Development of photo‐crosslinking reagents for protein kinase–substrate interactions

Parang, Keykavous; Kohn, Jeffrey A.; Saldanha, S. Adrian; Cole, Philip A.

doi:10.1016/s0014-5793(02)02778-3

Cited by 37 publications

(32 citation statements)

References 9 publications

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“…This approach was first described by Parang et al who designed a photoactivatable cross-linking reagent for c-Src. [62] Two azido groups were introduced into the inhibitor, resulting in a conjugate that is ATP-competitive and c-Src peptide substrate noncompetitive in the absence of UV. Upon UV irradiation, the reagent induced covalent cross-linking of the c-Src protein with the protein substrate that was competitively inhibited in the presence of ATP as revealed by Western blot analysis of the reaction mixture.…”

Section: A C H T U N G T R E N N U N G Recognition Of Pksmentioning

confidence: 99%

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

2009

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Bisubstrate inhibitors consist of two conjugated fragments, each targeted to a different binding site of a bisubstrate enzyme. The design of bisubstrate inhibitors presupposes the formation of the ternary complex in the course of the catalyzed reaction. The principle advantage of bisubstrate inhibitors is their ability to generate more interactions with the target enzyme that could result in improved affinity and selectivity of the conjugates, when compared with single-site inhibitors. Among phosphotransferases, the approach was first successfully used for adenylate kinase in 1973. Since then, several types of bisubstrate inhibitors have been developed for protein kinases, including conjugates of peptides with nucleotides, adenosine derivatives and potent ATP-competitive inhibitors. Earlier bisubstrate inhibitors had pharmacokinetic qualities that were unsuitable for cellular experiments and hence were mostly used for in vitro studies. The recently constructed conjugates of adenosine derivatives and D-arginine-rich peptides (ARCs) possess high kinase affinity, high biological and chemical stability and good cell plasma membrane penetrative properties that enable their application in the regulation of cellular protein phosphorylation balances in cell and tissue experiments.

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Section: A C H T U N G T R E N N U N G Recognition Of Pksmentioning

confidence: 99%

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

2009

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“…10–12 Building on the fact that adenosine-5′-triphosphate (ATP) ( 1 , Fig. 1A) acts as a universal phosphoryl donor, we developed an ATP-based crosslinking reagent that requires phosphorylation for covalent kinase–substrate linkage.…”

Section: Introductionmentioning

confidence: 99%

A comparative study of ATP analogs for phosphorylation-dependent kinase–substrate crosslinking

Garre

Senevirathne

Pflum

2014

Bioorganic & Medicinal Chemistry

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Kinase-catalyzed protein phosphorylation is an important post-translational modification that regulates a variety of cellular functions. Identification of the many substrates of a specific kinase is critical to fully characterize cell biology. Unfortunately, kinase–substrate interactions are often transient, which makes their identification challenging. Here, the transient kinase–substrate complex was stabilized by covalent crosslinking using γ-phosphate modified ATP analogs. Building upon prior use of an ATP-aryl azide photocrosslinking analog, we report here the creation of an ATP-benzophenone photocrosslinking analog. ATP-benzophenone displayed a higher conversion percentage but more diffuse crosslinking compared to the ATP-aryl azide analog. A docking study was also performed to rationalize the conversion and crosslinking data. In total, the photocrosslinking ATP analogs produced stable kinase–substrate complexes that are suitable for future applications characterizing cell signaling pathways.

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“…A number of adenine-based photoactivatable nucleotides have been described (MacMillan et al 1994;Parang et al 2002). Among them, 8-AzidoATP (8-N 3 ATP) has been widely used to map the active site of a variety of enzymes, and the nucleotide-binding domains of several proteins ( Fig.…”

Section: Introductionmentioning

confidence: 99%

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

Gopalakrishna¹,

Gusti²,

Nair³

et al. 2004

RNA

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UV-induced photochemical crosslinking is a powerful approach that can be used for the identification of specific interactions involving nucleic acid-protein and nucleic acid-nucleic acid complexes. 8-AzidoATP (8-N 3 ATP) is a photoaffinity-labeling agent which has been widely used to elucidate the ATP binding site of a variety of proteins. However, its true potential as a photoactivatable nucleotide analog could not be exploited due to the lack of 8-azidoadenosine phosphoramidite, a monomer used in the synthesis of RNA, and the inability of 8-N 3 ATP to serve as an efficient substrate for bacteriophage RNA polymerase. In this study, we explored the ability of SP6, T3, and T7 RNA polymerases and metal ion cofactors to catalyze the incorporation of 8-N 3 AMP into RNA. Whereas transcription buffer containing 2.0-2.5 mM Mn 2+ supports T7 RNA polymerase-mediated insertion of 8-N 3 AMP into RNA, a mixture of 2.5 mM Mn 2+ and 2.5 mM Mg 2+ further improves the yield of 8-N 3 AMP-containing transcript. In addition, both RNA transcription and reverse transcription proceed with high fidelity for the incorporation of 8-N 3 AMP and complementary residue, respectively. Finally, we show that a high-affinity MS2 coat protein binding sequence, in which adenosine residues were replaced by 8-azidoadenosine, crosslinks to the coat protein of the Escherichia coli phage MS2.

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Development of photo‐crosslinking reagents for protein kinase–substrate interactions

Cited by 37 publications

References 9 publications

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

A comparative study of ATP analogs for phosphorylation-dependent kinase–substrate crosslinking

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

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Development of photo‐crosslinking reagents for protein kinase–substrate interactions

Cited by 37 publications

References 9 publications

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

A comparative study of ATP analogs for phosphorylation-dependent kinase–substrate crosslinking

Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase

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Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase