Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain

Abstract

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIb or the RIIb subunit results in distinct neuronal phenotypes. Although RIb contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIb is enriched in dendrites and co-localizes with MAP2, whereas RIIb is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIb in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIb, but not of RIIb, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.

Switch-like PKA responses in the nucleus of striatal neurons

“…Striatal neurons exhibit higher levels of PKA than cortical neurons, in particular the RIIβ type (Brandon et al, 1998;Cadd and McKnight, 1989;Ilouz et al, 2017;Ventra et al, 1996), which we also see in our brain slice preparations from young mice (Fig. S1).…”

“…The nuclear responsiveness in D1 MSNs does not result from the existence of a nuclear pool of PKA cAMP can diffuse freely throughout the cell and in the nucleus (DiPilato et al, 2004;Haj Slimane et al, 2014;Yang et al, 2014) and other studies have reported the presence of PKA holoenzyme in the nuclear compartment, which might support the fast kinetics of the nuclear PKA responses (Ilouz et al, 2017;Sample et al, 2012;Zippin et al, 2004). We tested this hypothesis in our preparations by photo-releasing cAMP inside the neurons using a cell-permeant caged cAMP (4,5-dimethoxy-2-nitrobenzyl adenosine 3,5-cyclic monophosphate, DMNB-cAMP).…”

“…Regulatory PKA subunits have been reported to be localized in the nucleus, such as RIβ in the nucleus of cerebellar Purkinje neurons or hippocampal interneurons (Ilouz et al, 2017) and such 'pre-positioning' of PKA holoenzyme in the nucleus might speed up the transduction of the cytosolic cAMP signal into a nuclear response. However, the delayed nuclear response that we observed in striatal neurons does not fit with this hypothesis, since cAMP diffuses quickly through the nuclear pore (Sample et al, 2012) and should therefore have induced an instantaneous nuclear signal.…”

Interrogating cyclic AMP signaling using optical approaches

“…There are three mammalian genes encoding the PKA catalytic subunits (Cα, Cβ, and Cγ) and four encoding the regulatory subunits (RIα, RIβ, RIIα and RIIβ). These have differing tissue distributions, cAMP affinities, and subcellular localizations [25]. Moreover, different PKA subtypes can combine in multiple ways, generating ample diversity in the signaling outputs of the PKA hetero-tetramer.…”

Tau inhibits PKA by nuclear proteasome‐dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation

“…When in an inactive state, the pseudo‐substrate sequence of the PKA‐Rs blocks the active site on the PKA‐Cs. Three PKA‐Cs (C‐α, C‐β, and C‐γ) and two PKA‐Rs (RI and RII) families have been discovered, and all PKA‐Rs have different cAMP binding properties (Ilouz et al, ). The two R families have two isoforms: α and β (RI‐α, RI‐β, RII‐α, and RII‐β).…”