A Microarray Strategy for Mapping the Substrate Specificity of Protein Tyrosine Phosphatase

Köhn, Maja; Gutiérrez-Rodríguez, Marta; Jonkheijm, Pascal; Wetzel, Stefan; Wacker, Ron; Schroeder, Hendrik; Prinz, Heino; Niemeyer, Christof M.; Breinbauer, Rolf; Szedlacsek, Stefan E.; Waldmann, Herbert

doi:10.1002/anie.200701601

Cited by 75 publications

(47 citation statements)

References 28 publications

(22 reference statements)

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“…Because of its versatility, sensitivity, and the possibility of measuring phosphate release in real time, we think the MDCC-PBP sensor offers technical advantages over techniques that rely on phosphotyrosine-specific antibody (Kohn et al, 2007) or ProQDiamond (Sun et al, 2008) for the calculation of peptide dephosphorylation speed in high-throughput strategies. Furthermore, if necessary, the resolution and versatility of the strategy presented here can be increased using a single amino acid at each Z-scanning position and by designing a similar phosphotyrosinebased peptide library to study phosphotyrosine and dualspecific phosphatases.…”

Section: Discussionmentioning

confidence: 99%

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid inArabidopsis

et al. 2009

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The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.

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Section: Discussionmentioning

confidence: 99%

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid inArabidopsis

et al. 2009

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“…In general, each assay suitable for kinase profiling on peptide (micro)arrays could be used for analysis of phosphatase-mediated release of phosphate moieties from phosphopeptides, too. Surface-bound phosphopeptides could be generated either enzymatically [101,157] or chemically [75,101,[157][158][159][160][161]. Detection of phosphatase action on (micro)array bound phosphopeptides by signal decrease subsequent to treatment with anti-phosphotyrosine antibodies [101,159,160] or using phospho-specific dyes [75] was demonstrated.…”

Section: Assays and Detectionmentioning

confidence: 99%

Deciphering Enzyme Function Using Peptide Arrays

2011

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Enzymes are key molecules in signal-transduction pathways. However, only a small fraction of more than 500 human kinases, 300 human proteases and 200 human phosphatases is characterised so far. Peptide microarray based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimisation of enzyme substrates. Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases. Additionally, several examples for reliable identification of substrates for lysine methyl-transferases, histone deacetylases and SUMO-transferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples like cell lysates or lysates of complete organisms is described. All published examples of peptide arrays used for enzyme profiling are summarised comprehensively.

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“…IR spectra were recorded on a Nicolet 550 series II spectrometer. 1 H, 19 F, and 13 C NMR were recorded using a Bruker Avance 400 spectrometer. The proton and carbon chemical shifts are given in parts per million using CDCl 3 (d H 7.24 and 77.0) as internal standard.…”

Section: Synthesis Of the Orthogonally Protected Dipeptide Intermediamentioning

confidence: 99%

Development of activity-based probes with tunable specificity for protein tyrosine phosphatase subfamilies

et al. 2010

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A Microarray Strategy for Mapping the Substrate Specificity of Protein Tyrosine Phosphatase

Cited by 75 publications

References 28 publications

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid inArabidopsis

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid inArabidopsis

Deciphering Enzyme Function Using Peptide Arrays

Development of activity-based probes with tunable specificity for protein tyrosine phosphatase subfamilies

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