59 research outputs found
Investigation of mechanisms for restricting the activity of cyclic-AMP dependent protein kinase
Cyclic AMP (cAMP) is an ancient second messenger that is essential for many cellular processes including synaptic plasticity and control of heart rate and contractility. Cyclic AMP-dependent protein kinase (PKA) is the major intracellular receptor for cAMP. PKA consists of dimeric regulatory (R) subunits that bind and inhibit catalytic (C) subunits. PKA is activated upon binding of cAMP to the R subunits, which leads to the release of C subunits, and phosphorylation of intracellular protein substrates. An enduring challenge in cAMP research is to understand how PKA activity is directed to specific substrates, as the C subunits exhibit only limited substrate specificity in vitro. Elevations of cAMP are controlled in both space and time in the cell. This is achieved by the co-localization of enzymes for both the synthesis (cyclases) and breakdown (phosphodiesterases) of cAMP. Anchoring proteins are also essential for directing PKA to substrates in their immediate vicinity. However, a mechanism is yet to be established to explain how the activity of the C subunit of PKA is restrained following its dissociation from R subunits. This thesis details three parallel investigations that apply novel approaches with the shared aim of understanding how C subunit restraint is achieved. First, using quantitative immunoblotting in conjunction with purified PKA subunits, I investigated PKA subunit stoichiometry, finding that PKA R subunits typically outnumber C subunits by ~15-fold. Second, I developed a novel approach for monitoring R subunit isoform-specific association with C subunits in cells, with temporal precision. Comparative experiments using this approach and measurements with a fluorescent reporter of PKA activity show that only a small portion of C subunits need be dissociated to achieve high PKA activity. Third, I applied and developed a novel cross-linking coupled to mass spectrometry (XL-MS) protocol for analysis of the structure of PKA complexes. Insights include the likely orientation of PKA complexes that contain type II R (RII) subunits towards the membrane, and identification of a possible conformational change in PKA upon binding an anchoring protein. Together these experiments illuminate several aspects of PKA to show how the activity of this critical signalling enzyme is restrained within cells
CHANNELING & ALLOSTERY IN PDE-MEDIATED CYCLIC AMP SIGNAL TERMINATION
Ph.DDOCTOR OF PHILOSOPH
A novel interaction of the myosin light chain phosphatase with the regulatory subunits of protein kinase A in platelets
Platelet activation initiates a series of events, such as shape change, degranulation and aggregation, which results in the formation of a haemostatic plug and arrest of blood loss at the sites of vascular damage. Shape change is accompanied by remodelling of the actin cytoskeleton and formation of an acto-myosin contractile ring. This process is regulated by phosphorylation of the myosin light chain (MLC), which in turn is controlled by the relative activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). MLCP is a target of both inhibitory and activatory signalling molecules. Here we focussed on the control of MLCP activation by the cyclic adenosine 3', 5’ monophosphate (cAMP) signalling pathway. cAMP signalling is a major inhibitory pathway that inhibits platelet function through its effector protein kinase A (PKA). One of the downstream targets of PKA is MLCP. In vascular smooth muscle cells cAMP activates MLCP to dephosphorylate MLC and regulate cytoskeletal rearrangement. However, the relationship between cAMP signalling, MLCP and platelet function is unclear. Here we investigated the molecular and biochemical regulation of MLCP by cAMP signalling.We found that MYPT1, the targeting subunit of MLCP, is phosphorylated in platelets in response to cAMP elevating agents, suggesting that the phosphatase is a direct target for cAMP signalling. Our data suggests the possibility of at least two different splice variants of MYPT1 in platelets, but only one splice variant, possibly full length, was phosphorylated downstream of cAMP signalling. Using co-immunoprecipitation, cAMP pull-down assays and GST pull-down approaches we found that MYPT1 and PKA are part of the same complex and MYPT1 interacts with all four regulatory subunits of PKA (PKA-R) in platelets. Using a series of truncated proteins in HEK cells the interaction of MYPT1 and PKA-R was mapped to the central region of MYPT1 (aa501-706). Moreover, co-sedimentation assays with recombinant proteins confirmed the direct association of MYPT1 (aa501-706) with PKA-R. The conserved dimerisation and docking (D/D) domain of PKA-R, which facilitates interactions with A-kinase anchoring proteins (AKAP) was not required for the interaction. Consistent with this observation, the AKAP disruptor peptide Ht31 did not disrupt the interaction, indicating that the interaction of MYPT1 with PKA-R does not occur in an AKAP modus.These results were complemented with immunohistochemistry studies showing that MYPT1 co-localised with all four PKA regulatory subunits in both non-activated and spread platelets. In summary, our data identify MYPT1 as a novel PKA binding protein in platelets. The interaction of MYPT1 with PKA emerges as an important component of the signalling pathways that protect platelets from a hyperreactive state and may constitute a target towards preventing thrombogenic disorders
Substrate Identification of an Oncogenic Kinase: Elucidating the Pathogenesis of a Rare Liver Cancer
Fibrolamellar Hepatocellular Carcinoma (FLC) is a rare liver cancer with limited treatment options. This cancer primarily affects adolescents and young adults. Our lab has identified a new fusion gene in this cancer called DNAJB1-PRKACA that results from a break and re-fusion in chromosome 19. This chimeric gene results in a fusion kinase that acts as the driver of this cancer. While we have shown that the kinase activity of the fusion protein is essential for transformation, it is not currently known whether the oncogenic kinase that results from this fusion event, DNAJB1- PRKACA, phosphorylates the same substrates as PRKACA, the protein product of PRKACA. While the total phosphoproteome of a cancer can implicate critical pathway changes in the tumor versus healthy tissue, it cannot provide sufficient information on what kinase is directly responsible for the phosphorylations. Knowing which proteins DNAJB1-PRKACA is directly phosphorylating in the liver could help elucidate a stepwise mechanism for understanding the pathogenesis. Furthermore, it could provide new potential therapeutic options by targeting the downstream pathways of this oncogenic kinase. In this thesis, I will first describe my work to determine a method of directly identifying proteins that are substrates of DNAJB1-PRKACA and PRKACA. I first tested an approach developed by the Shokat Lab that uses an analog-sensitive (AS) kinase in combination with a selective adenosine triphosphate (ATP) analog to identify unique substrates of a kinase. However, serious concerns of substrate specificity of the AS-kinases were raised as I developed AS versions of DNAJB1-PRKACA and PRKACA. I pivoted to a method that kills the endogenous kinase activity of a lysate using 5’-(4-Fluorosulfonylbenzoyl)adenosine (FSBA); kinase reactions are performed using this kinase-inactive lysate with the purified active kinase of interest and an ATP analog that has a tag on the γ-phosphate. This results in substrates with a specific thiol tag. The ATP-γ-S analog I initially used for this method was effective in visualizing kinase activity changes via western blots, but the thiol-tags were not reliably identified using MS. With the improvement of phosphopeptide enrichment methods and encouragement from the Proteomics Resource Center, a pilot experiment was designed to enrich phosphopeptides from a kinase reaction using regular ATP, FSBAtreated mouse liver lysate, and either PRKACA or DNAJB1-PRKACA. The results of this pilot experiment showed promising differences in substrate specificity between PRKACA and DNAJB1-PRKACA so I moved forward with this assay using human hepatocyte lysate instead of mouse liver lysate. In the third chapter, I will discuss the results of the assay using kinase-inactive human hepatocyte lysate in kinase reactions to determine substrate differences between three kinases: DNAJB1-PRKACA, PRKACA, and PRKACA (L206R). PRKACA (L206R) is a PRKACA variant found in adrenal tumors of patients with Cushing’s disease. The L206R mutation is thought to block interaction with the regulatory subunit and pathogenesis of the adrenal tumors has been accepted to be the result of constitutive activity of this mutant catalytic subunit. Recently, two papers have suggested that there is an alteration in substrate specificity between PRKACA and PRKACA (L206R). My results demonstrate that there are differences in substrates that are directly phosphorylated by each of the three catalytic subunits: PRKACA, JPRKACA, and PRKACA (L206R). Finally, I will discuss how the results of a total phosphome study of FLC patient tumor and normal samples compared against my in vitro substrate identification assay. This comparison created a more patient-relevant and focused list of direct substrates of DNAJB1-PRKACA for further study. The thesis will conclude with discussion of the implications for the pathogenesis of FLC based on the direct substrates of interest found in these experiments and future experiments
When Just One Phosphate Is One Too Many: The Multifaceted Interplay between Myc and Kinases
Myc transcription factors are key regulators of many cellular processes, with Myc target genes crucially implicated in the management of cell proliferation and stem pluripotency, energy metabolism, protein synthesis, angiogenesis, DNA damage response, and apoptosis. Given the wide involvement of Myc in cellular dynamics, it is not surprising that its overexpression is frequently associated with cancer. Noteworthy, in cancer cells where high Myc levels are maintained, the overexpression of Myc-associated kinases is often observed and required to foster tumour cells' proliferation. A mutual interplay exists between Myc and kinases: the latter, which are Myc transcriptional targets, phosphorylate Myc, allowing its transcriptional activity, highlighting a clear regulatory loop. At the protein level, Myc activity and turnover is also tightly regulated by kinases, with a finely tuned balance between translation and rapid protein degradation. In this perspective, we focus on the cross-regulation of Myc and its associated protein kinases underlying similar and redundant mechanisms of regulation at different levels, from transcriptional to post-translational events. Furthermore, a review of the indirect effects of known kinase inhibitors on Myc provides an opportunity to identify alternative and combined therapeutic approaches for cancer treatment
Regulation of blood platelet function by nitric oxide
Upon vascular injury, platelets instantly adhere to the exposed extracellular matrix resulting in platelet activation and aggregation to form a haemostatic plug. This self-amplifying mechanism requires a tight control to prevent uncontrolled platelet aggregate formation that could occlude the vessel. Endothelial-derived nitric oxide (NO) and prostacyclin (PGI₂) are strong negative regulators that modulate platelet adhesion, activation, aggregation, secretion and shape change. In this study the effects of NO on Ca²+ dependent and independent pathways of activation were investigated. The data produced during the course of this study reveals new insights into the mechanisms by which NO regulates platelet responses via the activation of the AGC family of Ser/Thr protein kinases. NO inhibited platelet shape change in a concentration dependent manner. Platelet shape change is driven by phosphorylation of myosin light chain (MLC) and the experimental data shows that NO blocked this critical phosphorylation event. Phospho-MLC generated in response to platelet agonists occurs through a Ca²+ dependent and RhoA kinase (ROCK)-dependent mechanisms and NO differentially inhibits both pathways. Activation of the ROCK pathway via RhoA leads to the phosphorylation MLC phosphatase Threonine⁶⁹⁶⁄⁸⁵³, which inhibits enzyme activity. Experimental evidence in this thesis indicates that NO, acting through cGMP and protein kinase G, prevents this inhibitory phosphorylation of MLCP by at least two mechanisms, (i) inhibiting the ROCK pathway that phosphorylates MLCP, and (ii) directly phosphorylating MLCP at an independent site, Serine⁶⁹⁵. These original observations hint at a novel mechanism for platelet regulation by the NO-cGMP-signalling pathway
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
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
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