title: Monitoring native p38α:MK2/3 complexes via trans delivery of an ATP acyl phosphate probe.
abstract: 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.
authors.affiliation: ActivX Biosciences, Inc., 11025 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Eric S
authors.initials: ES
authors.lastname: Okerberg
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Brown
authors.forename: Lauro
authors.initials: L
authors.lastname: Minimo
authors.forename: Senait
authors.initials: S
authors.lastname: Alemayehu
authors.forename: Jonathan
authors.initials: J
authors.lastname: Rosenblum
authors.forename: Matt
authors.initials: M
authors.lastname: Patricelli
authors.forename: Tyzoon
authors.initials: T
authors.lastname: Nomanbhoy
authors.forename: John W
authors.initials: JW
authors.lastname: Kozarich
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D055785:Gene Knockdown Techniques; D018922:HL-60 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D000255:Adenosine Triphosphate; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C098416:MAP-kinase-activated kinase 3