Enzyme–substrate interactions are weak and occur only transiently and thus, a faithful analysis of these interactions typically requires elaborated biochemical methodology. The bimolecular-fluorescence complementation (BiFC) assay, also referred to as split YFP assay, is a powerful and straightforward tool to test protein–protein interactions. This system is commonly used due to many advantages and especially due to its simple ease of use. BIFC relies on the reconstitution of an N-terminal and C-terminal half of YFP into a functional, i.e., fluorescent protein. Noteworthy, the dissociation constant of the two YFP halves is much lower than the association constant leading to a stabilization of the protein–protein interaction to be monitored. Whereas this property is sometimes critical, it also increases the sensitivity of the detection system by stabilizing transient interactions. Here, we exploit this property to detect and monitor interaction between a kinase and its substrate. In particular, we characterize with the BiFC system kinase-variants that show an altered substrate binding.

Current standard cloning methods based on the use of restriction enzymes and ligase are very versatile, but are not well suited for high-throughput cloning projects or for assembly of many DNA fragments from several parental plasmids in a single step. We have previously reported the development of an efficient cloning method based on the use of type IIs restriction enzymes and restriction–ligation. Such method allows seamless assembly of multiple fragments from several parental plasmids with high efficiency, and also allows performing DNA shuffling if fragments prepared from several homologous genes are assembled together in a single restriction–ligation. Such protocol, called Golden Gate shuffling, requires performing the following steps: (1) sequences from several homologous genes are aligned, and recombination sites defined on conserved sequences; (2) modules defined by the position of these recombination sites are amplified by PCR with primers designed to equip them with flanking BsaI sites; (3) the amplified fragments are cloned as intermediate constructs and sequenced; and (4) finally, the intermediate modules are assembled together in a compatible recipient vector in a one-pot restriction–ligation. Depending on the needs of the user, and because of the high cloning efficiency, the resulting constructs can either be screened and analyzed individually, or, if required in larger numbers, directly used in functional screens to detect improved protein variants.

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.

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.

The growing number of fully annotated genomes of model and nonmodel plant species, such as Arabidopsis, Brachypodium, ­grapevine, maize, rice, rape seed, soybean, tomato, and others, has led to a tremendous increase in sequence information. The novel genome information has been translated into the putative proteomes and set the ground for a wealth of yet to be explored kinomes. At the same time, cellular pathways are now analyzed in model plants with an unprecedented depth revealing new ­mechanisms of regulation and integration of individual modules into regulatory networks. These studies are promoted by new developments in biochemical, molecular, and cell biological techniques of which we provide a comprehensive and state-of-the-art overview in the present methods book (Fig. 1).