Raf Kinase Inhibitory Protein regulates the cAMP-dependent protein kinase signaling pathway through a positive feedback loop

Jiyoung Lee, Cristina Olivieri, Colin Ong, Larry R Masterson, Suzana Gomes, Bok Soon Lee, Florian Schaefer, Kristina Lorenz, Gianluigi Veglia, Marsha Rich Rosner

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the β-adrenergic receptor (β-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes β-AR–activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the β-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.

Original languageEnglish (US)
Article numbere2121867119
JournalProceedings of the National Academy of Sciences of the United States of America
Volume119
Issue number25
DOIs
StatePublished - Jun 21 2022

Bibliographical note

Funding Information:
This work was supported by NIH grants GM087630 and GM121735 to M.R.R., NIH grant GM100310 to G.V., and the Deutsche Forschungsgemeinschaft (grants SFB1116/A09 and SFB/TR296/P10) to K.L. NMR experiments were carried out at the Minnesota NMR center. We thank Ya Chen and Arnold Satterthwait from the Sanford–Burnham Medical Research Institute, La Jolla, CA, for generously providing pS51-RKIP peptide for antibody isolation. We thank John Skinner and Jonggul Kim for helpful discussions and Long Nguyen for assistance with the figures.

Funding Information:
ACKNOWLEDGMENTS. This work was supported by NIH grants GM087630 and GM121735 to M.R.R., NIH grant GM100310 to G.V., and the Deutsche For-schungsgemeinschaft ( grants SFB1116/A09 and SFB/TR296/P10) to K.L. NMR experiments were carried out at the Minnesota NMR center. We thank Ya Chen and Arnold Satterthwait from the Sanford–Burnham Medical Research Institute, La Jolla, CA, for generously providing pS51-RKIP peptide for antibody isolation. We thank John Skinner and Jonggul Kim for helpful discussions and Long Nguyen for assistance with the figures.

Publisher Copyright:
Copyright © 2022 the Author(s). Published by PNAS.

Keywords

  • Raf Kinase Inhibitory Protein (RKIP)
  • nuclear magnetic resonance (NMR)
  • phosphatidylethanolamine binding protein (PEBP)
  • protein kinase A (PKA)

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