PKA Compartmentalization via AKAP220 and AKAP12 Contributes to Endothelial Barrier Regulation

cAMP-mediated PKA signaling is the main known pathway involved in maintenance of the endothelial barrier. Tight regulation of PKA function can be achieved by discrete compartmentalization of the enzyme via physical interaction with A-kinase anchoring proteins (AKAPs). Here, we investigated the role of AKAPs 220 and 12 in endothelial barrier regulation. Analysis of human and mouse microvascular endothelial cells as well as isolated rat mesenteric microvessels was performed using TAT-Ahx-AKAPis peptide, designed to competitively inhibit PKA-AKAP interaction. In vivo microvessel hydraulic conductivity and in vitro transendothelial electrical resistance measurements showed that this peptide destabilized endothelial barrier properties, and dampened the cAMP-mediated endothelial barrier stabilization induced by forskolin and rolipram. Immunofluorescence analysis revealed that TAT-Ahx-AKAPis led to both adherens junctions and actin cytoskeleton reorganization. Those effects were paralleled by redistribution of PKA and Rac1 from endothelial junctions and by Rac1 inactivation. Similarly, membrane localization of AKAP220 was also reduced. In addition, depletion of either AKAP12 or AKAP220 significantly impaired endothelial barrier function and AKAP12 was also shown to interfere with cAMP-mediated barrier enhancement. Furthermore, immunoprecipitation analysis demonstrated that AKAP220 interacts not only with PKA but also with VE-cadherin and ß-catenin. Taken together, these results indicate that AKAP-mediated PKA subcellular compartmentalization is involved in endothelial barrier regulation. More specifically, AKAP220 and AKAP12 contribute to endothelial barrier function and AKAP12 is required for cAMP-mediated barrier stabilization.     

Molecular basis of AKAP specificity for PKA regulatory subunits

Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.     

Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding

Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.     

Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1

Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1) as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults.     

Protein targeting and calcium signaling microdomains in neuronal cells

Over the last several years, a number of optical imaging, physiological, and molecular studies have clarified the mechanisms underlying differential calcium signaling in the postsynaptic neuron. These studies have revealed the existence of membrane-associated calcium microdomains, which are often specifically coupled to distinct protein signaling pathways. In this review, we discuss how these signaling microdomains are organized and regulated, emphasizing the structural and molecular features of synaptic protein complexes containing the metabotropic and N-methyl-D-aspartate (NMDA) glutamate receptors and the L-type voltage-dependent calcium channels (VDCCs). We conclude with a discussion of how these different signaling complexes may interact with one another, relationships which may be important in orchestrating the complex calcium signaling underlying developmental and activity-dependent changes in synaptic function.     

AKAP350 modulates microtubule dynamics

AKAP350 is a multiply spliced type II protein kinase A-anchoring protein that localizes to the centrosomes in most cells and the Golgi apparatus in epithelial cells. Multiple studies suggest that AKAP350 is involved in microtubule nucleation at the centrosome. Our previous studies demonstrated that AKAP350 was necessary for the maintenance of Golgi apparatus integrity. These data suggested that AKAP350 might be necessary for normal cytoskeletal interactions with the Golgi. To examine the relationship of AKAP350 with the microtubule cytoskeleton, we analyzed the effect of the depletion of AKAP350 on microtubule regrowth after nocodazole treatment in HeLa cells. The decrease in AKAP350 expression with short interfering RNA induced a delay in microtubule elongation with no effect on microtubule aster formation. In contrast, overexpression of the centrosomal targeting domain of AKAP350 elicited alterations in aster formation, but did not affect microtubule elongation. RNA interference for AKAP350 also induced an increase in cdc42 activity during microtubule regrowth. Our data suggest that AKAP350 has a role in the remodeling of the microtubule cytoskeleton.     

PACAP decides neuronal laminar fate via PKA signaling in the developing cerebral cortex

Laminar formation in the developing cerebral cortex requires the precisely regulated generation of phenotype-specified neurons. To test the possible involvement of pituitary adenylate cyclase-activating polypeptide (PACAP) in this formation, we investigated the effects of PACAP administered into the telencephalic ventricular space of 13.5-day-old mouse embryos. PACAP partially inhibited the proliferation of cortical progenitors and altered the position and gene-expression profiles of newly generated neurons otherwise expected for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex. The former and latter effects were seen only when the parent progenitor cells were exposed to PACAP in the later and in earlier G1 phase, respectively; and these effects were suppressed by co-treatment with a protein kinase A (PKA) inhibitor. These observations suggest that PACAP participates in the processes forming the neuronal laminas in the developing cortex via the intracellular PKA pathway.     

Regulation of biomolecular condensate dynamics by signaling

Biomolecular condensates are mesoscopic biomolecular assemblies devoid of long range order that contribute to important cellular functions. They form reversibly, are stabilized by numerous but relatively weak intermolecular interactions, and their formation can be regulated by various cellular signals including changes in local concentration, post-translational modifications, energy-consuming processes, and biomolecular interactions. Condensates formed by liquid–liquid phase separation are initially liquid but are metastable relative to hydrogels or irreversible solids that have been associated with protein aggregation diseases and are stabilized by stronger, more permanent interactions. As a consequence of this, a series of cellular mechanisms are available to regulate not only biomolecular condensation but also the physical properties of the condensates.     

Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation

In diverse neuronal processes ranging from neuronal survival to synaptic plasticity cyclic adenosine monophosphate (cAMP)-dependent signaling is tightly connected with the protein kinase B (PKB)/Akt pathway but the precise nature of this connection remains unknown. In the current study we investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons and HT-4 cells. We demonstrate that PKA activation leads to a reduction of PKB/Akt phosphorylation, whereas activation of Epac has the opposite effect. This effect of Epac on PKB/Akt phosphorylation was mediated by Rap activation. The increase in PKB/Akt phosphorylation after Epac activation could be blocked by pretreatment with Epac2 siRNA and to a somewhat smaller extent by Epac1 siRNA. PKA, PKB/Akt and Epac were all shown to establish complexes with neuronal A-kinase anchoring protein150 (AKAP150). Interestingly, activation of Epac increased phosphorylation of PKB/Akt complexed to AKAP150. From experiments using PKA-binding deficient AKAP150 and peptides disrupting PKA anchoring to AKAPs, we conclude that AKAP150 acts as a key regulator in the two cAMP pathways to control PKB/Akt phosphorylation.     

On the role of subthreshold dynamics in neuronal signaling

The role of subthreshold dynamics in neuronal signaling is examined using periodic pulse train stimulation of the Fitzhugh-Nagumo (FN) model of nerve membrane excitability and results from the squid giant axon as an experimental data base. For a broad range of stimulus conditions the first pulse in a pulse train elicited an action potential, whereas all subsequent pulses elicited subthreshold responses, both in the axon and in the FN model. These results are not well described by the Hodgkin and Huxley 1952 model. Various different patterns of subthreshold responses, including chaotic dynamics, can be observed in both systems-the FN model and the axon-depending upon stimulus conditions. For some conditions action potentials are occasionally interspersed among the subthreshold events with randomly occurring interspike intervals. The randomness is directly attributable to the underlying subthreshold chaos-deterministic chaos-rather than to a stochastic noise source. We conclude that this mechanism may contribute to multimodal interspike interval histograms which have been observed from individual neurons throughout the nervous system.     

Dynamics of signaling by PKA

The catalytic and regulatory subunits of cAMP-dependent protein kinase (PKA) are highly dynamic signaling proteins. In its dissociated state the catalytic subunit opens and closes as it moves through its catalytic cycle. In this subunit, the core that is shared by all members of the protein kinase family is flanked by N- and C-terminal segments. Each are anchored firmly to the core by well-defined motifs and serve to stabilize the core. Protein kinases are not only catalysts, they are also scaffolds. One of their major functions is to bind to other proteins. In addition to its interactions with the N- and C- termini, the catalytic subunit interacts with its inhibitor proteins, PKI and the regulatory subunits. Both bind with subnanomolar affinity. To achieve this tight binding requires docking of a substrate mimetic to the active site cleft as well as a peripheral docking site. The peripheral site used by PKI is distinct from that used by RIalpha as revealed by a recent structure of a C:RIalpha complex. Upon binding to the catalytic subunit, the linker region of RIalpha becomes ordered. In addition, cAMP-binding domain A undergoes major conformational changes. RIalpha is a highly malleable protein. Using small angle X-ray scattering, the overall shape of the regulatory subunits and corresponding holoenzymes have been elucidated. These studies reveal striking and surprising isoform differences.     

Differential PKA activation and AKAP association determines cell fate in cancer cells

Background

The dependence of malignant properties of colorectal cancer (CRC) cells on IGF1R signaling has been demonstrated and several IGF1R antagonists are currently in clinical trials. Recently, we identified a novel pathway in which cAMP independent PKA activation by TGFβ signaling resulted in the destabilization of survivin/XIAP complex leading to increased cell death. In this study, we evaluated the effect of IGF1R inhibition or activation on PKA activation and its downstream cell survival signaling mechanisms.

Methods

Small molecule IGF1R kinase inhibitor OSI-906 was used to test the effect of IGF1R inhibition on PKA activation, AKAP association and its downstream cell survival signaling. In a complementary approach, ligand mediated activation of IGF1R was performed and AKAP/PKA signaling was analyzed for their downstream survival effects.

Results

We demonstrate that the inhibition of IGF1R in the IGF1R-dependent CRC subset generates cell death through a novel mechanism involving TGFβ stimulated cAMP independent PKA activity that leads to disruption of cell survival by survivin/XIAP mediated inhibition of caspase activity. Importantly, ligand mediated activation of the IGF1R in CRC cells results in the generation of cAMP dependent PKA activity that functions in cell survival by inhibiting caspase activity. Therefore, this subset of CRC demonstrates 2 opposing pathways organized by 2 different AKAPs in the cytoplasm that both utilize activation of PKA in a manner that leads to different outcomes with respect to life and death. The cAMP independent PKA activation pathway is dependent upon mitochondrial AKAP149 for its apoptotic functions. In contrast, Praja2 (Pja2), an AKAP-like E3 ligase protein was identified as a key element in controlling cAMP dependent PKA activity and pro-survival signaling. Genetic manipulation of AKAP149 and Praja2 using siRNA KD had opposing effects on PKA activity and survivin/XIAP regulation.

Conclusions

We had identified 2 cytoplasmic pathways dependent upon the same enzymatic activity with opposite effects on cell fate in terms of life and death. Understanding the specific mechanistic functions of IGF1R with respect to determining the PKA survival functions would have potential for impact upon the development of new therapeutic strategies by exploiting the IGF1R/cAMP-PKA survival signaling in cancer.

     

Isoform-specific targeting of PKA to multivesicular bodies

Although RII protein kinase A (PKA) regulatory subunits are constitutively localized to discrete cellular compartments through binding to A-kinase-anchoring proteins (AKAPs), RI subunits are primarily diffuse in the cytoplasm. In this paper, we report a novel AKAP-dependent localization of RIα to distinct organelles, specifically, multivesicular bodies (MVBs). This localization depends on binding to AKAP11, which binds tightly to free RIα or RIα in complex with catalytic subunit (holoenzyme). However, recruitment to MVBs occurs only with the release of PKA catalytic subunit (PKAc). This recruitment is reversed by reassociation with PKAc, and it is disrupted by the presence of AKAP peptides, mutations in the RIα AKAP-binding site, or knockdown of AKAP11. Cyclic adenosine monophosphate binding not only unleashes active PKAc but also leads to the targeting of AKAP11:RIα to MVBs. Therefore, we show that the RIα holoenzyme is part of a signaling complex with AKAP11, in which AKAP11 may direct RIα functionality after disassociation from PKAc. This model defines a new paradigm for PKA signaling.     

Discovery of allostery in PKA signaling

Cyclic AMP (cAMP)-dependent protein kinase (PKA) was the second protein kinase to be identified, and the PKA catalytic (C)-subunit serves as a prototype for the large protein kinase superfamily that contains over 500 gene products. The protein kinases regulate many biological functions in eukaryotic cells and are now also a major therapeutic target. The discovery of PKA nearly 50 years ago was quickly followed by the identification of the regulatory subunits that bind cAMP and release the catalytic activity from the holoenzyme. Thus in PKA we see the convergence of two major signaling mechanisms—protein phosphorylation and second messenger signaling through cAMP. Crystallography provides a foundation for understanding function, and detailed knowledge of the structure of the isolated regulatory (R)- and catalytic (C)-subunits has been extremely informative. Yet it is the R₂C₂ holoenzyme that predominates in cells, and the allosteric features of PKA signaling can only be fully appreciated by seeing the full-length protein. The symmetry and the quaternary constraints that one R:C heterodimer exerts on the other in the holoenzyme simply are not present in the isolated subunits or even in the R:C heterodimer.     

Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (R_Select) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of R_Select proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.