Identification of protein interactors is ideally suited for the functional characterization of small molecules. 3′,5′-cAMP is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3′,5′-cAMP, we used a chemo-proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3′,5′-cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3′,5′-cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3′,5′-cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3′,5′-cAMP supplementation affected actin organization by inducing actin-bundling. Consistent with these results, the increase in 3′,5′-cAMP levels, obtained either by feeding or by chemical modulation of 3′,5′-cAMP metabolism, was sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. The observed rescue was specific to 3′,5′-cAMP, as demonstrated using a positional isomer 2′,3′-cAMP, and true for the nanomolar 3′,5′-cAMP concentrations reported for plant cells. In vitro characterization of the 3′,5′-cAMP–actin pairing argues against a direct interaction between actin and 3′,5′-cAMP. Alternative mechanisms by which 3′,5′-cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a specific resource, 3′,5′-cAMP interactome, as well as functional insight into 3′,5′-cAMP-mediated regulation in plants.
Bibliographical noteFunding Information:
MC is grateful to King Abdullah University of Science and Technology (KAUST) for funding. TGA thanks the Max Planck Society and Alexander von Humboldt Sofia Kovalevskaja program for funding. ASk acknowledges a National Science Foundation grant (NSF 2226270). We thank Dr Rene Schneider, Dr Sakutaru Kijima, Dr Taro Uyeda, Dr Jakub Sedzinski, Dr Manny Saluja, and Dr Lothar Willmitzer for their valuable scientific input. We are grateful to Dr Maria C. de Pinto and Dr Emanuela Blanco for sharing their cAMP sponge lines with us, Dr Michal Gorka for the input on proteomics, and Dr Ingrid Billault‐Chaumartin for help with the actin‐bundling analysis. We are also grateful to the KAUST core lab for running samples in their proteomics facility.
© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.
- calcium signaling
- thermal proteome profiling
ASJC Scopus subject areas
- Plant Science
- Cell Biology