Localization and quaternary structure of the PKA RIβ holoenzyme

Abstract

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)....

Using Markov State Models to Develop a Mechanistic Understanding of Protein Kinase A Regulatory Subunit RIα Activation in Response to cAMP Binding

“…This is consistent with recent publications (26,28). However, the diverse R 2 C 2 structures that various PKA isoforms exhibit suggest that although this model is consistent with current PKA-RI␣ experiments it would be expected that RI␤, RII␣, and RII␤ will exhibit significantly different mechanistic activations possibly with less interaction between the R-C heterodimers (13,29,30).…”

Dynamic architecture of a protein kinase

“…The importance of the C-lobe is emphasized, however, by the universal conservation of the α-helices G, H, and I, (GHI helical subdomain) in all EPKs. This region contains a binding site for regulatory subunits (43)(44)(45)(46), substrates (47), phosphatases (48), and as a dimerization site (49). One may speculate that the sensitivity of the C-lobe's community structure is mechanistically related to long-distance signaling between protein docking sites and the catalytic machinery.…”

Structural insights into mis-regulation of protein kinase A in human tumors

“…1, Table 1, and Table S1): the full-length wild-type C subunit (PKAc), the predominant point mutant (PKAc L205R), the predominant chimeric fusion protein (DnaJ-PKAc), and PKAc with exon 1 residues deleted (PKAc Δexon1). All structures were determined as complexes with ATP and the protein kinase inhibitor (PKI) peptide (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In each, ATP is bound with two metal ions within the cleft formed between the smaller N-terminal and larger C-terminal lobes of the C subunit, associated as previously described (14).…”

“…We further investigated possibilities that mis-regulation of PKA signaling is caused by mis-localization of the PKA fusion, a mechanism known to be associated with disease states (18). We did not expect interactions with canonical A-kinase anchoring proteins (AKAPs), which localize PKA through binding to the N-terminal dimerization domain of R subunits (7), to be affected by the fusion because this region is readily accessible in the structures of PKA holoenzymes (3)(4)(5)(6). AKIP1a, on the other hand, is an unconventional AKAP that binds to PKA near the N terminus of the C subunit (19).…”

“…Four isoforms of the R subunit (RIα, RIβ, RIIα, and RIIβ) are known. Structures of the R 2 :C 2 holoenzyme provide insight into mechanisms of allosteric control and reveal distinct, isoform-specific assemblies of the tetramers (3)(4)(5)(6), even though all R:C heterodimer associations are similar. Specificity of PKA activity is also controlled by localization.…”