Monitoring native p38α:MK2/3 complexes via trans delivery of an ATP acyl phosphate probe

Eric S Okerberg; Heidi E Brown; Lauro Minimo; Senait Alemayehu; Jonathan Rosenblum; Matt Patricelli; Tyzoon Nomanbhoy; John W Kozarich

doi:10.1021/ja4129907

Monitoring native p38α:MK2/3 complexes via trans delivery of an ATP acyl phosphate probe

J Am Chem Soc. 2014 Mar 26;136(12):4664-9. doi: 10.1021/ja4129907. Epub 2014 Mar 17.

Authors

Eric S Okerberg¹, Heidi E Brown, Lauro Minimo, Senait Alemayehu, Jonathan Rosenblum, Matt Patricelli, Tyzoon Nomanbhoy, John W Kozarich

Affiliation

¹ ActivX Biosciences, Inc., 11025 North Torrey Pines Road, La Jolla, California 92037, United States.

PMID: 24601623
DOI: 10.1021/ja4129907

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.

MeSH terms

Adenosine Triphosphate / chemistry*
Adenosine Triphosphate / metabolism*
Catalytic Domain
Gene Knockdown Techniques
HL-60 Cells
Humans
Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
Intracellular Signaling Peptides and Proteins / chemistry
Intracellular Signaling Peptides and Proteins / genetics
Intracellular Signaling Peptides and Proteins / metabolism
Models, Molecular
Molecular Probe Techniques*
Protein Kinase Inhibitors / chemistry
Protein Kinase Inhibitors / pharmacology
Protein Multimerization
Protein Serine-Threonine Kinases / antagonists & inhibitors
Protein Serine-Threonine Kinases / chemistry
Protein Serine-Threonine Kinases / genetics
Protein Serine-Threonine Kinases / metabolism
Protein Structure, Quaternary
Signal Transduction
p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
p38 Mitogen-Activated Protein Kinases / chemistry*
p38 Mitogen-Activated Protein Kinases / metabolism*

Substances

Intracellular Signaling Peptides and Proteins
Protein Kinase Inhibitors
Adenosine Triphosphate
MAP-kinase-activated kinase 2
MAP-kinase-activated kinase 3
Protein Serine-Threonine Kinases
p38 Mitogen-Activated Protein Kinases