A Multimodel Study of the Role of Novel PKC Isoforms in the DNA Integrity Checkpoint.

The protein kinase C (PKC) family plays important regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whereas in mammals, the PKC family comprises nine isoforms. Both Pkc1 and the novel isoform PKCδ are involved in the control of DNA integrity checkpoint activation, demonstrating that this mechanism is conserved from yeast to mammals. To explore the function of PKCδ in a non-tumor cell line, we employed CRISPR-Cas9 technology to obtain PKCδ knocked-out mouse embryonic stem cells (mESCs). This model demonstrated that the absence of PKCδ reduced the activation of the effector kinase CHK1, although it suggested that other isoform(s) might contribute to this function. Therefore, we used yeast to study the ability of each single PKC isoform to activate the DNA integrity checkpoint. Our analysis identified that PKCθ, the closest isoform to PKCδ, was also able to perform this function, although with less efficiency. Then, by generating truncated and mutant versions in key residues, we uncovered differences between the activation mechanisms of PKCδ and PKCθ and identified their essential domains. Our work strongly supports the role of PKC as a key player in the DNA integrity checkpoint pathway and highlights the advantages of combining distinct research models.

The CWI Pathway: A Versatile Toolbox to Arrest Cell-Cycle Progression.

Cell-signaling pathways are essential for cells to respond and adapt to changes in their environmental conditions. The cell-wall integrity (CWI) pathway of Saccharomyces cerevisiae is activated by environmental stresses, compounds, and morphogenetic processes that compromise the cell wall, orchestrating the appropriate cellular response to cope with these adverse conditions. During cell-cycle progression, the CWI pathway is activated in periods of polarized growth, such as budding or cytokinesis, regulating cell-wall biosynthesis and the actin cytoskeleton. Importantly, accumulated evidence has indicated a reciprocal regulation of the cell-cycle regulatory system by the CWI pathway. In this paper, we describe how the CWI pathway regulates the main cell-cycle transitions in response to cell-surface perturbance to delay cell-cycle progression. In particular, it affects the Start transcriptional program and the initiation of DNA replication at the G1/S transition, and entry and progression through mitosis. We also describe the involvement of the CWI pathway in the response to genotoxic stress and its connection with the DNA integrity checkpoint, the mechanism that ensures the correct transmission of genetic material and cell survival. Thus, the CWI pathway emerges as a master brake that stops cell-cycle progression when cells are coping with distinct unfavorable conditions.

The budding yeast Start repressor Whi7 differs in regulation from Whi5, emerging as a major cell cycle brake in response to stress.

Start is the main decision point in the eukaryotic cell cycle at which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional programme through the inactivation of Start transcriptional repressors: the retinoblastoma family in mammals, or Whi5 and its recently identified paralogue Whi7 (also known as Srl3) in budding yeast. Here, we provide a comprehensive comparison of Whi5 and Whi7 that reveals significant qualitative differences. Indeed, the expression, subcellular localization and functionality of Whi7 and Whi5 are differentially regulated. Importantly, Whi7 shows specific properties in its association with promoters not shared by Whi5, and for the first time, we demonstrate that Whi7, and not Whi5, can be the main contributor to Start inhibition such as it occurs in the response to cell wall stress. Our results help to improve understanding of the interplay between multiple differentially regulated Start repressors in order to face specific cellular conditions.

Genome wide DNA methylation profiling identifies specific epigenetic features in high-risk cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. Although most cSCCs have good prognosis, a subgroup of high-risk cSCC has a higher frequency of recurrence and mortality. Therefore, the identification of molecular risk factors associated with this aggressive subtype is of major interest. In this work we carried out a global-scale approach to investigate the DNA-methylation profile in patients at different stages, from premalignant actinic keratosis to low-risk invasive and high-risk non-metastatic and metastatic cSCC. The results showed massive non-sequential changes in DNA-methylome and identified a minimal methylation signature that discriminates between stages. Importantly, a direct comparison of low-risk and high-risk stages revealed epigenetic traits characteristic of high-risk tumours. Finally, a prognostic prediction model in cSCC patients identified a methylation signature able to predict the overall survival of patients. Thus, the analysis of DNA-methylation in cSCC revealed changes during the evolution of the disease through the different stages that can be of great value not only in the diagnosis but also in the prognosis of the disease.

Karyopherin Msn5 is involved in a novel mechanism controlling the cellular level of cell cycle regulators Cln2 and Swi5.

The yeast ß-karyopherin Msn5 controls the SBF cell-cycle transcription factor, responsible for the periodic expression of CLN2 cyclin gene at G1/S, and the nuclear export of Cln2 protein. Here we show that Msn5 regulates Cln2 by an additional mechanism. Inactivation of Msn5 causes a severe reduction in the cellular content of Cln2. This occurs by a post-transcriptional mechanism, since CLN2 mRNA level is not importantly affected in asynchronous cultures. Cln2 stability is not significantly altered in msn5 cells and inactivation of Msn5 causes a reduction in protein level even when Cln2 is stabilized. Therefore, the reduced amount of Cln2 in msn5 cells is mainly due not to a higher rate of protein degradation but to a defect in Cln2 synthesis. In fact, analysis of polysome profiles indicated that Msn5 inactivation causes a shift of CLN2 and SWI5 mRNAs from heavy-polysomal to light-polysomal and non-polysomal fractions, supporting a defect in Cln2 and Swi5 protein synthesis in the msn5 mutant. The analysis of truncated versions of Cln2 and of chimeric cyclins combining distinct domains from Cln2 and the related Cln1 cyclin identified an internal region in Cln2 from 181 to 225 residues that when fused to GFP is able to confer Msn5-dependent regulation of protein cellular content. Finally, we showed that a high level of Cln2 is toxic in the absence of Msn5. In summary, we described that Msn5 is required for the proper protein synthesis of specific proteins, introducing a new level of control of cell cycle regulators.

Periodic expression of cell-cycle regulators: A laboratory experiment proposal for students in molecular and cell biology.

This article describes a laboratory exercise designed for undergraduate students in the subject of "Regulation of cell proliferation" which allows the students to carry out a research experiment in an important field such as cell cycle control, and to be introduced to a widely used technique in molecular biology laboratories such as the western blot. The cell cycle is regulated by the succession of cyclin-CDK kinase activities. Activation and inactivation of different cyclin-CDK complexes depend on the control of their positive and negative regulators, cyclins and CDK inhibitors (CKIs), respectively. In this experiment, fluctuations in the level of mitotic cyclin Clb2 and CDK inhibitor Sic1 throughout the cell cycle of Saccharomyces cerevisiae are analyzed, particularly in the context of the control of mitotic exit and Start, two of the most important cell cycle transitions. In order to do this, a cdc15 mutant strain is used to block cells in telophase and, upon release from this blocking, the variation in the levels of Clb2 and Sic1 proteins are analyzed by western blot. Progress along the cell cycle is also evaluated by microscopic analysis of cell morphology and nuclear staining. This practical illustrates the experimental basis of theoretical concepts worked in the classroom and it is a good framework for an in-depth discussion of these concepts based on experimental data analysis. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):527-535, 2018.

Whi7 is an unstable cell-cycle repressor of the Start transcriptional program.

Start is the main decision point in eukaryotic cell cycle in which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional program by G1 CDK-cyclin complexes through the inactivation of Start transcriptional repressors, Whi5 in yeast or Rb in mammals. Here we provide novel keys of how Whi7, a protein related at sequence level to Whi5, represses Start. Whi7 is an unstable protein, degraded by the SCFGrr1 ubiquitin-ligase, whose stability is cell cycle regulated by CDK1 phosphorylation. Importantly, Whi7 associates to G1/S gene promoters in late G1 acting as a repressor of SBF-dependent transcription. Our results demonstrate that Whi7 is a genuine paralog of Whi5. In fact, both proteins collaborate in Start repression bringing to light that yeast cells, as occurs in mammalian cells, rely on the combined action of multiple transcriptional repressors to block Start transition.The commitment of cells to a new cycle of division involves inactivation of the Start transcriptional repressor Whi5. Here the authors show that the sequence related protein Whi7 associates to G1/S gene promoters in late G1 and collaborates with Whi5 in Start repression.

Chimeric proteins tagged with specific 3xHA cassettes may present instability and functional problems.

Epitope-tagging of proteins has become a widespread technique for the analysis of protein function, protein interactions and protein localization among others. Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo. Different systems have been developed during years in the yeast Saccharomyces cerevisiae. In the present study, we analysed systematically a set of yeast proteins that were fused to different tags. Analysis of the tagged proteins revealed an unexpected general effect on protein level when some specific tagging module was used. This was due in all cases to a destabilization of the proteins and caused a reduced protein activity in the cell that was only apparent in particular conditions. Therefore, an extremely cautious approach is required when using this strategy.

A comparative study of the degradation of yeast cyclins Cln1 and Cln2.

The yeast cyclins Cln1 and Cln2 are very similar in both sequence and function, but some differences in their functionality and localization have been recently described. The control of Cln1 and Cln2 cellular levels is crucial for proper cell cycle initiation. In this work, we analyzed the degradation patterns of Cln1 and Cln2 in order to further investigate the possible differences between them. Both cyclins show the same half-life but, while Cln2 degradation depends on ubiquitin ligases SCFGrr1 and SCFCdc4, Cln1 is affected only by SCFGrr1. Degradation analysis of chimeric cyclins, constructed by combining fragments from Cln1 and Cln2, identifies the N-terminal sequence of the proteins as responsible of the cyclin degradation pattern. In particular, the N-terminal region of Cln2 is required to mediate degradation by SCFCdc4. This region is involved in nuclear import of Cln1 and Cln2, which suggests that differences in degradation may be due to differences in localization. Moreover, a comparison of the cyclins that differ only in the presence of the Cln2 nuclear export signal indicates a greater instability of exported cyclins, thus reinforcing the idea that cyclin stability is influenced by their localization.

Protein kinase C controls activation of the DNA integrity checkpoint.

The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.

Molecular basis of the functional distinction between Cln1 and Cln2 cyclins.

Cln1 and Cln2 are very similar but not identical cyclins. In this work, we tried to describe the molecular basis of the functional distinction between Cln1 and Cln2. We constructed chimeric cyclins containing different fragments of Cln1 and Cln2 and performed several functional analysis that make it possible to distinguish between Cln1 or Cln2. We identified that region between amino acids 225 and 299 of Cln2 is not only necessary but also sufficient to confer Cln2 specific functionality compared with Cln1. We also studied Cln1 and Cln2 subcellular localization identifying additional differences between them. Both cyclins are distributed between the nucleus and the cytoplasm, but Cln1 shows stronger nuclear accumulation. Nuclear import of both cyclins is mediated by the classical nuclear import pathway and by sequences in the N-terminal end of the proteins. For Cln2, but not for Cln1, a nuclear export mechanism mediated by karyopherin Msn5 has been identified. Strikingly, Cln2 export depends on a Msn5-dependent NES between amino acids 225 and 299. In fact, the introduction of this region confers to Cln1 an export mechanism dependent on Msn5; importantly, this causes the gain of Cln2-specific cytosolic functions and the impairment of nuclear function. In short, a region from Cln2 controlling an Msn5-dependent nuclear export mechanism confers a specific functionality to Cln2 compared with Cln1.

Regulation of cell cycle transcription factor Swi5 by karyopherin Msn5.

Inactivation of S. cerevisiae ß-karyopherin Msn5 causes hypersensitivity to the overexpression of mitotic cyclin Clb2 and aggravates growth defects of many mutant strains in mitotic exit, suggesting a connection between Msn5 and mitotic exit. We determined that Msn5 controlled subcellular localization of the mitotic exit transcription factor Swi5, since it was required for Swi5 nuclear export. Msn5 physically interacted with the N-terminal end of Swi5. Inactivation of Msn5 caused a severe reduction in cellular levels of Swi5 protein. This effect occurred by a post-transcriptional mechanism, since SWI5 mRNA levels were not affected. The reduced amount of Swi5 in msn5 mutant cells was not due to an increased protein degradation rate, but to a defect in Swi5 synthesis. Despite the change in localization and protein level, Swi5-regulated transcription was not defective in the msn5 mutant strain. However, a high level of Swi5 was toxic in the absence of Msn5. This deleterious effect was eliminated when Swi5 nuclear import was abrogated, suggesting that nuclear export by Msn5 is important for cell physiology, because it prevents toxic Swi5 nuclear accumulation.

Yeast karyopherins Kap123 and Kap95 are related to the function of the cell integrity pathway.

The characterization of mutant strains in the gene encoding karyopherin Kap123 has revealed several morphogenetic defects. Inactivation of KAP123 caused alterations in the actin cytoskeleton, resulting in hyperpolarization and resistance to the actin polymerization inhibitor latrunculin B. In fact, the level of actin filaments is increased in kap123 mutant cells. In addition to the defect in actin cytoskeleton, the kap123 mutant cells showed a weakened cell wall, cell lysis and a growth defect in either the presence of sodium dodecyl sulfate or at high temperatures, which is alleviated by osmotic stabilizers. These defects in cell integrity and the actin cytoskeleton suggested a relationship with the protein kinase C (PKC) cell integrity pathway. Slt2, the mitogen-activated protein kinase of the PKC cell integrity pathway, is constitutively activated in the absence of Kap123, which is consistent with the existence of cell integrity defects. Analysis of the subcellular localization of nuclear proteins involved in cell wall gene expression indicated that the localization of the Slt2 kinase and the transcription factors Rlm1, Swi6 and Paf1 was not affected by Kap123. Finally, we identified karyopherin Kap95 as the transport factor responsible for the nuclear import of Slt2 and Rlm1.