Structure and Allostery of the PKA RIIβ Tetrameric Holoenzyme

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Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP3), not stimulatory G protein (Gαs), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gαi/o) signaling but rather Ca2+ entry through a Gd3+-sensitive channel. Therefore, PIP3 sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca2+ entry.

“…The high basal cAMP in cilia that we measured (Fig. 3 C and D) is sufficient to activate PKA (30), and indeed there is higher PKA activity in cilia compared with the whole cell (Fig. 1B).…”

Section: Shh Increases Camentioning

confidence: 90%

“…By directly measuring cAMP in cilia, we have established that the basal concentration of cAMP in cilia is not only higher than in the whole cell but also high enough to sustain PKA activity (30). Primary cilia of MEFs are capable of responding to signals from stimulatory GPCRs (Fig.…”

Section: Discussionmentioning

confidence: 99%

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

Moore

Stepanchick

Tewson

et al. 2016

109

133

“…5). In the recently solved structure of the RIIβ holoenzyme (18), as well as in the model of the RIα holoenzyme, the two Csubunits do not touch one another, in contrast to the RIβ holoenzyme (Fig. 5), and the interfaces between the two heterodimers in each isoform are completely different.…”

Section: Discussionmentioning

confidence: 99%

“…This structure provides a plausible model explaining how a dimer of heterodimers can be assembled. Most recently, the structure described for the RIIβ holoenzyme shows that its quaternary structure differs dramatically from the model of the RIα holoenzyme (18) and shows how differently the C-subunit can be packaged by two functionally nonredundant R-subunits. However, none of the D/D domains of the R-dimer in the holoenzyme complex have been visualized, and the full allosteric potential that is embedded in each holoenzyme structure remains elusive.…”

mentioning

confidence: 99%

Localization and quaternary structure of the PKA RIβ holoenzyme

Ilouz

Bubis

Wu³

et al. 2012

Self Cite

Specificity for signaling by cAMP-dependent protein kinase (PKA) is achieved by both targeting and isoform diversity. The inactive PKA holoenzyme has two catalytic (C) subunits and a regulatory (R) subunit dimer (R 2 :C 2 ). Although the RIα, RIIα, and RIIβ isoforms are well studied, little is known about RIβ. We show here that RIβ is enriched selectively in mitochondria and hypothesized that its unique biological importance and functional nonredundancy will correlate with its structure. Small-angle X-ray scattering showed that the overall shape of RIβ 2 :C 2 is different from its closest homolog, RIα 2 :C 2 . The full-length RIβ 2 :C 2 crystal structure allows us to visualize all the domains of the PKA holoenzyme complex and shows how isoform-specific assembly of holoenzyme complexes can create distinct quaternary structures even though the R 1 :C 1 heterodimers are similar in all isoforms. The creation of discrete isoform-specific PKA holoenzyme signaling "foci" paves the way for exploring further biological roles of PKA RIβ and establishes a paradigm for PKA signaling.structural biology | signal transduction | allostery C yclic AMP-dependent protein kinase (PKA) regulates a plethora of biological events that extend from development and differentiation to memory, ion transport, and metabolism. The inactive PKA holoenzyme is composed of a regulatory (R) subunit dimer and two catalytic (C) subunits. Binding of cAMP to the R-subunits unleashes the C-subunits, thereby allowing phosphorylation of PKA substrates. Specificity of PKA signaling is achieved in large part by the isoform diversity of its R-subunits. There are two classes of R-subunits, RI and RII, which are subclassified into α and β subtypes. Each isoform is encoded by a unique gene and is functionally nonredundant. RIα-null mice display early embryonic lethality (1), whereas RIIβ-null mice have a lean phenotype and are resistant to diet-induced diabetes (2). Specific isoforms also are expressed differently in cells and tissues. The RIβ isoform is expressed abundantly in brain and spinal cord (3-5). Hippocampal slices from RIβ-null mice show a severe deficit in long-term potentiation and also in long-term depression and memory. Although RIα protein levels are increased in these mice, the hippocampal function is not rescued, suggesting a unique role for RIβ, which is the least studied Rsubunit (6, 7). Most localization studies have focused on the differences between RI and RII and showed that RI isoforms are diffused mainly in the cytoplasm, whereas RIIs typically are associated with membranous organelles. More than 50 A kinase-anchoring proteins (AKAPs) have been identified as targeting PKA to a specific subcellular location (8). The only potential AKAP that binds preferentially to the RIβ isoform identified so far is the neurofibromatosis 2 tumor suppressor protein, merlin (9).All R isoforms share the same domain organization, which includes a docking and dimerization (D/D) domain followed by an inhibitor sequence and two cAMP-binding domains (Fig. 1E)....

“…cAMP binding to the PKA regulatory subunits (R) induces dissociation of the tetrameric PKA holoenzyme, resulting in active PKA catalytic subunits (PKAc; Fig. 1C) (7,8). To ensure substrate specificity, PKA is tethered to distinct subcellular compartments through physical interaction with A-kinase anchoring proteins (AKAPs; refs.…”

mentioning

confidence: 99%

Reciprocal regulation of PKA and Rac signaling

Bachmann

Riml

Huber

et al. 2013

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac-bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTPRac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA-boosted activation of Raf-Mek-Erk. Consequently GTPRac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac-mediated activation of nuclear Erk1/2 signaling. signal transduction | cross-talk S ignal transduction cascades coordinate the plethora of extracellular stimuli into biological responses within cells. The specificity of receptor-initiated signaling responses is encoded by spatial and temporal dynamics of downstream signaling networks (1). These networks, initiating from e.g., the G protein-coupled receptor (GPCR) superfamily and receptor tyrosine kinases (RTK), tightly regulate signaling pathways at several critical points via feedback loops and cross-talk among other pathways (2-5). A large number of GPCR signaling cascades uses cAMP as an intracellular second messenger (3, 6). In response to hormone binding to distinct GPCRs, cAMP is produced and binds to its canonical effector, the cAMP-dependent protein kinase A (PKA). cAMP binding to the PKA regulatory subunits (R) induces dissociation of the tetrameric PKA holoenzyme, resulting in active PKA catalytic subunits (PKAc; Fig. 1C) (7, 8). To ensure substrate specificity, PKA is tethered to distinct subcellular compartments through physical interaction with A-kinase anchoring proteins (AKAPs; refs. 9 and 10). It has been long regarded that the...