Monitoring native p38α:MK2/3 complexes via trans delivery of an ATP acyl phosphate probe
| Autor: | Eric Okerberg, Jonathan S. Rosenblum, Heidi E. Brown, Senait Alemayehu, Lauro Minimo, John W. Kozarich, Matthew P. Patricelli, Tyzoon K. Nomanbhoy |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Molecular Probe Techniques HL-60 Cells Protein Serine-Threonine Kinases Proteomics Biochemistry p38 Mitogen-Activated Protein Kinases Catalysis chemistry.chemical_compound Colloid and Surface Chemistry Adenosine Triphosphate RNA interference Catalytic Domain Humans Protein Structure Quaternary Protein Kinase Inhibitors Kinase Intracellular Signaling Peptides and Proteins A protein General Chemistry Phosphate Binding constant In vitro chemistry Gene Knockdown Techniques Signal transduction Protein Multimerization Signal Transduction |
| Zdroj: | Journal of the American Chemical Society. 136(12) |
| ISSN: | 1520-5126 |
| Popis: | Here we describe a chemical proteomics strategy using ATP acyl phosphates to measure the formation of a protein:protein complex between p38α and mapkap kinases 2 and/or 3. Formation of the protein:protein complex results in a new probe labeling site on p38α that can be used to quantify the extent of interaction in cell lysates and the equilibrium binding constant for the interaction in vitro. We demonstrate through RNA interference that the labeling site is dependent on formation of the protein:protein complex in cells. Further, we identify that active-site-directed, small-molecule inhibitors of MK2/3 selectively inhibit the heterodimer-dependent probe labeling, whereas p38α inhibitors do not. These findings afford a new method to evaluate p38α and MK2/3 inhibitors within native biological systems and a new tool for improved understanding of p38α signaling pathways. |
| Databáze: | OpenAIRE |
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