Substrate Analysis of Arabidopsis PP2C-Type Protein Phosphatases
Protein phosphorylation by protein kinases can be reversed by the action of protein phosphatases. In plants, the Ser/Thr-specific phosphatases dominate among the protein phosphatase families with the type 2C protein phosphatases (PP2Cs) being the most abu
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. Introduction Reversible protein phosphorylation, performed by protein kinases (PKs) and protein phosphatases (PPs), is one of the most common mechanisms to control protein function and thereby regulating 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_8, © Springer Science+Business Media, LLC 2011
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multiple processes in plants. Phosphorylation, performed by PKs, can be reversed by the action of PPs, leading to the dephosphorylation of substrate proteins. A variety of proteins can be targets for the reversible protein phosphorylation, including protein kinases themselves. PKs are represented by 1,126 members in Arabidopsis and 1,496 in rice (Oryza sativa) according to the Kinomer v. 1.0 database (http://www.compbio.dundee.ac.uk/kinomer/) (1) and the Rice Kinase Database (http://rkd.ucdavis.edu/) (2), respectively (see Note 1). Interestingly, PPs are represented only by 150 members in Arabidopsis (3, 4) and 194 phosphatase loci in rice (Rice Genome Annotation; see Note 1; (2, 5)). The discrepancy in gene numbers between kinases and phosphatases might create the impression that PPs are “pleiotropic” enzymes that would dephosphorylate protein substrates unspecifically. However, physiological studies have shown that different stimuli modulate a subset of phosphorylation events to regulate selected aspects of mammalian cell physiology (6) and plant PP2Cs show specificity in substrate recognition and dephosphorylation (7, 8). Our results demonstrate that even members from the same phosphatase family retain strict specificity toward the protein substrate in vivo and in vitro (7, 8), underlining the applicability for testing enzyme specificity already by in vitro assays. The specificity toward the protein substrate may be attained through gene expression, protein localization, regulatory proteins, and specific domains in protein structure. According to the structure and specificity to substrate phospho-amino acids (phosphoserine, phosphothreonine, and phosphotyrosine), eukaryotic PPs are classified into several families (9, 10). Protein tyrosine phosphatases (PTPs) dephosphorylate phosphotyrosine (tyrosine-specific PTPs) or all three amino acids (dual specificity protein phosphatases; DSPs), whereas phosphoprotein phosphatase (PPP), metallodependent protein phosphatase (PPM), and aspartate-based phosphatases (FCP-like and HAD-family phosphatases) dephosphorylate phosphoserine and phosphothreonine residues. PPP and PPM families represent the majority of eukaryotic serine/threonine phosphatases. They contain structurally similar protein catalytic sites, but the sequence divergence suggests that these families evolved independently (4). The PPM family is represented by the type 2C PPs (PP2Cs) that act as monomers, require Mn2+ or Mg2+ for their activity, and are insensitive to known phosphatase inhibitors, including okadaic acid (OA). Recently, compounds inhibiting the mammalian PP2Ca in vitro at
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