Evolution of opposing regulatory interactions underlies the emergence of eukaryotic cell cycle checkpoints

In eukaryotes the entry into mitosis is initiated by activation of cyclin-dependent kinases (CDKs), which in turn activate a large number of protein kinases to induce all mitotic processes. The general view is that kinases are active in mitosis and phosphatases turn them off in interphase. Kinases activate each other by cross- and self-phosphorylation, while phosphatases remove these phosphate groups to inactivate kinases. Crucial exceptions to this general rule are the interphase kinase Wee1 and the mitotic phosphatase Cdc25. Together they directly control CDK in an opposite way of the general rule of mitotic phosphorylation and interphase dephosphorylation. Here we investigate why this opposite system emerged and got fixed in almost all eukaryotes. Our results show that this reversed action of a kinase-phosphatase pair, Wee1 and Cdc25, on CDK is particularly suited to establish a stable G2 phase and to add checkpoints to the cell cycle. We show that all these regulators appeared together in LECA (Last Eukaryote Common Ancestor) and co-evolved in eukaryotes, suggesting that this twist in kinase-phosphatase regulation was a crucial step happening at the emergence of eukaryotes.

Kinetochore phosphatases suppress autonomous Polo-like kinase 1 activity to control the mitotic checkpoint

Local phosphatase regulation is needed at kinetochores to silence the mitotic checkpoint (a.k.a. spindle assembly checkpoint [SAC]). A key event in this regard is the dephosphorylation of MELT repeats on KNL1, which removes SAC proteins from the kinetochore, including the BUB complex. We show here that PP1 and PP2A-B56 phosphatases are primarily required to remove Polo-like kinase 1 (PLK1) from the BUB complex, which can otherwise maintain MELT phosphorylation in an autocatalytic manner. This appears to be their principal role in the SAC because both phosphatases become redundant if PLK1 is inhibited or BUB–PLK1 interaction is prevented. Surprisingly, MELT dephosphorylation can occur normally under these conditions even when the levels or activities of PP1 and PP2A are strongly inhibited at kinetochores. Therefore, these data imply that kinetochore phosphatase regulation is critical for the SAC, but primarily to restrain and extinguish autonomous PLK1 activity. This is likely a conserved feature of the metazoan SAC, since the relevant PLK1 and PP2A-B56 binding motifs have coevolved in the same region on MADBUB homologues.

Parallel siRNA screens to identify kinase and phosphatase modulators of NF-κB activity following DNA damage

DNA damage, such as that experienced by people undergoing chemotherapy, can directly activate NF-κB signalling which in turn can lead to resistance to genotoxic stress. NF-κB signalling is highly regulated by phosphorylation, but the enzymes required for these processes remain largely unknown. Identifying those enzymes responsible for regulating NF-κB activity may yield attractive targets for new clinical therapies, as well as provide the basis for better understanding of signalling network crosstalk. Here we present datasets from two independent RNAi screens using a stable NF-κB reporter U2OS cell line with the aim of identifying enzymes that alter NF-κB activity in response to DNA damage following etoposide and ionising radiation treatments. Although we observed high internal validity and specificity to NF-κB modulation within the screens, there was a striking dissimilarity between the results of the two different screens. These data therefore provide a cautionary lesson regarding the use of RNAi screening but also provide new candidates for kinase and phosphatase regulation of NF-κB activity in response to genotoxic stress.

Kinetochore phosphatases suppress autonomous kinase activity to control the spindle assembly checkpoint

Local phosphatase regulation is critical for determining when phosphorylation signals are activated or deactivated. A typical example is the spindle assembly checkpoint (SAC) during mitosis, which regulates kinetochore PP1 and PP2A-B56 activities to switch-off signalling events at the correct time. In this case, kinetochore phosphatase activation dephosphorylates MELT motifs on KNL1 to remove SAC proteins, including the BUB complex. We show here that, surprisingly, neither PP1 or PP2A are required to dephosphorylate the MELT motifs. Instead, they remove polo-like kinase 1 (PLK1) from the BUB complex, which can otherwise maintain MELT phosphorylation in an autocatalytic manner. This is their principle role in the SAC, because both phosphatases become redundant if PLK1 is inhibited or BUB-PLK1 interaction is prevented. Therefore, phosphatase regulation is critical for the SAC, but primarily to restrain and extinguish autonomous kinase activity. We propose that these circuits have evolved to generate a semi-autonomous SAC signal that can be synchronously silenced following kinetochore-microtubule tension.

Quantitative Proteomic Analysis of buffalo ovaries reveals the regulation of kinase and phosphatase during the Formation and Regression of Corpus Luteum

Background: Reproductive characteristics are made up of many complicated physiology procedures that have significant effects on the growth of the buffalo industry and are affected by numerous factors. The ovary is the most important sexual organ of female mammals, and during the oestrous cycles, its corpus luteum (CL) plays a significant part in mammalian reproduction. During the development and regression of corpus luteum, however, the differentially expressed proteins are less defined. In this study, we used a 6-plex tandem mass tag (TMT) strategy for quantitative proteomic comparison of three distinct ovary phases (corpus hemorrhagicum, corpus luteum and corpus hemorrhagicum) in buffalo. Results: A total of 148 differentially expressed proteins were identified, 32 of these proteins were identified as differentially expressed in the group CH (corpus hemorrhagicum) and 116 were identified as differentially expressed in the group CF (corpus fibrosum), with the group CL (corpus luteum) serving as the control group. Notably, we discovered that quite some enzymes such as kinase and phosphatase, are upregulated in the ovary CL phase, and three upregulated enzymes and proteins in the CL phase (PLK1, PGP, and HGS) were verified using Western blotting, quantitative RT-PCR, and immunohistochemistry analysis. The results of these validations were consistent with the quantitative analysis of the TMT-label, which indicated that they could play a crucial role during the CL's reproductive cycle. These buffalo results during the formation and regression of the corpus luteum are also shown to be a substantial reference value for comparable human and mouse studies after analyzing homologous BLAST cross-species. The expression information is accessible with the PXD009957 identifier via ProteomeXchange. Conclusions: Our research gives a deeper understanding of CL formation and regression during the oestrous cycles, which shows kinase and phosphatase regulation, and indicates some potential enzymes and proteins that may influence buffalo fertility. Keywords: corpus luteum, quantitative proteomic, oestrous cycles, ovary, cross-species