Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches

Abstract

Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average ∼17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled to mass spectrometry. Quantitative comparison of cross-linking within a complex of RIIβ and Cβ, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIβ is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane.

Postsynaptic localization and regulation of AMPA receptors and Cav1.2 by β2 adrenergic receptor/PKA and Ca 2+ /CaMKII signaling

“…A recent publication seems to confirm these earlier studies as both R and C co-immunoprecipitate with the A kinase anchor protein AKAP5 from cell lysate prepared after strong b-adrenergic stimulation of the cells (Smith et al, 2017). Yet, followup work suggests that the co-immunoprecipitation is due to re-association of C with R upon cell lysis as cAMP becomes diluted (Walker- Gray et al, 2017). Phosphotransfer and substrate release is modestly fast for most kinases (~500/s) (Zhou & Adams, 1997;Shaffer & Adams, 1999).…”

Aspects of astrocytic cAMP signaling with an emphasis on the putative power of compartmentalized signals in health and disease

“…Extending on the concept of compartmentalization, the intracellular diffusion of cAMP has been shown to occur at a rate substantially slower than in aqueous solution (Agarwal et al, ). Clearly, cAMP will bind to its effector proteins throughout the cell and in this respect, it is noteworthy that the regulatory subunit of the main effector of cAMP, PKA, is an abundant protein and outnumbers the catalytic subunit in a wide range of tissues including brain (Walker‐Gray, Stengel, & Gold, ); incidentally, these authors also make a solid case for the idea that the catalytic subunit dissociates from the regulatory subunit upon binding of cAMP, a long‐held notion that was recently challenged (Smith et al, ; Smith et al, ). On a final note on PKA's contribution to compartmentalization of cAMP‐mediated responses, the findings of Walker‐Gray et al also suggest that active PKA catalytic subunits do not travel far before they are recaptured by regulatory subunits (Walker‐Gray et al, ).…”

“…Clearly, cAMP will bind to its effector proteins throughout the cell and in this respect, it is noteworthy that the regulatory subunit of the main effector of cAMP, PKA, is an abundant protein and outnumbers the catalytic subunit in a wide range of tissues including brain (Walker‐Gray, Stengel, & Gold, ); incidentally, these authors also make a solid case for the idea that the catalytic subunit dissociates from the regulatory subunit upon binding of cAMP, a long‐held notion that was recently challenged (Smith et al, ; Smith et al, ). On a final note on PKA's contribution to compartmentalization of cAMP‐mediated responses, the findings of Walker‐Gray et al also suggest that active PKA catalytic subunits do not travel far before they are recaptured by regulatory subunits (Walker‐Gray et al, ). In conclusion, spatially and functionally distinct cAMP signals may be generated within the same cell at any given time as a consequence of one or more extracellular cues or indeed, as we will discuss below, in response to an intracellular cue.…”

“…Clearly, cAMP will bind to its effector proteins throughout the cell and in this respect, it is noteworthy that the regulatory subunit of the main effector of cAMP, PKA, is an abundant protein and outnumbers the catalytic subunit in a wide range of tissues including brain (Walker-Gray, Stengel, & Gold, 2017); incidentally, these authors also make a solid case for the idea that the catalytic subunit dissociates from the regulatory subunit upon binding of cAMP, a long-held notion that was recently challenged (Smith et al, 2013;Smith et al, 2017). On a final note on PKA's contribution to compartmentalization of cAMP-mediated F I G U R E 1 Directly and indirectly receptor-coupled cAMP signaling.…”

“…AKAPs can also bind calmodulin (CaM), calcineurin (CaN), and protein kinase C (PKC); all proteins involved in Ca 2+ signaling, representing another entry point for the interplay between Ca 2+ and cAMP signaling. Membrane-bound receptors and ion channels (indicated by a question mark), such as GPCR and L-type Ca 2+ channels have also been observed to participate in such AKAP complexes (Wild & Dell'Acqua, 2018) raising the question whether they are involved in cAMP dynamics in astrocytes responses, the findings of Walker-Gray et al also suggest that active PKA catalytic subunits do not travel far before they are recaptured by regulatory subunits (Walker-Gray et al, 2017). In conclusion, spatially and functionally distinct cAMP signals may be generated within the same cell at any given time as a consequence of one or more extracellular cues or indeed, as we will discuss below, in response to an intracellular cue.…”