification controlling a wide range of signalling events involved in virtually all cellular processes. Almost one third of cellular proteins become phosphorylated, underlining the immense importance of this tightly regulated signalling network. To control the modification of that many targets, cells encode around 400 kinases with differing specificities. In contrast, protein phosphatases, which reverse the phosphorylation of proteins, were long thought to be unspecific enzymes due to the rather small number of genes encoding for these enzymes. Furthermore, the purified catalytic entities display a fairly non-discriminating activity towards their substrates in in vitro assays. However, research from recent years demonstrated that many phosphatases are obligatory oligomeric enzymes, forming a large number of holoenzymes with distinct substrate specificities. Protein phosphatase 1 (PP1) accounts for the majority of serine/threonine dephosphorylation events in eukaryotic cells. To date, approximately 200 regulatory subunits of PP1 are identified, which determine its specificity and subcellular localization. Despite their tremendous importance in controlling cellular signalling, little is known about the biology of specific PP1 holoenzymes. During my stay in the Bertolotti lab, I plan to identify and characterize new phosphatase holoenzymes, playing a major role in the regulation and termination of cellular stress signalling. For this purpose, I aim to investigate the PP1 interactome during endoplasmic reticulum (ER) and cytosolic stress conditions to identify regulatory subunits, which are involved in the regulation and signal termination of these pathways. Additionally, I aim to elucidate the signalling events involved in the dephosphorylation of the ubiquitin conjugating enzyme UBE2J1, which is a key player in protein homeostasis at the ER. UBE2J1 is phosphorylated upon different stresses and this modification appears to be essential for cellular survival. In a third project, I will focus on the characterization of a specific PP1 regulatory subunit potentially involved in cytosolic stress signalling. Altogether, the identification and characterization of new PP1 holoenzymes will deepen our understanding of stress response pathways and related diseases.