Discovery of a Cushing's syndrome protein kinase A mutant that biases signaling through type I AKAPs.

Adrenal Cushing's syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a Cushing's driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAcW196G is unexpectedly distinct from other described Cushing's variants, exhibiting retained association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of tryptophan-196 with glycine creates a 653-cubic angstrom cleft between the catalytic core of PKAcW196G and type II regulatory subunits (RII), but only a 395-cubic angstrom cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAcW196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing's syndrome.

Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.

The DNAJ-PKAc fusion kinase is a defining feature of fibrolamellar carcinoma (FLC). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. By combining proximity proteomics, biochemical analyses, and live-cell photoactivation microscopy, we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates, including proteins involved in translation and the anti-apoptotic factor Bcl-2-associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Tissue samples from patients with FLC exhibit increased levels of BAG2 in primary and metastatic tumors. Furthermore, drug studies implicate the DNAJ-PKAc/Hsp70/BAG2 axis in potentiating chemotherapeutic resistance. We find that the Bcl-2 inhibitor navitoclax enhances sensitivity to etoposide-induced apoptosis in cells expressing DNAJ-PKAc. Thus, our work indicates BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.

Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.

The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Immunoblot and immunohistochemical analyses of FLC patient samples correlate increased levels of BAG2 with advanced disease and metastatic recurrences. BAG2 is linked to Bcl-2, an anti-apoptotic factor that delays cell death. Pharmacological approaches tested if the DNAJ- PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in AML12 DNAJ-PKAc hepatocyte cell lines using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12 DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a biomarker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.

Classification of Cushing's syndrome PKAc mutants based upon their ability to bind PKI.

Cushing's syndrome is an endocrine disorder caused by excess production of the stress hormone cortisol. Precision medicine strategies have identified single allele mutations within the PRKACA gene that drive adrenal Cushing's syndrome. These mutations promote perturbations in the catalytic core of protein kinase A (PKAc) that impair autoinhibition by regulatory subunits and compartmentalization via recruitment into AKAP signaling islands. PKAcL205R is found in ∼45% of patients, whereas PKAcE31V, PKAcW196R, and L198insW and C199insV insertion mutants are less prevalent. Mass spectrometry, cellular, and biochemical data indicate that Cushing's PKAc variants fall into two categories: those that interact with the heat-stable protein kinase inhibitor PKI, and those that do not. In vitro activity measurements show that wild-type PKAc and W196R activities are strongly inhibited by PKI (IC50 < 1 nM). In contrast, PKAcL205R activity is not blocked by the inhibitor. Immunofluorescent analyses show that the PKI-binding variants wild-type PKAc, E31V, and W196R are excluded from the nucleus and protected against proteolytic processing. Thermal stability measurements reveal that upon co-incubation with PKI and metal-bound nucleotide, the W196R variant tolerates melting temperatures 10°C higher than PKAcL205. Structural modeling maps PKI-interfering mutations to a ∼20 Šdiameter area at the active site of the catalytic domain that interfaces with the pseudosubstrate of PKI. Thus, Cushing's kinases are individually controlled, compartmentalized, and processed through their differential association with PKI.

Proximity biotinylation to define the local environment of the protein kinase A catalytic subunit in adrenal cells.

Mutant protein kinase A catalytic subunit (PKAc) drives adrenal Cushing's syndrome, though its signaling interactions remain unclear. This protocol details steps to use live-cell proximity labeling to identify subcellular compartments and proteins closely associated with variants of PKAc in human adrenal cells. We include instructions for clonal cell line generation, live biotin labeling of proximal proteins, isolation of biotinylated proteins, and sample processing for proteomic analysis using the biotin ligase miniTurbo with wild-type and mutant PKAc.1,2 For complete details on the use and execution of this protocol, please refer to Omar et al. (2022).3.

Mislocalization of protein kinase A drives pathology in Cushing's syndrome.

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals.

Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes.

Generation of the prototypic second messenger cAMP instigates numerous signaling events. A major intracellular target of cAMP is Protein kinase A (PKA), a Ser/Thr protein kinase. Where and when this enzyme is activated inside the cell has profound implications on the functional impact of PKA. It is now well established that PKA signaling is focused locally into subcellular signaling "islands" or "signalosomes." The A-Kinase Anchoring Proteins (AKAPs) play a critical role in this process by dictating spatial and temporal aspects of PKA action. Genetically encoded biosensors, small molecule and peptide-based disruptors of PKA signaling are valuable tools for rigorous investigation of local PKA action at the biochemical level. This chapter focuses on approaches to evaluate PKA signaling islands, including a simple assay for monitoring the interaction of an AKAP with a tunable PKA holoenzyme. The latter approach evaluates the composition of PKA holoenzymes, in which regulatory subunits and catalytic subunits can be visualized in the presence of test compounds and small-molecule inhibitors.

Targeting an anchored phosphatase-deacetylase unit restores renal ciliary homeostasis.

Pathophysiological defects in water homeostasis can lead to renal failure. Likewise, common genetic disorders associated with abnormal cytoskeletal dynamics in the kidney collecting ducts and perturbed calcium and cAMP signaling in the ciliary compartment contribute to chronic kidney failure. We show that collecting ducts in mice lacking the A-Kinase anchoring protein AKAP220 exhibit enhanced development of primary cilia. Mechanistic studies reveal that AKAP220-associated protein phosphatase 1 (PP1) mediates this phenotype by promoting changes in the stability of histone deacetylase 6 (HDAC6) with concomitant defects in actin dynamics. This proceeds through a previously unrecognized adaptor function for PP1 as all ciliogenesis and cytoskeletal phenotypes are recapitulated in mIMCD3 knock-in cells expressing a phosphatase-targeting defective AKAP220-ΔPP1 mutant. Pharmacological blocking of local HDAC6 activity alters cilia development and reduces cystogenesis in kidney-on-chip and organoid models. These findings identify the AKAP220-PPI-HDAC6 pathway as a key effector in primary cilia development.

AKAP Signaling Islands: Venues for Precision Pharmacology.

Regulatory enzymes often have different roles in distinct subcellular compartments. Yet, most drugs indiscriminately saturate the cell. Thus, subcellular drug-delivery holds promise as a means to reduce off-target pharmacological effects. A-kinase anchoring proteins (AKAPs) sequester combinations of signaling enzymes within subcellular microdomains. Targeting drugs to these 'signaling islands' offers an opportunity for more precise delivery of therapeutics. Here, we review mechanisms that bestow protein kinase A (PKA) versatility inside the cell, appraise recent advances in exploiting AKAPs as platforms for precision pharmacology, and explore the impact of methodological innovations on AKAP research.

An acquired scaffolding function of the DNAJ-PKAc fusion contributes to oncogenic signaling in fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric enzyme consisting of a chaperonin-binding domain fused to the Cα subunit of protein kinase A. Biochemical analyses of clinical samples reveal that a unique property of this fusion enzyme is the ability to recruit heat shock protein 70 (Hsp70). This cellular chaperonin is frequently up-regulated in cancers. Gene-editing of mouse hepatocytes generated disease-relevant AML12DNAJ-PKAc cell lines. Further analyses indicate that the proto-oncogene A-kinase anchoring protein-Lbc is up-regulated in FLC and functions to cluster DNAJ-PKAc/Hsp70 sub-complexes with a RAF-MEK-ERK kinase module. Drug screening reveals Hsp70 and MEK inhibitor combinations that selectively block proliferation of AML12DNAJ-PKAc cells. Phosphoproteomic profiling demonstrates that DNAJ-PKAc biases the signaling landscape toward ERK activation and engages downstream kinase cascades. Thus, the oncogenic action of DNAJ-PKAc involves an acquired scaffolding function that permits recruitment of Hsp70 and mobilization of local ERK signaling.

Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins.

A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18γ isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca2+ currents in situ. Live-cell imaging of AKAP18γ V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18ε: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms.

CNS Neurons Deposit Laminin α5 to Stabilize Synapses.

Synapses in the developing brain are structurally dynamic but become stable by early adulthood. We demonstrate here that an α5-subunit-containing laminin stabilizes synapses during this developmental transition. Hippocampal neurons deposit laminin α5 at synapses during adolescence as connections stabilize. Disruption of laminin α5 in neurons causes dramatic fluctuations in dendritic spine head size that can be rescued by exogenous α5-containing laminin. Conditional deletion of laminin α5 in vivo increases dendritic spine size and leads to an age-dependent loss of synapses accompanied by behavioral defects. Remaining synapses have larger postsynaptic densities and enhanced neurotransmission. Finally, we provide evidence that laminin α5 acts through an integrin α3ß1-Abl2 kinase-p190RhoGAP signaling cascade and partners with laminin ß2 to regulate dendritic spine density and behavior. Together, our results identify laminin α5 as a stabilizer of dendritic spines and synapses in the brain and elucidate key cellular and molecular mechanisms by which it acts.

Corticosteroid-induced dendrite loss and behavioral deficiencies can be blocked by activation of Abl2/Arg kinase.

Stressor exposure induces neuronal remodeling in specific brain regions. Given the persistence of stress-related illnesses, key next steps in determining the contributions of neural structure to mental health are to identify cell types that fail to recover from stressor exposure and to identify "trigger points" and molecular underpinnings of stress-related neural degeneration. We evaluated dendrite arbor structure on hippocampal CA1 pyramidal neurons before, during, and following prolonged exposure to one key mediator of the stress response - corticosterone (cortisol in humans). Basal dendrite arbors progressively simplified during a 3-week exposure period, and failed to recover when corticosterone was withdrawn. Corticosterone exposure decreased levels of the dendrite stabilization factor Abl2/Arg nonreceptor tyrosine kinase and phosphorylation of its substrates p190RhoGAP and cortactin within 11days, suggesting that disruption of Arg-mediated signaling may trigger dendrite arbor atrophy and, potentially, behavioral abnormalities resulting from corticosterone exposure. To test this, we administered the novel, bioactive Arg kinase activator, 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin, 5-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,4-imidazolidinedione (DPH), in conjunction with corticosterone. We found that repeated treatment corrected CA1 arbor structure, otherwise simplified by corticosterone. DPH also corrected corticosterone-induced errors in a hippocampal-dependent reversal learning task and anhedonic-like behavior. Thus, pharmacological compounds that target cytoskeletal regulators, rather than classical neurotransmitter systems, may interfere with stress-associated cognitive decline and mental health concerns.

Long-Term Live-Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI-SiR.

Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR. Using DiI-SiR, we visualized filopodia dynamics in HeLa cells over 25 min at 0.5 s temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 s temporal resolution. HIDE probes such as DiI-SiR are non-toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super-resolution over biologically relevant timescales.

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2.

Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.

Extracellular matrix control of dendritic spine and synapse structure and plasticity in adulthood.

Dendritic spines are the receptive contacts at most excitatory synapses in the central nervous system. Spines are dynamic in the developing brain, changing shape as they mature as well as appearing and disappearing as they make and break connections. Spines become much more stable in adulthood, and spine structure must be actively maintained to support established circuit function. At the same time, adult spines must retain some plasticity so their structure can be modified by activity and experience. As such, the regulation of spine stability and remodeling in the adult animal is critical for normal function, and disruption of these processes is associated with a variety of late onset diseases including schizophrenia and Alzheimer's disease. The extracellular matrix (ECM), composed of a meshwork of proteins and proteoglycans, is a critical regulator of spine and synapse stability and plasticity. While the role of ECM receptors in spine regulation has been extensively studied, considerably less research has focused directly on the role of specific ECM ligands. Here, we review the evidence for a role of several brain ECM ligands and remodeling proteases in the regulation of dendritic spine and synapse formation, plasticity, and stability in adults.