Ronit Ilouz

Assembly of allosteric macromolecular switches: lessons from PKA.

Protein kinases are dynamic molecular switches that have evolved to be only transiently activated. Kinase activity is embedded within a conserved kinase core, which is typically regulated by associated domains, linkers and interacting proteins. Moreover, protein kinases are often tethered to large macromolecular complexes to provide tighter spatiotemporal control. Thus, structural characterization of kinase domains alone is insufficient to explain protein kinase function and regulation in vivo. Recent progress in structural characterization of cyclic AMP-dependent protein kinase (PKA) exemplifies how our knowledge of kinase signalling has evolved by shifting the focus of structural studies from single kinase subunits to macromolecular complexes.

Inhibition of glycogen synthase kinase-3β by bivalent zinc ions: insight into the insulin-mimetic action of zinc

Zinc is an important trace element found in most body tissues as bivalent cations and has essential roles in human health. The insulin-like effect of zinc cations raises the possibility that they inhibit glycogen synthase kinase-3beta (GSK-3beta), a serine/threonine protein kinase linked with insulin resistance and type 2 diabetes. Here we show that physiological concentrations of zinc ions directly inhibit GSK-3beta in vitro in an uncompetitive manner. Treatment of HEK-293 cells with zinc enhanced glycogen synthase activity and increased the intracellular levels of beta-catenin, providing evidence for inhibition of endogenous GSK-3beta by zinc. Moreover, zinc ions enhanced glucose uptake 3-fold in isolated mouse adipocytes, an increase similar to activation with saturated concentrations of insulin. We propose that the in vivo insulin-mimetic actions of zinc are mediated via direct inhibition of endogenous GSK-3beta.

Challenges and opportunities with glycogen synthase kinase-3 inhibitors for insulin resistance and Type 2 diabetes treatment

The role of the serine/threonine protein kinase, glycogen synthase kinase-3 (GSK-3), in attenuating the insulin signalling pathway has led to the concept that inhibition of GSK-3 may have therapeutic benefits in the treatment of insulin resistance and Type 2 diabetes. Indeed, various selective GSK-3 inhibitors have been developed recently and have proven to promote insulin-like effects and to act as insulin sensitisers in both in vitro and in vivo systems. GSK-3 inhibition may thus present a new, effective approach for the treatment of insulin resistance and Type 2 diabetes. This review describes the qualifications of GSK-3 as a novel drug-discovery target for Type 2 diabetes and discusses the strategies and challenges in developing small-molecule inhibitors for this important protein kinase.

Localization and quaternary structure of the PKA RIβ holoenzyme

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 (R2:C2). 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 RIβ2:C2 is different from its closest homolog, RIα2:C2. The full-length RIβ2:C2 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 R1:C1 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.

Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling

Significance At the plasma membrane an array of more than 800 G protein-coupled receptors (GPCRs) receive, convert, amplify, and transmit incoming signals. Activated GPCRs team-up with intracellular scaffolding proteins to compartmentalize signal transmission. Scaffolds, such as β-arrestin and A-kinase anchoring proteins (AKAPs), function as a physical nexus between receptors and molecular switches. Typically, these receptor-bound AKAPs recruit protein kinase A (PKA) to assemble dedicated polyvalent signaling complexes that are spatially and temporally confined. Here, we report that the orphan GPCR, Gpr161, is a PKA substrate and also has an AKAP motif embedded in its C-terminal tail. Our results suggest that Gpr161, by directly recruiting type I PKA holoenzymes to the receptor, creates a cAMP-sensing signalosome. Furthermore, we propose that Gpr161 plays a role in recruiting isoform-specific PKA complexes to primary cilia. Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

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

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ 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. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization. DOI: http://dx.doi.org/10.7554/eLife.17681.001

D‐AKAP2:PKA RII:PDZK1 ternary complex structure: Insights from the nucleation of a polyvalent scaffold

A‐kinase anchoring proteins (AKAPs) regulate cAMP‐dependent protein kinase (PKA) signaling in space and time. Dual‐specific AKAP2 (D‐AKAP2/AKAP10) binds with high affinity to both RI and RII regulatory subunits of PKA and is anchored to transporters through PDZ domain proteins. Here, we describe a structure of D‐AKAP2 in complex with two interacting partners and the exact mechanism by which a segment that on its own is disordered presents an α‐helix to PKA and a β‐strand to PDZK1. These two motifs nucleate a polyvalent scaffold and show how PKA signaling is linked to the regulation of transporters. Formation of the D‐AKAP2: PKA binary complex is an important first step for high affinity interaction with PDZK1, and the structure reveals important clues toward understanding this phenomenon. In contrast to many other AKAPs, D‐AKAP2 does not interact directly with the membrane protein. Instead, the interaction is facilitated by the C‐terminus of D‐AKAP2, which contains two binding motifs—the D‐AKAP2AKB and the PDZ motif—that are joined by a short linker and only become ordered upon binding to their respective partner signaling proteins. The D‐AKAP2AKB binds to the D/D domain of the R‐subunit and the C‐terminal PDZ motif binds to a PDZ domain (from PDZK1) that serves as a bridging protein to the transporter. This structure also provides insights into the fundamental question of why D‐AKAP2 would exhibit a differential mode of binding to the two PKA isoforms.

The crystal structures of PKG Iβ (92-227) with cGMP and cAMP reveal the molecular details of cyclic-nucleotide binding - eScholarship

Although we have learned a great deal about the protein kinase superfamily from our structure/function studies of the free PKA catalytic (C) subunit and the free cAMP-bound regulatory (R) subunits, we did not understand how the C-subunit was inhibited in the holoenzyme complexes, nor did we understand at the molecular level how the holoenzymes were activated by cAMP. There are four functionally non-redundant R-subunits (RIα, RIβ, RIIα, and RIIβ) and each is assembled as a dimer. The N-terminal dimerization/docking (D/D) is fllowed by a flexible linker and two tandem camp binding domains. The D/D domain also serves as the docking site for A Kinase Anchoring Proteins (AKAPs) which serve as scaffolds that target the PKA holoenzyme to specific sites in the cell in close proximity to specific substrates. Isoform diversity of the R-subunits is a major mechanism for achieving specificity in PKA signaling. We describe here, for the first time, how PKA is assembled into diverse tetrameric holoenzymes and this allows us to begin to fully appreciate the role of the regulatory subunits in creating novel tetramers that are then recruited in unique ways to supramolecular complexes. How these scaffolds are then assembled as part of polyvalent PKA signaling scaffolds is our next challenge. By showing how the RII subunit is anchored to a PDZ domain through a dual specific AKAP, DAKAP2, we are beginning to understand how larger scaffolds are assembled and anchored to the C-termini of ion transporters. We are also exploring PKA signaling scaffolds at the mitochondria.Author(s): Taylor, Susan S; Ilouz, Ronit; Darshi, Manjula; Sarma, Ganapathy; Kornev, Alexandr; Zhang, Ping

The crystal structures of PKG Iβ (92-227) with cGMP and cAMP reveal the molecular details of cyclic-nucleotide binding

Although we have learned a great deal about the protein kinase superfamily from our structure/function studies of the free PKA catalytic (C) subunit and the free cAMP-bound regulatory (R) subunits, we did not understand how the C-subunit was inhibited in the holoenzyme complexes, nor did we understand at the molecular level how the holoenzymes were activated by cAMP. There are four functionally non-redundant R-subunits (RIα, RIβ, RIIα, and RIIβ) and each is assembled as a dimer. The N-terminal dimerization/docking (D/D) is fllowed by a flexible linker and two tandem camp binding domains. The D/D domain also serves as the docking site for A Kinase Anchoring Proteins (AKAPs) which serve as scaffolds that target the PKA holoenzyme to specific sites in the cell in close proximity to specific substrates. Isoform diversity of the R-subunits is a major mechanism for achieving specificity in PKA signaling. We describe here, for the first time, how PKA is assembled into diverse tetrameric holoenzymes and this allows us to begin to fully appreciate the role of the regulatory subunits in creating novel tetramers that are then recruited in unique ways to supramolecular complexes. How these scaffolds are then assembled as part of polyvalent PKA signaling scaffolds is our next challenge. By showing how the RII subunit is anchored to a PDZ domain through a dual specific AKAP, DAKAP2, we are beginning to understand how larger scaffolds are assembled and anchored to the C-termini of ion transporters. We are also exploring PKA signaling scaffolds at the mitochondria.Author(s): Taylor, Susan S; Ilouz, Ronit; Darshi, Manjula; Sarma, Ganapathy; Kornev, Alexandr; Zhang, Ping

PKA: assembly of dynamic macromolecular signaling.

Although we have learned a great deal about the protein kinase superfamily from our structure/function studies of the free PKA catalytic (C) subunit and the free cAMP-bound regulatory (R) subunits, we did not understand how the C-subunit was inhibited in the holoenzyme complexes, nor did we understand at the molecular level how the holoenzymes were activated by cAMP. There are four functionally non-redundant R-subunits (RIα, RIβ, RIIα, and RIIβ) and each is assembled as a dimer. The N-terminal dimerization/docking (D/D) is fllowed by a flexible linker and two tandem camp binding domains. The D/D domain also serves as the docking site for A Kinase Anchoring Proteins (AKAPs) which serve as scaffolds that target the PKA holoenzyme to specific sites in the cell in close proximity to specific substrates. Isoform diversity of the R-subunits is a major mechanism for achieving specificity in PKA signaling. We describe here, for the first time, how PKA is assembled into diverse tetrameric holoenzymes and this allows us to begin to fully appreciate the role of the regulatory subunits in creating novel tetramers that are then recruited in unique ways to supramolecular complexes. How these scaffolds are then assembled as part of polyvalent PKA signaling scaffolds is our next challenge. By showing how the RII subunit is anchored to a PDZ domain through a dual specific AKAP, DAKAP2, we are beginning to understand how larger scaffolds are assembled and anchored to the C-termini of ion transporters. We are also exploring PKA signaling scaffolds at the mitochondria.Author(s): Taylor, Susan S; Ilouz, Ronit; Darshi, Manjula; Sarma, Ganapathy; Kornev, Alexandr; Zhang, Ping