Major progress has been made in unravelling of regulatory mechanisms in eukaryotic cells. However, until today, only few in vivo substrates for few protein kinases have been identified in plants. Thus, the understanding of kinase machineries, signalling pathways and their wiring remains fragmented. In the yeast and animal fields, large-scale and high-throughput approaches to study complex signalling networks have been developed and led to improvement in plant-related technologies (1).
1. Aims and Scope.
Novel techniques in genomics, bioinformatics, and systems biology along with a growing number of fully annotated genomes of model and non-model plant species, such as for Arabidopsis, maize, rice, soybean, rape seed, tomato, grapevine, and others have led to a tremendous increase in sequence information. The novel genome information was translated into the putative proteomes setting the ground for a wealth in yet to be explored kinomes. This exploration is promoted by new developments in biochemical, molecular and cell biological techniques of which we provide a comprehensive and state-of-the-art overview in this methods book (Figure 1).
2. This Book.
The book Plant Kinases from the Methods in Molecular Biology series is intended to be a benchtop manual and guide for both those new to the techniques and also for established workers who wish to use a method for the first time. By providing molecular and cell biological, biochemical, spectroscopic, and proteomic methods with special emphasis on the plant field, this volume aims at scientists who desire to further test their results and link their data with a broad range of methodologies.
Since the major goal is to provide the experimentalist with a full account of the practical steps necessary for carrying out each protocol successfully, the Materials as well as the Methods sections contain detailed step-by-step descriptions of all procedures. Moreover, each chapter opens with an Introduction which describes the basic theory behind both the biology and the method being used to spotlight each technique in the context of a certain biological question. The final Notes section complements the material with invaluable hands-on comments peculiar to the particular method as suggested by experienced users.
3. The Chapters.
Plant Kinases is divided into four major sections comprising a total of 17 chapters: (I) an detailed introductory chapter where we review general structural and functional aspects of protein kinase function highlighting several regulatory signaling networks related to this methods book. In addition, we provide a collection of databases and web tools for plant kinase research. Part (II) covers expression, purification, activity monitoring and reporter gene assays of plant protein kinases; part (III) address the study of kinase function at the cellular level by chemical genetics, microscopy- and spectroscopy-based techniques; and part (V) provides information about the identification of kinase substrates and phosphorylation-site detection via interaction assays, substrate labelling protocols and proteomics.
The book contains several protocols in Chapter 3 and 4 that deal with protein expression and purification techniques, Chapter 4, 5, 6, 8, and 9 describe immunoprecipitation-and affinity-coupled kinase and phosphatase assays including inhibitor studies. Also reporter gene assays and promoter studies use of promoter -- reporter fusions belong to the standard repertoire not only in kinase research. In this book, we present various techniques to study kinase properties such as bacterial assays (Chapter 7) and classical in planta reporter gene assays (Chapter 5; Figure 1).
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4. Specific Applications for Biological Questions.
Due to the problematically low abundance of some kinases and substrates of interest (or transgenically engineered kinase variants), in vitro protein production in and purification from cell-free systems (reticulocyte and wheat germ lysates), bacterial and viral expression systems have been adapted to plant target proteins and also plant suspension cultures are used for protein production and determination of substrate preference.
Both this low abundance and stability of the modification by phosphorylation are the reasons for recent developments of techniques that either allow to 1) specifically mark substrates of kinase X amongst the pool of other kinases, to 2) specifically enrich phosphorylated polypeptides for further analysis, and to 3) show kinase-interactor or -substrate interactions directly in the cell in situ. These last three issues are also in the main focus of this methods book (Figure 1):
1) Recent advances make extensively use of inhibitor studies and chemical genetics combining mainly the use of ATP-analogous and -competitive molecules. These approaches involve mutant inhibitor-sensitive protein kinase variants replacing the wild type copy or those selectively accepting bulky non-standard ATP-derivatives and thus the label which has revealed many novel potential substrates (2, 3). These alleles were used to selectively inhibit kinases (4) or, important in terms of substrate screening, specifically mark substrates (e.g. (3) and many more; Figure 1).
In Plant Kinases, the Chapters 10 and 15 are focused on chemical genetics and explain both the application of ATP-competitive molecules and the engineering of inhibitor-sensitive protein kinase variants.
2) The preservation, detection and identification of the actual site(s) of phosphorylation, however, is experimentally difficult but fundamentally important for functional characterization of a kinase cascade. This issue still represents a major bottleneck. Therefore, large-scale strategies for both the isolation of plant phosphopeptides and their identification by mass spectrometry have been developed (5). Modifications of existing MS-based protocols such as Metal Oxide Affinity Chromatography (MOAC), isobaric Tags for Relative and Absolute Quantitation (iTRAQ), and Stable Isotope Labelling (SILAC) allow to further purify and enrich the phosphorylated target sequences for subsequent spectrometric analysis. Lab-on-a-chip methodologies such as kinome analysis and profiling with protein and peptide arrays or chips (6, 7) and phosphopeptide mapping (8) are en vogue and help to combine the experimental needs for both a high-troughput with the starting point of having a couple of hundreds of kinases and much more putative substrate sequences present. Furthermore, quantitative phosphoproteomic methods using antibody microarrays (9), gel-based approaches (10) and gel-free methods lead to the identification of novel in vivo phospho-sites (11-14). More recent advances in the field of MS-based detection of post-translational modification of plant proteins have been acquired not only in phosphoproteomics (15-17) but also on the role of protein phosphorylation in regulatory mechanisms (18).
In Plant Kinases, Chapters 16 and 17 solely spotlight on phosphoproteomics, dealing especially with current protocols such as MOAC, iTRAQ, and SILAC in the plant-specific context in combination with Combined with 2D-PAGE and MS-related technologies (Figure 1).
3) Highly sensitive microscopical techniques have been successfully applied to localize and quantify fluoescent fusion proteins by standard or Confocal Laser Scanning fluorescence Microscopy (CLSM) and to detect transient associations of proteins with each other by using Bimolecular Fluorescence Complementation (BiFC or "Split-YFP", described in Chapter 5 and 14), Förster/Fluorescence Resonance Energy Transfer (FRET), and Fluorescence Lifetime Imaging Microscopy (FLIM). These techniques were further specialized and new spectroscopical methods evolved such as Fluorescence Fluctuation Analysis (FFA), Fluorescence Correlation or Cross-Correlation Spectroscopy (FCS/FCCS), Photon Counting Histogram Analysis (PCH), and Fluorescence Recovery after Photobleaching (FRAP). All these techniques are capable to get closer to explanatins on pair-wise interactions, i.e. if and how proteins interact under different experimental conditions (Figure 1).
In Plant Kinases, Chapters 11, 12, and 13 put special emphasis on the explanation of fluorescence microscopy-based applications.
