Proximity biotinylation to define the local environment of the protein kinase A catalytic subunit in adrenal cells

Abstract

Summary: Mutant protein kinase A catalytic subunit (PKAc) drives adrenal Cushing’s syndrome, though its signaling interactions remain unclear. This protocol details steps to use live-cell proximity labeling to identify subcellular compartments and proteins closely associated with variants of PKAc in human adrenal cells. We include instructions for clonal cell line generation, live biotin labeling of proximal proteins, isolation of biotinylated proteins, and sample processing for proteomic analysis using the biotin ligase miniTurbo with wild-type and mutant PKAc.1,2For complete details on the use and execution of this protocol, please refer to Omar et al. (2022).3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell culture
• Signal Transduction
• Molecular/Chemical Probes
• Protein Biochemistry
• Proteomics
• Mass Spectrometry