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Publikation

Wijnker, E.; Harashima, H.; Müller, K.; Parra-Nuñez, P.; de Snoo, C. B.; van de Belt, J.; Rajjou, L.; Bayer, M.; Pradillo, M.; Schnittger, A.; The Cdk1/Cdk2 homolog CDKA;1 controls the recombination landscape in Arabidopsis Proc. Natl. Acad. Sci. U.S.A. 116, 12534-12539, (2019) DOI: 10.1073/pnas.1820753116

Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in Arabidopsis. Arabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.
Publikation

Faden, F.; Ramezani, T.; Mielke, S.; Almudi, I.; Nairz, K.; Froehlich, M. S.; Höckendorff, J.; Brandt, W.; Hoehenwarter, W.; Dohmen, R. J.; Schnittger, A.; Dissmeyer, N.; Phenotypes on demand via switchable target protein degradation in multicellular organisms Nat. Commun. 7, 12202, (2016) DOI: 10.1038/ncomms12202

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation.
Publikation

Weimer, A. K.; Nowack, M. K.; Bouyer, D.; Zhao, X.; Harashima, H.; Naseer, S.; De Winter, F.; Dissmeyer, N.; Geldner, N.; Schnittger, A.; RETINOBLASTOMA RELATED1 Regulates Asymmetric Cell Divisions in Arabidopsis Plant Cell 24, 4083-4095, (2012) DOI: 10.1105/tpc.112.104620

Formative, also called asymmetric, cell divisions produce daughter cells with different identities. Like other divisions, formative divisions rely first of all on the cell cycle machinery with centrally acting cyclin-dependent kinases (CDKs) and their cyclin partners to control progression through the cell cycle. However, it is still largely obscure how developmental cues are translated at the cellular level to promote asymmetric divisions. Here, we show that formative divisions in the shoot and root of the flowering plant Arabidopsisthaliana are controlled by a common mechanism that relies on the activity level of the Cdk1 homolog CDKA;1, with medium levels being sufficient for symmetric divisions but high levels being required for formative divisions. We reveal that the function of CDKA;1 in asymmetric cell divisions operates through a transcriptional regulation system that is mediated by the Arabidopsis Retinoblastoma homolog RBR1. RBR1 regulates not only cell cycle genes, but also, independent of the cell cycle transcription factor E2F, genes required for formative divisions and cell fate acquisition, thus directly linking cell proliferation with differentiation. This mechanism allows the implementation of spatial information, in the form of high kinase activity, with intracellular gating of developmental decisions.
Bücher und Buchkapitel

Dissmeyer, N.; Schnittger, A.; The Age of Protein Kinases (Dissmeyer, N. & Schnittger, A., eds.). Methods Mol. Biol. 779, 7-52, (2011) ISBN: 978-1-61779-264-9 DOI: 10.1007/978-1-61779-264-9_2

Major progress has been made in unravelling of regulatory mechanisms in eukaryotic cells. Modification of target protein properties by reversible phosphorylation events has been found to be one of the most prominent cellular control processes in all organisms. The phospho-status of a protein is dynamically controlled by protein kinases and counteracting phosphatases. Therefore, monitoring of kinase and phosphatase activities, identification of specific phosphorylation sites, and assessment of their functional significance are of crucial importance to understand development and homeostasis. Recent advances in the area of molecular biology and biochemistry, for instance, mass spectrometry-based phosphoproteomics or fluorescence spectroscopical methods, open new possibilities to reach an unprecidented depth and a proteome-wide understanding of phosphorylation processes in plants and other species. In addition, the growing number of model species allows now deepening evolutionary insights into signal transduction cascades and the use of kinase/phosphatase systems. Thus, this is the age where we move from an understanding of the structure and function of individual protein modules to insights how these proteins are organized into pathways and networks. In this introductory chapter, we briefly review general definitions, methodology, and current concepts of the molecular mechanisms of protein kinase function as a foundation for this methods book. We briefly review biochemistry and structural biology of kinases and provide selected examples for the role of kinases in biological systems.
Bücher und Buchkapitel

Dissmeyer, N.; Schnittger, A.; Use of Phospho-Site Substitutions to Analyze the Biological Relevance of Phosphorylation Events in Regulatory Networks (Dissmeyer, N. & Schnittger, A., eds.). Methods Mol. Biol. 779, 93-138, (2011) ISBN: 978-1-61779-264-9 DOI: 10.1007/978-1-61779-264-9_6

Biological information is often transmitted by phosphorylation cascades. However, the biological relevance of specific phosphorylation events is often difficult to determine. An invaluable tool to study the effect of kinases and/or phosphatases is the use of phospho- and dephospho-mimetic substitutions in the respective target proteins. Here, we present a generally applicable procedure of how to design, set-up, and carry out phosphorylation modulation experiments and subsequent monitoring of protein activities, taking ­cyclin-dependent kinases (CDKs) as a case study. CDKs are key regulators of cell cycle progression in all eukaryotic cells. Consequently, CDKs are controlled at many levels and phosphorylation of CDKs ­themselves is used to regulate their kinase activity. We describe in detail complementation experiments of a mutant in CDKA;1, the major cell cycle kinase in Arabidopsis, with phosphorylation-site variants of CDKA;1. CDKA;1 versions were generated either by mimicking a phosphorylated amino acid by replacing the respective residue with a negatively charged amino acid, e.g., aspartate or glutamate, or by mutating it to a non-phoshorylatable amino acid, such as alanine, valine, or phenylalanine. The genetic complementation studies were accompanied by the isolation of these kinase variants from plant extract and subsequent kinase assays to determine changes in their activity levels. This work allowed us to judge the importance of ­posttranslational regulation of CDKA;1 in plants and has shown that the molecular mechanistics of CDK function are apparently conserved across the kingdoms. However, the regulatory wiring of CDKs is ­strikingly different between plants, animals, and yeast.
Publikation

Dissmeyer, N.; Weimer, A. K.; De Veylder, L.; Novak, B.; Schnittger, A.; The regulatory network of cell-cycle progression is fundamentally different in plants versus yeast or metazoans Plant Signal Behav. 5, 1613-1618, (2010) DOI: 10.4161/psb.5.12.13969

Plant growth and proliferation control is coming into a global focus due to recent ecological and economical developments. Plants represent not only the largest food supply for mankind but also may serve as a global source of renewable energies. However, plant breeding has to accomplish a tremendous boost in yield to match the growing demand of a still rapidly increasing human population. Moreover, breeding has to adjust to changing environmental conditions, in particular increased drought. Regulation of cell-cycle control is a major determinant of plant growth and therefore an obvious target for plant breeding. Furthermore, cell-cycle control is also crucial for the DNA damage response, for instance upon irradiation. Thus, an in-depth understanding of plant cell-cycle regulation is of importance beyond a scientific point of view. The mere presence of many conserved core cell-cycle regulators, e.g. CDKs, cyclins, or CDK inhibitors, has formed the idea that the cell cycle in plants is exactly or at least very similarly controlled as in yeast or human cells. Here together with a recent publication we demonstrate that this dogma is not true and show that the control of entry into mitosis is fundamentally different in plants versus yeast or metazoans. Our findings build an important base for the understanding and ultimate modulation of plant growth not only during unperturbed but also under harsh environmental conditions.
Publikation

Dissmeyer, N.; Nowack, M. K.; Pusch, S.; Stals, H.; Inze, D.; Grini, P. E.; Schnittger, A.; T-Loop Phosphorylation of Arabidopsis CDKA;1 Is Required for Its Function and Can Be Partially Substituted by an Aspartate Residue Plant Cell 19, 972-985, (2007) DOI: 10.1105/tpc.107.050401

As in other eukaryotes, progression through the cell cycle in plants is governed by cyclin-dependent kinases. Phosphorylation of a canonical Thr residue in the T-loop of the kinases is required for high enzyme activity in animals and yeast. We show that the Arabidopsis thaliana Cdc2+/Cdc28 homolog CDKA;1 is also phosphorylated in the T-loop and that phosphorylation at the conserved Thr-161 residue is essential for its function. A phospho-mimicry T161D substitution restored the primary defect of cdka;1 mutants, and although the T161D substitution displayed a dramatically reduced kinase activity with a compromised ability to bind substrates, homozygous mutant plants were recovered. The rescue by the T161D substitution, however, was not complete, and the resulting plants displayed various developmental abnormalities. For instance, even though flowers were formed, these plants were completely sterile as a result of a failure of the meiotic program, indicating that different requirements for CDKA;1 function are needed during plant development.
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