A 10,000 Member PNA-Encoded Peptide Library for Profiling Tyrosine Kinases

Pouchain, Delphine; Díaz‐Mochón, Juan José; Bialy, Laurent; Bradley, Mark

doi:10.1021/cb700199k

Cited by 33 publications

(19 citation statements)

References 55 publications

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“…Accordingly, much effort has been directed toward identifying these phosphorylation site sequence motifs by peptide library screening (3,41,(43)(44)(45)(46). However, short amino acid sequence motifs can occur at high frequency in proteomes.…”

Section: Discussionmentioning

confidence: 99%

Substrate Discrimination among Mitogen-activated Protein Kinases through Distinct Docking Sequence Motifs

Sheridan

Kong

Parker

et al. 2008

Journal of Biological Chemistry

133

138

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Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.Mitogen-activated protein kinases (MAPKs) 4 lie at the bottom of conserved three-component phosphorylation cascades that integrate cellular responses to a wide variety of extracellular stimuli, including growth factors, cytokines, UV irradiation, and oxidative stress (1). Canonical MAPKs are classified into three major subfamilies, extracellular signal-regulated kinases (ERK), p38 MAPK (p38), and c-Jun N-terminal kinases (JNK), based on sequence homology, shared upstream kinases, and activating stimuli. In addition, the different MAPK subfamilies phosphorylate a distinct set of protein substrates. These substrates act as the critical effectors that enable cells to mount the appropriate responses to varied stimuli.Analysis of reported MAPK substrate phosphorylation sites suggests a nearly absolute requirement for a Pro residue immediately downstream of the Ser or Thr phosphoacceptor. Extensive peptide library studies on ERK1, ERK2, and p38␣ have defined a common phosphorylation site motif for these MAPKs. In addition to the requirement for a Pro residue at the ϩ1 position relative to the phosphorylation site, this motif includes a weak preference for Pro and other aliphatic residues at the Ϫ2 position (2-4). Mutagenesis experiments with the JNK substrate JunB have also suggested additional specificity for JNK family MAPKs at positions downstream of the phosphorylation site (5). MAPKs have affinities for short peptide substrates that are at least an order of magnitude lower than for full-length protein substrates (4, 6 -10). Thus although phosphorylation site motifs likely serve to direct the MAPK to phosphorylate a specific serine or threonine residue within a protein, they are insufficient to fully account for protein substrate recognition. Although mechanisms such as subcellular localization and the use of scaffolding proteins can contribute to kinase substrate targeting in vivo, MAPKs also exhibit a high degree of target specificity in vitro (11-13).For MAPKs, substrate specificity is ensured through the use of docking...

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Section: Discussionmentioning

confidence: 99%

Substrate Discrimination among Mitogen-activated Protein Kinases through Distinct Docking Sequence Motifs

Sheridan

Kong

Parker

et al. 2008

Journal of Biological Chemistry

133

138

View full text Add to dashboard Cite

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“…All these favourable features have allowed PNAs to be applied to various research areas such as antisense [6][7][8] and antigene [9][10][11] therapies (see Figure 2 for a schematic representation), biosensing 12 and in the high-throughput screening of enzymes using a PNA library. 13 In order to modify the intrinsic properties of PNAs or in order to add new functionalities and/or spectroscopic properties to PNA oligomers, metal complexes have been synthetically attached to these non-natural DNA analogues. [14][15][16] In this perspective article, we are reporting on the recent advances in both the preparation and use of metal-containing PNAs.…”

Section: Introductionmentioning

confidence: 99%

Metal-containing peptide nucleic acid conjugates

2011

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Peptide Nucleic Acids (PNAs) are non-natural DNA/RNA analogues with favourable physicochemical properties and promising applications. Discovered nearly 20 years ago, PNAs have recently re-gained quite a lot of attention. In this Perspective article, we discuss the latest advances on the preparation and utilisation of PNA monomers and oligomers containing metal complexes. These metalconjugates have found applications in various research fields such as in the sequence-specific detection of nucleic acids, in the hydrolysis of nucleic acids and peptides, as radioactive probes or as modulators of PNA˙DNA hybrid stability, and last but not least as probes for molecular and cell biology.

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“…Additionally, for protein-tyrosine kinase p60 c-src only incorporation of the long and hydrophilic 1-amino-4,7,10-trioxa-13-tridecanamine succinimic acid building block spacer allowed effective phosphorylation of the glass surface-bound peptides [26]. A similar linker structure was used to space apart Peptide Nucleic Acid (PNA) tags from potential protease substrates [66], protein serine/threonine kinase substrates [29,30] or tyrosine kinase substrates [67]. Inamori et al reported that insertion of a PEG spacer between chemoselective attachment point and the kinase substrate sequence improved phosphorylation efficiency by c-Src but not by PKA [35].…”

Section: Chemistry Of Peptide Array Preparationmentioning

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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A 10,000 Member PNA-Encoded Peptide Library for Profiling Tyrosine Kinases

Cited by 33 publications

References 55 publications

Substrate Discrimination among Mitogen-activated Protein Kinases through Distinct Docking Sequence Motifs

Substrate Discrimination among Mitogen-activated Protein Kinases through Distinct Docking Sequence Motifs

Metal-containing peptide nucleic acid conjugates

Deciphering Enzyme Function Using Peptide Arrays

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