Local protein kinase A action proceeds through intact holoenzymes

Abstract

Hormones can transmit signals through adenosine 3′,5′-monophosphate (cAMP) to precise intracellular locations. The fidelity of these responses relies on the activation of localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory type II (RII) subunits with A-kinase—anchoring proteins (AKAPs) confers location, and catalytic (C) subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that AKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets. Native mass spectrometry established that these macromolecular assemblies incorporated stoichiometric amounts of cAMP. Chemical-biology— and live cell—imaging techniques revealed that catalytically active PKA holoenzymes remained intact within the cytoplasm. These findings indicate that the parameters of anchored PKA holoenzyme action are much more restricted than originally anticipated.

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

“…C subunit activity is suppressed by a pseudosubstrate segment on R, which binds to the catalytic site on C and is released by cAMP (Brandon et al, 1997), although C does not have to be fully released from R in order to catalyze phosphorylation as indicated by correlating full dissociation of C from R or better a lack thereof with full enzymatic activity (Johnson et al, 1993;Yang et al, 1995). 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).…”

Cardiac function modulation depends on the A‐kinase anchoring protein complex

“…cAMP production in response to physiological effectors of GPCR signalling appears not to promote catalytic subunit release from anchored PKA holoenzymes. 25 But this result has been challenged recently by Gray's group, 26 which proved that catalytic subunits are released from regulatory subunits by cAMP, and during cAMP activation, tether to R subunits does not restrict C subunit activity. These views remain controversial and have yet to be explored.…”

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, 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.…”