Adenylate control in cAMP signaling: Implications for adaptation in signalosomes

Nikhil K. Tulsian, Abhijeet Ghode, Ganesh S. Anand

Research output: Contribution to journalArticlepeer-review

Abstract

In cAMP-Protein Kinase A (PKA) signaling, A-kinase anchoring protein scaffolds assemble PKA in close proximity to phosphodiesterases (PDE), kinase-substrates to form signaling islands or 'signalosomes'. In its basal state, inactive PKA holoenzyme (R2:C2) is activated by binding of cAMP to regulatory (R)-subunits leading to dissociation of active catalytic (C)-subunits. PDEs hydrolyze cAMP-bound to the R-subunits to generate 50-AMP for termination and resetting the cAMP signaling. Mechanistic basis for cAMP signaling has been derived primarily by focusing on the proteins in isolation. Here, we set out to simulate cAMP signaling activation-termination cycles in a signalosome-like environment with PDEs and PKA subunits in close proximity to each other. Using a combination of fluorescence polarization and amide hydrogen exchange mass spectrometry with regulatory (RIα), C-subunit (Cα) and PDE8 catalytic domain, we have tracked movement of cAMP through activation-termination cycles. cAMP signaling operates as a continuum of four phases: (1) Activation and dissociation of PKA into R- and C-subunits by cAMP and facilitated by substrate (2) PDE recruitment to R-subunits (3) Hydrolysis of cAMP to 50-AMP (4) Reassociation of C-subunit to 50-AMP-bound-RIα in the presence of excess ATP to reset cAMP signaling to form the inactive PKA holoenzyme. Our results demonstrate that 50-AMP is not merely a passive hydrolysis end-product of PDE action. A 'ligand-free' state R subunit does not exist in signalosomes as previously assumed. Instead the R-subunit toggles between cAMP- or 50-AMP bound forms. This highlights, for the first time, the importance of 50-AMP in promoting adaptation and uncovers adenylate control in cAMP signaling.

Original languageEnglish (US)
Pages (from-to)2981-2998
Number of pages18
JournalBiochemical Journal
Volume477
Issue number16
DOIs
StatePublished - Aug 2020

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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