Protein Kinase A Substrates Research Articles

ARPP19 phosphorylation site evolution and the switch in cAMP control of oocyte maturation in vertebrates.

cAMP-PKA signaling initiates the crucial process of oocyte meiotic maturation in many animals, but inhibits it in vertebrates. To address this 'cAMP paradox', we exchanged the key PKA substrate ARPP19 between representative species, the vertebrate Xenopus and the cnidarian Clytia, comparing its phosphorylation and function. We found that, as in Xenopus, Clytia maturing oocytes undergo ARPP19 phosphorylation on a highly conserved Gwl site, which inhibits PP2A and promotes M-phase entry. In contrast, despite a PKA phosphorylation signature motif recognizable across most animals, Clytia ARPP19 was only poorly phosphorylated by PKA in vitro and in vivo. Furthermore, unlike Xenopus ARPP19, exogenous Clytia ARPP19 did not delay Xenopus oocyte maturation. We conclude that, in Clytia, ARPP19 does not intervene in oocyte maturation initiation because of both poor recognition by PKA and the absence of effectors that mediate vertebrate oocyte prophase arrest. We propose that ancestral ARPP19 phosphorylated by Gwl has retained a key role in M-phase across eukaryotes and has acquired new functions during animal evolution mediated by enhanced PKA phosphorylation, allowing co-option into oocyte maturation regulation in the vertebrate lineage.

CABS1 Is Essential for Progressive Motility and the Integrity of Fibrous Sheath in Mouse Epididymal Spermatozoa.

The calcium-binding protein spermatid-associated 1 (CABS1) localizes to the principal piece of mature sperm flagella. Deletion of CABS1 results in subfertility in male mice, possibly due to an impaired annulus in the sperm flagella. However, it is unknown whether there are other mechanisms by which CABS1 affects male fertility. Our current investigation has uncovered that CABS1 is located in the midsection of the flagellum in testicular sperm and the principal piece in epididymal sperm. Moreover, male mice lacking CABS1 exhibit a defect in the progressive motility of sperm. Furthermore, the regulation of calcium levels, which has been reported to have a significant impact on sperm motility, capacitation, and the acrosome reaction, is also affected when sperm are exposed to alkalized high-salt buffer (pH 8.0) and progesterone (100 μM) in Cabs1-null spermatozoa. This alteration in calcium response may contribute to changes in the phosphorylation of PKA substrates and subsequent phosphorylation of tyrosine residues. Additionally, the absence of CABS1 leads to a defective fibrous sheath and abnormal configuration of doublet microtubules in post-testicular sperm. These findings indicate that the absence of CABS1 also disrupts the structural integrity of the fibrous sheath, resulting in male subfertility. The highly conserved nature of CABS1 in humans suggests that it could potentially be a contributing factor to asthenozoospermia in men.

A-Kinase-Anchoring-Protein Subtypes Differentially Regulate GPCR Signaling and Function in Human Airway Smooth Muscle.

A-kinase-anchoring proteins (AKAPs) act as scaffold proteins that anchor the regulatory subunits of the cAMP-dependent protein kinase A (PKA) to coordinate and compartmentalize signaling elements and signals downstream of Gs-coupled G protein-coupled receptors (GPCRs). The beta-2-adrenoceptor (β2AR), as well as the Gs-coupled EP2 and EP4 receptor subtypes of the E-prostanoid (EP) receptor subfamily, are effective regulators of multiple airway smooth muscle (ASM) cell functions whose dysregulation contributes of asthma pathobiology. Here, we identify specific roles of the AKAPs Ezrin and Gravin, in differentially regulating PKA substrates downstream of the β2AR, EP2 receptor (EP2R) and EP4 receptor (EP4R). Knockdown of Ezrin, Gravin, or both in primary human ASM cells caused differential phosphorylation of the PKA substrates vasodilator-stimulated phosphoprotein (VASP) and heat shock protein 20 (HSP20). Ezrin knockdown, as well as combined Ezrin + Gravin knockdown significantly reduced the induction of phospho-VASP and phospho-HSP20 by β2AR, EP2R, and EP4R agonists. Gravin knockdown inhibited the induction of phospho-HSP20 by β2AR, EP2R, and EP4R agonists. Knockdown of Ezrin, Gravin, or both also attenuated histamine-induced phosphorylation of MLC20. Moreover, knockdown of Ezrin, Gravin or both suppressed the inhibitory effects of Gs-coupled receptor agonists on cell migration in ASM cells. These findings demonstrate the role of AKAPs in regulating Gs-coupled GPCR signaling and function in ASM, and suggest the therapeutic utility of targeting specific AKAP family members in the management of asthma.

Targeted Degradation of Protein Kinase A via a Stapled Peptide PROTAC.

Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that bind and recruit an E3 ubiquitin ligase to a targeted protein of interest, often through the utilization of a small molecule inhibitor. To expand the possible range of kinase targets that can be degraded by PROTACs, we sought to develop a PROTAC utilizing a hydrocarbon-stapled peptide as the targeting agent to bind the surface of a target protein of interest. In this study, we describe the development of a proteolysis-targeting chimera, dubbed Stapled Inhibitor Peptide - PROTAC or StIP-TAC, linking a hydrocarbon-stapled peptide with an E3 ligase ligand for targeted degradation of Protein Kinase A (PKA). This StIP-TAC molecule stimulated E3-mediated protein degradation of PKA, and this effect could be reversed by the addition of the proteasomal inhibitor MG-132. Further, StIP-TAC treatment led to a significant reduction in PKA substrate phosphorylation. Since many protein targets of interest lack structural features that make them amenable to small molecule targeting, development of StIP-TACs may broaden the potential range of protein targets using a PROTAC-mediated proteasomal degradation approach.

Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

Ketogenic diet administration later in life improves memory by modifying the synaptic cortical proteome via the PKA signaling pathway in aging mice

Aging compromises brain function leading to cognitive decline. A cyclic ketogenic diet (KD) improves memory in aged mice after long-term administration; however, short-term effects later in life and the molecular mechanisms that govern such changes remain unclear. Here, we explore the impact of a short-term KD treatment starting at elderly stage on brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings reveal that KD improves working memory and hippocampal LTP. Furthermore, the synaptosome proteome of aged mice fed a KD long-term evidence changes predominantly at the presynaptic compartment associated to the protein kinase A (PKA) signaling pathway. These findings were corroborated invivo by western blot analysis, with high BDNF abundance and PKA substrate phosphorylation. Overall, we show that a KD modifies brain function even when it is administered later in life and recapitulates molecular features of long-term administration, including the PKA signaling pathway, thus promoting synaptic plasticity at advanced age.

Conservation of Protein Kinase A Substrates in the Cnidarian Coral Spermatozoa Among Animals and Their Molecular Evolution.

The coral Acropora spp., known for its reef-building abilities, is a simultaneous hermaphroditic broadcast spawning species. Acropora spp. release gametes into seawater, activating sperm motility. This activation is mediated by adenylyl cyclase (AC) and protein kinase A (PKA). Notably, membrane-permeable cAMP (8-bromo-cAMP) promotes sperm motility activation of Acropora florida. While the signal transduction for PKA-dependent motility activation is highly conserved among animals, the downstream signaling of PKA remains unclear. In this study, we used mass spectrometry (MS) analyses to identify sperm proteins in the coral Acropora digitifera, as well as the serine/threonine residues of potential PKA substrates, and then, we investigated the conservation of these proteins from corals to vertebrates. We identified 148 sperm proteins of A. digitifera with typical PKA recognition motifs, namely RRXT and RRXS. We subsequently used ORTHOSCOPE to screen for orthologs encoding these 148 proteins from corals to vertebrates. Among the isolated orthologs, we identified positive selection in 48 protein-encoding genes from 18 Acropora spp. Subsequently, we compared the conservation rates of the PKA phosphorylation motif residues between the orthologs under positive and purifying selections. Notably, the serine residues of the orthologs under positive selection were more conserved. Therefore, adaptive evolution might have occurred in the orthologs of PKA substrate candidates from corals to vertebrates, accompanied by phosphorylation residue conservation. Collectively, our findings suggest that while PKA signal transduction, including substrates in sperm, may have been conserved, the substrates may have evolved to adapt to diverse fertilization conditions, such as synchronous broadcast spawning.

Phosphorylation of the compartmentalized PKA substrate TAF15 regulates RNA-protein interactions.

Spatiotemporal-controlled second messengers alter molecular interactions of central signaling nodes for ensuring physiological signal transmission. One prototypical second messenger molecule which modulates kinase signal transmission is the cyclic-adenosine monophosphate (cAMP). The main proteinogenic cellular effectors of cAMP are compartmentalized protein kinase A (PKA) complexes. Their cell-type specific compositions precisely coordinate substrate phosphorylation and proper signal propagation which is indispensable for numerous cell-type specific functions. Here we present evidence that TAF15, which is implicated in the etiology of amyotrophic lateral sclerosis, represents a novel nuclear PKA substrate. In cross-linking and immunoprecipitation experiments (iCLIP) we showed that TAF15 phosphorylation alters the binding to target transcripts related to mRNA maturation, splicing and protein-binding related functions. TAF15 appears to be one of multiple PKA substrates that undergo RNA-binding dynamics upon phosphorylation. We observed that the activation of the cAMP-PKA signalingaxis caused a change in the composition of a collection of RNA species that interact with TAF15. This observation appears to be a broader principle in the regulation of molecular interactions, as we identified a significant enrichment of RNA-binding proteins within endogenous PKA complexes. We assume that phosphorylation of RNA-binding domains adds another layer of regulation to binary protein-RNAs interactions with consequences to RNA features including binding specificities, localization, abundance and composition.

Protein kinase A is a functional component of focal adhesions

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify 53 high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3)-a well-established molecular scaffold, regulator of cell migration, and a component of focal and fibrillar adhesions-as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

The focal adhesion protein talin is a mechanically gated A-kinase anchoring protein

Protein kinase A (PKA) is a ubiquitous, promiscuous kinase whose activity is specified through subcellular localization mediated by A-kinase anchoring proteins (AKAPs). PKA has complex roles as both an effector and a regulator of integrin-mediated cell adhesion to extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), suggesting the existence of one or more FA AKAPs. Using a promiscuous biotin ligase fused to PKA type-IIα regulatory (RIIα) subunits and subcellular fractionation, we identify the archetypal FA protein talin1 as an AKAP. Talin is a large, mechanosensitive scaffold that directly links integrins to actin filaments and promotes FA assembly by recruiting additional components in a force-dependent manner. The rod region of talin1 consists of 62 α-helices bundled into 13 rod domains, R1 to R13. Direct binding assays and NMR spectroscopy identify helix41 in the R9 subdomain of talin as the PKA binding site. PKA binding to helix41 requires unfolding of the R9 domain, which requires the linker region between R9 and R10. Experiments with single molecules and in cells manipulated to alter actomyosin contractility demonstrate that the PKA-talin interaction is regulated by mechanical force across the talin molecule. Finally, talin mutations that disrupt PKA binding also decrease levels of total and phosphorylated PKA RII subunits as well as phosphorylation of VASP, a known PKA substrate, within FA. These observations identify a mechanically gated anchoring protein for PKA, a force-dependent binding partner for talin1, and a potential pathway for adhesion-associated mechanotransduction.

Dibutyl phthalate disrupts [Ca2+]i, reactive oxygen species, [pH]i, protein kinases and mitochondrial activity, impairing sperm function

To explore the mechanism of sperm dysfunction caused by dibutyl phthalate (DBP), the effects of DBP on intracellular [Ca2+] and [pH], reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, phosphorylation of protein kinase A (PKA) substrate proteins and phosphotyrosine (p-Tyr) proteins, sperm motility, spontaneous acrosome reaction, and tail bending were examined in mouse spermatozoa. At 100 µg/mL, DBP significantly increased tail bending and [Ca2+]i. Interestingly, DBP showed biphasic effects on [pH]i. DBP at 10–100 µg/mL significantly decreased sperm motility. Similarly, Ca2+ ionophore A23187 decreased [pH]i sperm motility, suggesting that DBP-induced excessive [Ca2+]i decreased sperm motility. DBP significantly increased ROS and LPO. DBP at 100 µg/mL significantly decreased mPTP closing, MMP, and ATP levels in spermatozoa, as did H2O2, indicative of ROS-mediated mitochondrial dysfunction caused by DBP. DBP as well as H2O2 increased p-Tyr sperm proteins and phosphorylated PKA substrate sperm proteins. DBP at 1–10 µg/mL significantly increased the spontaneous acrosome reaction, suggesting that DBP can activate sperm capacitation. Altogether, DBP showed a biphasic effect on intracellular signaling in spermatozoa. At concentrations relevant to seminal ortho-phthalate levels, DBP activates [pH]i, protein tyrosine kinases and PKA via physiological levels of ROS generation, potentiating sperm capacitation. DBP at high doses excessively raises [Ca2+]i and ROS and disrupts [pH]i, impairing the mitochondrial function, tail structural integrity, and sperm motility.

CLCF1 inhibits energy expenditure via suppressing brown fat thermogenesis.

Brown adipose tissue (BAT) is the main site of nonshivering thermogenesis which plays an important role in thermogenesis and energy metabolism. However, the regulatory factors that inhibit BAT activity remain largely unknown. Here, cardiotrophin-like cytokine factor 1 (CLCF1) is identified as a negative regulator of thermogenesis in BAT. Adenovirus-mediated overexpression of CLCF1 in BAT greatly impairs the thermogenic capacity of BAT and reduces the metabolic rate. Consistently, BAT-specific ablation of CLCF1 enhances the BAT function and energy expenditure under both thermoneutral and cold conditions. Mechanistically, adenylate cyclase 3 (ADCY3) is identified as a downstream target of CLCF1 to mediate its role in regulating thermogenesis. Furthermore, CLCF1 is identified to negatively regulate the PERK-ATF4 signaling axis to modulate the transcriptional activity of ADCY3, which activates the PKA substrate phosphorylation. Moreover, CLCF1 deletion in BAT protects the mice against diet-induced obesity by promoting BAT activation and further attenuating impaired glucose and lipid metabolism. Therefore, our results reveal the essential role of CLCF1 in regulating BAT thermogenesis and suggest that inhibiting CLCF1 signaling might be a potential therapeutic strategy for improving obesity-related metabolic disorders.

AKAP3-mediated type I PKA signaling is required for mouse sperm hyperactivation and fertility†.

The protein kinase A (PKA) signaling pathway, which mediates protein phosphorylation, is important for sperm motility and male fertility. This process relies on A-kinase anchoring proteins that organize PKA and its signalosomes within specific subcellular compartments. Previously, it was found that the absence of A-kinase anchoring protein 3 (AKAP3) leads to multiple morphological abnormalities in mouse sperm. But how AKAP3 regulates sperm motility is yet to be elucidated. AKAP3 has two amphipathic domains, here named dual and RI, in its N-terminus. These domains are responsible for binding regulatory subunits I alpha (RIα) and II alpha (RIIα) of PKA and for RIα only, respectively. Here, we generated mutant mice lacking the dual and RI domains of AKAP3. It was found that the deletion of these domains caused male mouse infertile, accompanied by mild defects in the fibrous sheath of sperm tails. Additionally, the levels of serine/threonine phosphorylation of PKA substrates and tyrosine phosphorylation decreased in the mutant sperm, which exhibited a defect in hyperactivation under capacitation conditions. The protein levels of PKA subunits remained unchanged. But, interestingly, the regulatory subunit RIα was mis-localized from principal piece to midpiece of sperm tail, whereas this was not observed for RIIα. Further protein-protein interaction assays revealed a preference for AKAP3 to bind RIα over RIIα. Collectively, our findings suggest that AKAP3 is important for sperm hyperactivity by regulating type-I PKA signaling pathway mediated protein phosphorylation via its dual and RI domains.

Paralog-specific TTC30 regulation of Sonic hedgehog signaling

The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A–IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly.

Inhibition of PKA/CREB1 pathway confers sensitivity to ferroptosis in non-small cell lung cancer

Ferroptosis is a type of regulated cell death characterized by iron accumulation and lipid peroxidation. The molecular mechanisms underlying ferroptosis regulation in non-small cell lung cancer (NSCLC) are poorly understood. In this study, we found that protein kinase A (PKA) inhibition enhanced ferroptosis susceptibility in NSCLC cells, as evidenced by reduced cell viability and increased lipid peroxidation. We further identified cAMP-responsive element protein 1 (CREB1), a transcription factor and a substrate of PKA, as a key regulator of ferroptosis. Knockdown of CREB1 sensitized NSCLC cells to ferroptosis inducers (FINs) and abolished the effects of PKA inhibitor and agonist, revealing the pivotal role of CREB1 in ferroptosis regulation. Using a high-throughput screening approach and subsequent validation by chromatin immunoprecipitation (ChIP) and dual-luciferase assays, we discovered that CREB1 transcriptionally activated stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids. SCD conferred ferroptosis resistance by decreasing the availability of polyunsaturated fatty acids for lipid peroxidation, and its overexpression rescued the effect of CREB1 knockdown on ferroptosis in vitro. Besides, CREB1 knockdown suppressed xenograft tumor growth in the presence of Imidazole Ketone Erastin (IKE), a potent FIN, and this effect was reversed by SCD. Finally, we showed that high expression of CREB1 was associated with poor prognosis in NSCLC patients from public datasets and our institution. Collectively, this study illustrates the effect of PKA/CREB1/SCD axis in regulating ferroptosis of NSCLC, targeting this pathway may provide new strategies for treating NSCLC patients.

Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide.

The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

THU507 Tumor Activin A Mediates Cachexia Primarily Through White Adipose Tissue Hydrolysis In Pancreatic Ductal Adenocarcinoma

Abstract Disclosure: S. Yu: None. Y. Luan: None. R. Dong: None. A. Abazarikia: None. S. Kim: None. Pancreatic ductal adenocarcinoma (PDAC) commonly harbors mutations in KRAS and TP53 genes and its overall 5-year survival rate is only 10% in the US. Among risk factors associated with the dismal PDAC prognosis, cachexia accounts for about 30% of PDAC-related death and is present in nearly 80% of PDAC patients. Significant adipose tissue loss followed by skeletal muscle loss characterizes PDAC-related cachexia in humans and mice. Accumulated evidence indicates that activin A, a homodimer of inhibin βA that is encoded by the INHBA gene, is a recognized key cachexia mediator as supported by mitigation of weight loss by activin A antagonists in PDAC-bearing mice. However, much remains unclear regarding the primary source cells of activin A and the mechanisms by which activin A mediates cachexia in PDAC. To address the unknowns, we employed CRISPR/Cas9 system for Inhba gene knock-down (KD) in mouse KPC (KrasG12D and Trp53R172H) PDAC cells and generated orthotopic mice using Inhba KD or control KPC cells. Sham group which received the same operation except for cell injection was included. Body weight was measured on a daily basis, and blood and white adipose tissues (WATs) and skeletal muscle tissues were harvested on post-surgery day 12. We found that, when compared to the Sham group, injection of control KPC cells significantly elevated serum activin A levels up to 6 ng/mL and temporally decreased the average body weight in the orthotopic mice (Ctrl group), whereas Inhba gene KD significantly reduced serum activin A levels by ∼ 2 ng/mL and prevented weight loss in the orthotopic mice (KD group). When compared to the Sham and KD groups, notable reductions in organ weights of epididymal, mesenteric or subcutaneous WATs were found in the Ctrl group, however, weights for gastrocnemius and plantaris or soleus muscle tissues were statistically similar between the Ctrl and KD groups at post-surgery day 12. Correspondingly, the average serum free fatty acids levels were higher in the Ctrl group than the KD group. Histological observations support that dramatic adipocyte size reductions in WATs were prevented by serum activin A suppression. Immunofluorescence analysis showed that WATs from the Ctrl group had considerably higher protein expression of hormone-sensitive or adipose triglyceride lipase than the KD and Sham groups. Furthermore, phosphorylation of SMAD3 and phosphorylated PKA substrate which regulate adipocyte lipolysis were intensely stained in WATs from the Ctrl group when compared to the KD and Sham groups. Collectively, our findings identify PDAC cancer cells as the major source of activin A in PDAC and suggest systemic activin A as a potential mediator of PDAC-related adipose tissue loss. The current findings warrant further investigation to address clinical relevance. Presentation: Thursday, June 15, 2023

S14-Phosphorylated RPN6 Mediates Proteasome Activation by PKA and Alleviates Proteinopathy.

A better understanding of the regulation of proteasome activities can facilitate the search for new therapeutic strategies. A cell culture study shows that PKA (cAMP-dependent protein kinase or protein kinase A) activates the 26S proteasome by pS14-Rpn6 (serine14-phosphorylated Rpn6), but this discovery and its physiological significance remain to be established in vivo. Male and female mice with Ser14 of Rpn6 (regulatory particle non-ATPase 6) mutated to Ala (S14A [Rpn6/Psmd11S14A]) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created and used along with cells derived from them. cAMP/PKA were manipulated pharmacologically. Ubiquitin-proteasome system functioning was evaluated with the GFPdgn (green fluorescence protein with carboxyl fusion of the CL1 degron) reporter mouse and proteasomal activity assays. Impact of S14A and S14D on proteotoxicity was tested in mice and cardiomyocytes overexpressing the misfolded protein R120G-CryAB (R120G [arginine120 to glycine missense mutant alpha B-crystallin]). PKA activation increased pS14-Rpn6 and 26S proteasome activities in wild-type but not S14A embryonic fibroblasts (mouse embryonic fibroblasts), adult cardiomyocytes, and mouse hearts. Basal 26S proteasome activities were significantly greater in S14D myocardium and adult mouse cardiomyocytes than in wild-type counterparts. S14D::GFPdgn mice displayed significantly lower myocardial GFPdgn protein but not mRNA levels than GFPdgn mice. In R120G mice, a classic model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with nontransgenic littermates, which was not always associated with reduction of other phosphorylated PKA substrates. Cultured S14D neonatal cardiomyocytes displayed significantly faster proteasomal degradation of R120G than wild-type neonatal cardiomyocytes. Compared with R120G mice, S14D/S14D::R120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates, and of aberrant CryAB (alpha B-crystallin) protein aggregates, less fetal gene reactivation, and cardiac hypertrophy, and delays in cardiac malfunction. This study establishes in animals that pS14-Rpn6 mediates the activation of 26S proteasomes by PKA and that the reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifying a new therapeutic target to reduce cardiac proteotoxicity.

Prorelaxant E-type Prostanoid Receptors Functionally Partition to Different Procontractile Receptors in Airway Smooth Muscle.

Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction-magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively-to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 × 10-7 vs. 2.2 × 10-6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259-induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.

1593-P: Insulin-Degrading Enzyme (IDE) Regulates Glucagon Signalling and Mitochondrial Respiration in Hepatocytes

IDE is a ubiquitous metalloprotease with cytosolic and mitochondrial subcellular localization that degrades insulin and glucagon. People with T2D and diet-induced obese mice show lower hepatic IDE levels. We revealed a key role of IDE on the insulin-mediated repression of hepatic gluconeogenesis, but its function on glucagon-dependent activation of gluconeogenesis and mitochondrial respiration in hepatocytes remains completely unknown. Here, we aim to elucidate the role of IDE on glucagon signalling and its impact on gluconeogenesis and energy metabolism in hepatocytes. Liver homogenized and primary hepatocytes obtained from L-IDE-KO mice (deletion of IDE in liver) showed decreased expression of glucagon receptor (~60%), CREB protein (~40%), and lower phosphorylation of CREB (~50%) upon glucagon stimulation compared to controls. Similar results were found in AML12-shRNA-Ide cells, in which IDE protein levels were reduced by ~50%. Additionally, glucagon stimulation resulted in lower (~30%) cAMP levels and diminished phosphorylation of PKA substrates in AML12-shRNA-Ide. Surprisingly, these alterations in glucagon signalling paralleled with ~20-fold increases in the expression of the gluconeogenic genes G6p6 and Pck1. Of note, basal and uncoupler-stimulated respiration increased ~4-fold in AML12-shRNA-Ide in parallel with a ~2-fold increment of mitochondrial and glycolytic ATP production. Finally, similar mitochondrial phenotype was found in human hepatocytes lacking IDE (HepG2-IDE-KO cells), which exhibited higher FoxO1 levels and fragmented mitochondria. The effects on mitochondrial respiration were independent of IDE's proteolytic activity. In summary, reduced IDE expression in hepatocytes has a deleterious effect on glucagon signalling leading to up-regulated gluconeogenesis and mitochondrial respiration. We conclude that IDE is a mechanistic link to couple hepatic gluconeogenesis with mitochondrial energy production. Disclosure C.M. González-Casimiro: None. P. Cámara-Torres: None. B. Merino: None. J. Santo-Domingo: None. M.A. de la Fuente: None. A. Alonso: None. I. Cozar-Castellano: None. G. Perdomo: None. Funding Ministerio de Ciencia e Innovación-Spain (PID2019-110496RB-C22)