Use of Phospho-Site Substitutions to Analyze the Biological Relevance of Phosphorylation Events in Regulatory Networks
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
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Introduction Modulation of protein activities through phosphorylation and de-phosphorylation is a recurrent theme in most if not all regulatory cascades. Typically, this modulation is repeated such as protein
N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_6, © Springer Science+Business Media, LLC 2011
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N. Dissmeyer and A. Schnittger
A phosphorylates protein B, which becomes activated by this and subsequently phosphorylates protein C (a well-studied example are MAP kinase cascades, see also Chapters 2, 4, 7, 8, 9, and 14). The consecutive change in protein activities serves many purposes. Among others, it allows information to be transmitted from one compartment, e.g., from the cell membrane to the nucleus. In addition, the initial information becomes amplified since one upstream kinase might activate several downstream kinases. Moreover, especially long cascades allow the integration of several information pathways and different inputs can be compared with each other, resulting in a fine-tuned response. These cascades in turn might be coupled to threshold levels of protein activities, resulting in very sensitive biological switches. A paradigm for such a wiring of phosphorylation cascades is cell cycle regulation in which extrinsic (environmental) cues such as temperature or light are integrated with intrinsic (developmental) information such as tissue or organ cues. 1.1. Regulation of CDK Activity During Cell Cycle Progression by Phosphorylation
The prototypical mitotic cell cycle in plants and in other eukaryotes is composed of four phases: DNA synthesis or S phase when replication events take place, M phase or mitosis when chromosomes are separated and cells subsequently divide, and gap phases G1 (after M) and G2 (after S), in which most of the cell-physiological processes take place. The progression through the mitotic cell cycle is regulated by the protein kinase activity of a heterodimeric complex formed by a substrate-specific cyclin protein and a catalytically active cyclin-dependent kinase (Cdk) subunit. The small 34-kDa serine/threonine protein kinase in yeast designated p34, Cdc2+ (Schizosaccharomyces pombe), and CDC28p (Saccharomyces cerevisiae), or the homologous cyclin-dependent kinase 1 (Cdk1) in humans is a key regulator of the eukaryotic cell cycle, and controls entry into S- and M-phase (for a general review, see (1)). Similarly, the Arabidopsis homolog of Cdk1, designated CDKA;1, was found to be involved in the regulation of both cell cycle checkpoints (2, 3). Studies in yeast and human cell cultures have demonstrated that monomeric CDKs have no detectable activity and need to bind their cyclin cofactors (1). Different cyclins can form complexes with CDKs that are implicated at specific cell cycle phases such as, for instance, B-type cyclins for mitosis. CDK activity is also negatively regulated by cofactors, called CDK inhibitors (CKIs) that are – like cyclins – themselves under strict
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