Reverse protein phosphorylation is a highly conserved mechanism for post-transla-tional regulation of protein function, which has been found in all prokaryotes and eukaryotes examined (see also Chapter 29). Phosphorylation is mediated by protein kinases, which transfer the y-phosphate of ATP on amino acid residues of proteins, and can be reversed by protein phosphatases. The phosphorylation state of a protein has often profound effects on its activity, stability, structure, and interaction with other proteins or biomolecules . Virtually all cellular processes are regulated in one or multiple ways by phosphorylation and dephosphorylation. Phosphorylation plays a central role, especially in cell signaling in diverse organisms including plants . The phosphorylation of proteins in plants has been found to be connected with the reaction of the organism toward different internal and external factors, such as light, invasion of pathogens, hormones, temperature stress, and shortage of nutrition .
Current annotation of protein families in Arabidopsis predicts approximately 1053 kinases, representing about 4% of all Arabidopsis proteins (Pfam; http://www.sanger. ac.uk/cgibin/Pfam/genome_view,pl?ncbi=3702) , In contrast, the human genome with approximately the same number of genes as that of Arabidopsis is estimated to encode for approximately 500 protein kinases, accounting for only almost 2.0% of its genome [5, 6]. Why do plants need a comparable high number of protein kinases? Analysis of these kinases, knowing which kinases are activated during a biological response and identifying their specific substrates, is important for a better understanding of the regulation of a wide range of biological processes in plants and to increase our knowledge of what makes plants different from other organisms.
Despite recent advances that have been made in phosphoproteomics  (see Chapter 29), we know very little about kinase activity pattern, substrates, and the elements mediating specificity in plants, compared to the status in mammals. Therefore, the assignment of phosphoproteins to specific kinases and the search for novel substrates of plant kinases are great challenges.
Several criteria for establishing that a protein is a substrate of a given kinase were defined by Berwick and Tavare  comprising the ability of the kinase to phos-phorylate the substrate in vitro and the dependence of substrate phosphorylation on kinase activity in vivo. In a recent paper, Peck  described very detailed strategies for monitoring kinase activity, investigating kinase-substrate specificity, examining phosphorylation in planta and determining phosphorylation sites in a protein. It is underlined that it is very important to establish, by different assays and strategies, links in the following chain of a signal transduction: (i) environmental or internal triggers (e.g., specific abiotic or biotic stresses), (ii) activation of a kinase, (iii) specific phosphorylation of a substrate by the kinase, and (iv) specific cellular response. To demonstrate that a kinase is involved in a signal transduction, it is essential to demonstrate that the activity of the kinase increases during the biological response. One way is to immunoprecipitate the kinase from plant cell extracts, preferably using a specific antibody against the native kinase. Then the activity of the immunoprecip-itated kinase is assayed using a reporter protein—for example, a "generic substrate" such as MBP. Once a kinase has been determined to be activated during a response, the hunt begins for substrates of this kinase . Because the identification of kinase substrates in vivo continues to be laborious and time-consuming, it is more efficient to identify substrate candidates in an efficient manner in vitro before proceeding to the in vivo step. In vitro-identified substrate candidates and their phosphorylation sites have to be confirmed in vivo. Different in vitro and in vivo methods and strategies used in search for kinase substrates, their increasing application in the plant field, and the obtained results will be reviewed in this chapter.
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