RNA aptamers as pathway-specific MAP kinase inhibitors.
BACKGROUND
In eukaryotic cells, many intracellular signaling pathways have closely related mitogen activated protein kinase (MAPK) paralogs as central components. Although MAPKs are therefore obvious targets to control the cellular responses resulting from the activation of these signaling pathways, the development of inhibitors which target specific cell signaling pathways involving MAPKs has proven difficult.


RESULTS
We used an RNA combinatorial approach to isolate RNAs that inhibit the in vitro phosphorylation activity of extracellular regulated kinase 2 (ERK2). These inhibitors block phosphorylation by ERK1 and ERK2, but do not inhibit Jun N-terminal kinase or p38 MAPKs. Kinetic analysis indicates these inhibitors function at high picomolar concentrations through the steric exclusion of substrate and ATP binding. In one case, we identified a compact RNA structural domain responsible for inhibition.


CONCLUSIONS
RNA reagents can selectively recognize and inhibit MAPKs involved in a single signal transduction pathway. The methodology described here is readily generalizable, and can be used to develop inhibitors of MAPKs involved in other signal transduction pathways. Such reagents may be valuable tools to analyze and distinguish homologous effectors which regulate distinct signaling responses.
Seiwert
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SD
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309-0215, USA. scott.siewert@colorado.edu
Stines Nahreini
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T
Aigner
S
S
Ahn
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NG
Uhlenbeck
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OC
D000818:Animals; D001483:Base Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004268:DNA-Binding Proteins; D015723:Gene Library; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D008024:Ligands; D020935:MAP Kinase Signaling System / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; C494114:Elk1 protein, rat; D008024:Ligands; D015221:Potassium Channels; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D012313:RNA; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases