Involvement of calcium-dependent protein kinases and phosphatases in sperm capacitation-associated events

ABSTRACT To acquire fertilizing ability, mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation1,2. At the molecular level, capacitation is associated with a fast bicarbonate (HCO3-)-dependent activation of a unique type of soluble adenyl cyclase (sAC) and a consequent increase in cyclic AMP (cAMP) levels and PKA activation3. Activation of a cAMP/PKA pathway results in the phosphorylation of PKA substrates, which in turn initiates activation of several signaling cascades ultimately leading to an increase in phosphorylation on tyrosine residues (P-Tyr) of sperm axonemal proteins4,5. Increase in P-Tyr has been associated to sperm hyperactivation, an asymmetric and whip-like motion of the sperm tail necessary for fertilization4,6. Although the increase in protein phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remains largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites7–9. As a part of the work on Chapter 2, we found that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is highly phosphorylated in capacitated mouse sperm. Phylogenetic analysis showed that Rsph6a gene contains six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a. Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum. Besides HCO3-, the role of Ca2+ in capacitation pathway is indispensable. Ca2+ regulates cAMP/PKA pathway through direct stimulation of sAC3. Ca2+ also binds to another binding partner calmodulin (CaM) and regulates activity of multiple enzymes such as phosphodiesterases (PDEs), protein phosphatase 2B (PP2B, also known as calcineurin), and to protein kinases; Ca2+/CaM-dependent kinase II (CaMKII) and Ca2+/CaM-dependent kinase IV (CaMKIV)10. The role of Ca2+ and Ca2+/CaM in sperm capacitation was demonstrated by genetic and pharmacological approaches. Mice lacking CatSper, a sperm specific Ca2+ channel, failed to hyperactive and displayed aberrant/premature P-Tyr of sperm axonemal proteins, and consequently were infertile11–14. Consistently, inhibition of CaM inhibitors also inhibited onset of sperm hyperactivation15. Nonetheless, the role of downstream targets of Ca2+/CaM in capacitation process is not well understood. cAMP controls several signaling events during sperm capacitation and its levels during this process are quite dynamic. Depending on the need to fine tune PKA signaling, its levels are regulated by constant synthesis and degradation events. Such dynamics in cAMP levels can be explained by crosstalk between cAMP and Ca2+ signaling pathways. On one hand, Ca2+ positively promotes cAMP synthesis by direct stimulation of sAC3,16; on the other hand, Ca2+ binds to calmodulin (CaM), which activates a phosphodiesterase (PDE) and promotes cAMP degradation17–19. Besides PDE, PKA has been shown to control cAMP synthesis in a negative feedback loop by direct or indirect phosphorylation of sAC20. However, there is no clear understanding as to how this feedback loop is regulated, and how it fine-tunes PKA signaling during capacitation. As a part of this work on Chapter 3, we showed that calcineurin, a Ser/Thr phosphatase, is involved in this process. Inhibition of calcineurin by calcineurin inhibitors such as Cyclosporin A (CsA) and FK506 negatively PKA substrate phosphorylation. By measuring PKA and sAC activity in vitro in the presence of calcineurin inhibitors, we demonstrated that inhibition of PKA substrate phosphorylation is not due to off-target effect of the pharmacological drugs. Furthermore, we hypothesized that calcineurin being a Ser/Thr phosphatase, is able to activate sAC and thus regulates cAMP levels during sperm capacitation. Consistently, calcineurin inhibitors significantly reduced intracellular cAMP levels of the sperm incubated under capacitating conditions, which suggested a mechanistic role of calcineurin in a PKA feedback loop. Moreover, built on the rationale of the significance of Ca2+/CaM signaling, as a part of our work on Chapter 4, we characterized the presence and localization of Ca2+ signaling molecules involved in mouse and human sperm. In addition, we provided evidence that CaMKII is regulated during capacitation and is associated to cAMP/PKA pathway. Taken together, our data provide evidence for the role of novel signaling molecules in sperm capacitation. Understanding of the underlying mechanisms how these molecules regulate sperm capacitation will provide new insights into the development of novel contraceptive methods for men

Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy

Background: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac β-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. Methods: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with β-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. Results: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. Conclusions: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors

A NOVEL PIPELINE FOR DRUG DISCOVERY IN NEUROPSYCHIATRIC DISORDERS USING HIGH-CONTENT SINGLE-CELL SCREENING OF SIGNALLING NETWORK RESPONSES EX VIVO

The current work entails the development of a novel high content platform for the measurement of kinetic ligand responses across cell signalling networks at the single-cell level in distinct PBMC subtypes ex vivo. Using automated sample preparation, fluorescent cellular barcoding and flow cytometry the platform is capable of detecting 21, 840 parallel cell signalling responses in each PBMC sample. We apply this platform to characterize the effects of neuropsychiatric treatments and CNS ligands on the T cell signalling repertoire. We apply it to define cell signalling network abnormalities in PBMCs from drug-naïve first-onset schizophrenia patients (n=12) relative to healthy controls (n=12) which are subsequently normalized in PBMCs from the same patients (n=10) after a six week course of clinical treatment with the atypical antipsychotic olanzapine. We then validate the abnormal cell signalling responses in PBMCs from an independent cohort of drug-naïve first-onset schizophrenia patients (n=25) relative to controls (n=25) and investigate the specificity of the abnormal PBMC responses in schizophrenia as compared to major depression (n=25), bipolar disorder (n=25) and autism spectrum disorder (n=25). Subsequently we conduct a phenotypic drug screen using the US Food and Drug Administration (FDA) approved compound library, in addition to experimental neuropsychiatric drug candidates and nutraceuticals, to identify compounds which selectively normalize the schizophrenia-associated cell signalling response. Finally these candidate compounds are characterized using structure-activity relationships to reveal specific chemical moieties implicated in the putative therapeutic effect.EPSR

Stiimulitundlike ja kovalentsete bisubstraatsete proteiinkinaasi inhibiitorite arendamine

Väitekirja elektrooniline versioon ei sisalda publikatsiooneProteiinkinaasid on laialdaselt levinud ensüümid, mis katalüüsivad valkude fosforüülimist. Valkude fosforüülimine mõjutab arvukate mehhanismide kaudu pea kõiki raku funktsioone. Proteiinkinaaside talitlushäirete korral võib nende aktiivsus muutuda liigseks, mis on seotud erinevate haigustega, nt vähkkasvajatega. Proteiinkinaaside liigse aktiivsuse mahasurumiseks arendatakse inhibiitoreid, mille ülesandeks on tugevalt proteiinkinaasiga seonduda ning see seeläbi inaktiveerida. Viimase kahekümne aasta jooksul on ravimina kasutusele võetud ligi sada proteiinkinaasi inhibiitorit, mida rakendatakse enamasti vähkkasvajate raviks. Tartu Ülikoolis, arendatakse bisubstraatseid proteiinkinaasi inhibiitoreid, mida nimetatakse ARC-inhibiitoriteks. Bisubstraatsus väljendub inhibiitori võimes seonduda korraga kahte proteiinkinaasi sidumispiirkonda, kuhu peaksid muidu seonduma substraadid. Tänu sellele saavutavad ARC-inhibiitorid väga tugeva seondumisvõime ja hea selektiivsuse. Selles töös demonstreeriti, et optimeeritud struktuurimodifikatsioone kasutades oli ARC-inhibiitoritele võimalik lisada täiendavaid omadusi. Nimelt konstrueeriti kõrge seondumisvõimega proteiinkinaasi inhibiitorid, mille seondumisvõime ilmneb või kaob välise stiimuli rakendamisel (näiteks LED-valgustiga valgustamisel või redutseeriva keskkonna loomisel). Proteiinkinaasi inhibiitorite aktiivsuse kaudne juhtimine võiks viia tulevikus näiteks valgustundlike vähiravimiteni, mida saab kehas lokaalselt aktiveerida. Samuti töötati välja kovalentsed ARC-inhibiitorid, mis moodustavad proteiinkinaasiga seondumisel pöördumatu keemilise ehk kovalentse sideme. Kovalentse mehhanismi abil töötavad ravimid võimaldavad haigust tekitavaid sihtmärke teistest paremini eristada. Uute inhibiitorite struktuuride disainimisel oli oluline osa varasemalt mõõdetud valgu-inhibiitori kristallstruktuuridel, mis võimaldasid ennustada proteiinkinaasi ja inhibiitori vahelisi interaktsioone. Töö illustreerib bisubstraatsete inhibiitorite modifitseerimise paindlikkust uute omaduste lisamiseks.Protein kinases are enzymes, which catalyse protein phosphorylation. Protein phosphorylation affects almost all cell functions via a myriad of mechanisms. Dysregulation of protein kinases may lead to excess protein kinase activity, which is related to different kinds of diseases, including cancer. Inhibitors, which bind protein kinases and deactivate them, are developed. During the past twenty years around 100 protein kinase inhibitors have been approved as drugs. ARC-inhibitors are bisubstrate protein kinase inhibitors, which have been developed in the University of Tartu for the past 30 years. Bisubstrate inhibitors bind two protein kinase binding sites simultaneously and thus prevent the substrates from binding. Therefore, ARC inhibitors have a very strong binding affinity and good selectivity towards their protein kinase targets. However, the structures of bisubstrate inhibitors are usually larger and more complicated. It was demonstrated that special features can be added to ARC-inhibitors by incorporating optimized structural modifications. Namely, high affinity stimuli-responsive bisubstrate protein kinase inhibitors were constructed, which could be activated or deactivated on command using different stimuli (e.g., by illuminating with LED light or producing a reductive environment). For example, the ability to remotely regulate protein kinase inhibitors could lead to light-sensitive cancer drugs, which could be locally activated. Further, covalent ARC-inhibitors were developed, which form an irreversible covalent bond to their target protein kinase. The inhibitors which function via a covalent mechanism can be more selective towards their target. Previously measured crystal structures of protein-inhibitor complexes were an important element in the design of new inhibitors. This work illustrates the flexibility of bisubstrate protein kinase inhibitor structures for integrating new features.https://www.ester.ee/record=b552784

Extracellular Phosphorylation of the Amyloid β-Peptide Promotes Aggregation

Alzheimer’s disease (AD) is the most prevalent, progressive neurodegenerative disorder and its incidence is increasing with the aging population worldwide. The presence of extracelluar neuritic plaques containing amyloid beta (Aβ) and intracellular neurofibrillary tangles (NFT) in the brain are pathological hallmarks of the disease. Aβ is generated by proteolytic processing, involving sequential cleavages of the amyloid precursor protein (APP) by beta (β) and gamma (γ) secretase. Aggregation of Aβ is believed to be critical for its neurotoxicity and pathogenesis of AD. Mutations that lead to amino acid substitutions within the Aβ can cause early onset AD and promote the formation of neurotoxic Aβ assemblies. However, such mutations are very rare and account for only a very small number of cases. Mechanisms that increase the aggregation of wild-type Aβ and cause the more common sporadic forms of AD are largely unknown. It is plausible that the aggregation of Aβ in AD might be induced by unknown post-translational modification. The role of post-translational modification of Aβ in its aggregation and the pathogenesis of AD are unclear. The amino acid sequence of Aβ contains three potential phosphorylation sites at serine residue 8, tyrosine residue 10 and serine residue 26. In silico and in vitro studies indicated that Aβ can undergo phosphorylation by different protein kinases. In vivo and ex vivo phosphorylation experiments using cultured cells, mouse cerebellar neurons and in cerebrospinal fluid (CSF) of AD patients showed the presence of extracellular PKs activity. Extracellular Aβ is phosphorylated by protein kinase A (PKA) that is secreted by or localized at the cell surface of culture neurons. In addition, the presence of PKA-like kinase activity was identified in CSF of AD patients. Different biophysical methods such as Circular dichroism, NMR, Thioflavin T, Congo red binding, Dynamic light scattering and Electron microscope using synthetic phosphorylated and non-phosphorylated variants of Aβ elucidated the role of phosphorylation on Aβ conformation, oligomerization and aggregation. Polyclonal phosphorylation-state Aβ specific antibody was generated to study the occurrence of phosphorylated Aβ in vivo. Biochemical and immunohistological stainings using brains of APP transgenic mice and human AD patients employing phosphorylation-state Aβ specific antibodyshowed the specific detection of phosphorylated and non-phosphorylated Aβ species. Importantly, phosphorylated Aβ was detected in small deposits in the brains of young transgenic mice and showed age-dependent accumulation in plaques. Phosphorylated Aβ was also detected in the core of neuritic plaques and associated with dystrophic neurites in human AD brains. In summary, the undertaken study shows that extracellular Aβ is phosphorylated by PKs present at the cell surface and in the CSF of the human brain. The phosphorylation at serine residue 8 increases the propensity of Aβ to adopt b-sheet conformation and promotes the formation of small oligomeric aggregates that could seed aggregation into larger oligomeric and fibrillar assemblies. The specific detection of phosphorylated and non-phosphorylated Aβ species in APP transgenicmice and human AD brain indicates the preferential aggregation of phosphorylated Aβ in vivo. The combined data demonstrate that extracellular phosphorylation of Aβ strongly promotes its assembly into neurotoxic species and thus might represent an important molecular mechanism in the pathogenesis of the most common sporadic AD. Thus, targeting extracellular phosphorylation of Aβ could be explored for therapeutic or preventive strategies to decrease Aβ aggregation in sporadic AD

Development and Characterization of Tool Compounds Targeting the Runt Domain’s interaction With Cbfβ

RUNX1 and CBFβ, which encode subunits of the core binding factor, are frequent targets of chromosomal aberrations in hematological malignancies. We previously determined that CBFβ (encoded by CBFB) is important for the transforming activity of the chimeric protein AML1-ETO protein (RUNX1-RUNX1T1) generated by the t(8;21), and other studies showed that normal Runx1 functions are essential for survival and maintenance of some leukemias lacking RUNX1 or CBFB mutations. Thus, we hypothesized that we could achieve therapeutic efficacy in multiple leukemias by targeting the Runx1:CBFβinteraction with small molecules. Using the structural information of the DNA binding Runt domain (RD) of Runx1 and its interface with CBFβ, we employed a computational screen for a library of 78,000 drug-like compounds, and further optimized our leads. The Runt domain inhibitors (RDIs) bind directly to the RD and disrupt its interaction with CBFβ. We showed that the RDIs reduced growth and induced apoptosis of t(8;21) acute myeloid leukemia (AML) cell lines, and reduced the progenitor activity of mouse and human leukemia cells harboring the t(8;21), but not normal bone marrow cells. The RDIs had similar effects on murine and human T cell acute lymphocytic leukemia (T-ALL) cell lines that did not harbor the t(8;21). Furthermore, our inclusion of a structurally related and weakly active compound as a control strongly support that the efficacies we observed were due to on target inhibition of RUNX functions. Our results confirmed that the RDIs might prove efficacious in various AMLs, and that a therapeutic window is available to specifically target malignant cells. We developed a pro-drug AI-9-59 with improved solubility and pharmacokinetic properties and assessed whether it has any in vivo efficacies in mouse leukemia models. Our results showed that the pro-drug was toxic to mice at dosage above 50 mg/kg and had no observable growth inhibitory effect on leukemia cells, suggesting that the concentration of the pro-drug necessary to inhibit endogenous core binding factor activity exceeds the maximum tolerated dose in mice. However, the expansion of granulocyte macrophage progenitors, and the gastrointestinal toxicity phenotype we observed suggested that the effects could be from on-target repression of RUNX proteins functions

Functional characterisation of phosphodiesterase 4D7 in prostate cancer

3’,5’-cyclic adenosine monophosphate (cAMP) is the best studied intracellular second messenger. Adenylyl cyclase (AC) catalyses the synthesis of cAMP from ATP following the stimulation of a G protein coupled receptor (GPCR), and its degradation is catalysed by cAMP phosphodiesterases (PDEs) to allow cessation of signal. cAMP can act to bring about a multitude of varying and often opposing cellular responses, which depend on the stimulus received by the GPCR, the cell type, the cell cycle stage, and the complement of downstream effector molecules within that cell. The cAMP PDE subfamilies express multiple splice variants, which possess unique N-termini and non-redundant functional roles. By virtue of this, they are targeted to specific and discrete subcellular locations, where they may form highly specific interactions with scaffold proteins and other enzymes. Here, in these discrete locales, PDEs act to hydrolyse local cAMP, thereby underpinning the spatial and temporal compartmentalisation of cAMP gradients. This fine-tuned balance of synthesis and degradation is paramount for the dynamic cellular responses to extracellular stimuli, allowing differing signal transduction cascades to occur simultaneously in the crowded macromolecular environment of the cell. The compartmentalisation of cAMP signalling is, thus, essential for maintaining cellular homeostasis, and is subject to perturbation in various diseases, including prostate cancer (PC). Despite the wealth of literature implicating cAMP signalling in the progression of PC, little work has been done on the expression or function of PDE splice variant in this disease. Our group, in collaboration with Philips Research and the Prostate Cancer and Molecular Medicine (PCMM) group in the Netherlands, set out to investigate the changes in cAMP signalling during PC progression by studying the expression of cAMP PDE isoforms, with the aim of identifying a novel PC biomarker, as the current standard biomarker (PSA) is not disease-specific and leads to much over-diagnosis and over-treatment of otherwise non-life threatening prostate tumours. Interestingly, we found PDE4D7 to be dramatically downregulated as PC progresses from an androgen sensitive (AS) to an androgen insensitive (AI) state, and, indeed, this enzyme is showing promise as a novel, disease-specific PC biomarker. In this thesis, I report my efforts to characterise a function of PDE4D7 within prostate cancer. Firstly, I report the raising of a novel highly specific PDE4D7 antibody and describe the differential expression of this isoform, at the protein level, between AS and AI PC cell models. I present evidence to suggest that PDE4D7 mediates PC cell growth and migration, and that its loss may play a role in PC progression. I propose that an altered epigenome plays a role in the downregulation of PDE4D7 expression. I then report on the raising of a novel phospho-specific antibody and present evidence to show that PDE4D7 is regulated by PKA phosphorylation within its unique N-terminal region, and that this event confers negative regulation on enzyme activity. Finally, I describe my endeavours to elucidate a PDE4D7 protein-protein interaction that may help transduce PDE4D7-specific signals and maintain the enzymes cellular location

Identification and Characterization of Ethanol Responsive Genes in Acute Ethanol Behaviors in Caenorhabditis elegans

Alcohol abuse and dependence are complex disorders that are influenced by many genetic and environmental factors. Acute behavioral responses to ethanol have predictive value for determining an individual’s long-term susceptibility to alcohol abuse and dependence. These behavioral responses are strongly influenced by genetics. Here, we have explored the role of genetic influences on acute behavioral responses to ethanol using the nematode worm, Caenorhabditis elegans. First, we explored the role of ethanol metabolism in acute behavior responses to ethanol. Natural variation in human ethanol metabolism machinery is one of the most reported and reproducible associations found to alter drinking behavior. Ethanol metabolism is conserved across phyla and alteration in this pathway alters acute behavioral responses to ethanol in humans, mice, rats, and flies. We have extended these findings to the worm and have shown that loss of either alcohol dehydrogenase or aldehyde dehydrogenase results in an increase in sensitivity to the acute effects of ethanol. Second, we explored the influence of differences in basal and ethanol-induced gene expression in ethanol responsive behaviors. We identified a set of candidate genes using the basal gene expression differences in npr-1(ky13) mutant animals to enrich for genes involved in AFT. This analysis revealed ethanol changes to the expression of genes involved in a variety of biological processes including lipid metabolism. We focused on a gene involved in the metabolism of fatty acids, acs-2. acs-2 encodes an acyl-CoA synthetase that activates fatty acids for mitochondrial beta-oxidation. Animals carrying mutant acs-2 have significantly reduced AFT and we explored the role of genes in the mitochondria beta-oxidation pathway for alterations in ethanol responsive behaviors. We have shown that knockdown of ech-6, an enoyl-CoA hydratase, enhances the development of AFT. This work has uncovered a role for fatty acid utilization pathways in acute ethanol responses and we suggest that natural variation in these pathways in humans may impact the acute alcohol responses to alcohol that in turn influence susceptibility to alcohol abuse and dependence

Initiation and termination of cAMP signalling in PANC-1 cells: interplay between cAMP and Ca2+ signalling cascades.

Pancreatic ductal adenocarcinoma (PDAC) is noted for its resistance to therapy and poor prognosis. PDAC is characterised by extensive local invasion and metastases at distant organs. Having previously shown that the cAMP cascade had regulatory effects on cell migration/invasion in PDAC cells, which are vital processes in metastasis formation, we decided to characterise the cAMP signalling machinery in PANC-1 cells. In this study we adopted a live-cell imaging approach taking advantage of genetically engineered probes that use Fӧrster resonance energy transfer (FRET) to reveal cAMP concentration and PKA activity at a single cell level. Using the Epac-based cAMP sensor H134 we found that PDE3 and PDE4 are the principal cAMP destroyers, whereas β-adrenoceptors are the main physiological cAMP-cascade activators, in PANC-1 cells. Downstream, using Boyden chamber assays we found that PDE3 has the biggest role in cell migration under ‘basal’ conditions, whereas PDE4 has a bigger role in the presence of isoproterenol. However, isoproterenol on its own did not influence PANC-1 cell migration despite having the ability to increase cAMP concentration inside the cell. This part of the study puts forward PDE3 and PDE4 as potential targets for reducing migration/invasion of PDAC. In the second part of the study, we found that these cells have an efficient Ca2+ signalling system; in which ORAI1 is the main mediator of store-operated Ca2+ entry (SOCE) in PANC-1 cells. To explore the possibility of a Ca2+-cAMP crosstalk in PANC-1 cells, we used the AKAR4 probe to measure PKA activity during Ca2+ responses. The application of neurotensin (NT), a well-known IP3-producing agonist in this cell type, induced a Ca2+ response which was accompanied by an increase in PKA activity. SOCE is likely to play an important role in this process since the activation of SOCE by thapsigargin-mediated store depletion consistently and robustly increased PKA activity. To further investigate the downstream roles of PKA signalling in PANC-1 cells we utilised immunofluorescence to visualise the distribution of PKA activity. Using antibodies specific for phosphorylated PKA substrates, we found that phosphorylated PKA substrates are preferentially concentrated in the nucleus; and notably at the leading edges of PANC-1 cells displaying a migratory phenotype. At the leading edges phosphorylated PKA substrates colocalised with actin-rich ruffles. Interestingly, utilising the rapamycin-inducible heterodimerisation system to reveal endogenous ER-PM junctions in migrating PANC-1 cells, we found that ER-PM junctions are also situated in close proximity to the leading edge. These results suggest that SOCE activates PKA responses in the region strategically important for PDAC cell migration

Prediction of peptides binding to the PKA RIIα subunit using a hierarchical strategy

Motivation: Favorable interaction between the regulatory subunit of the cAMP-dependent protein kinase (PKA) and a peptide in A-kinase anchoring proteins (AKAPs) is critical for translocating PKA to the subcellular sites where the enzyme phosphorylates its substrates. It is very hard to identify AKAPs peptides binding to PKA due to the high sequence diversity of AKAPs