Browsing by Subject "kinase"

Now showing 1 - 4 of 4
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    Allostery governs Cdk2 activation and differential recognition of CDK inhibitors
    (2021-05) Majumdar, Abir
    Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. Using a series of DEER and NMR experiments, we studied the activation process of the G1/S kinase Cdk2 in solution. We show that catalytically inactive Cdk2 readily adopts multiple active-like states for efficient dephosphorylation, and that regulatory phosphorylation on the activation loop enhances allosteric coupling with the cyclin subunit. We then used DEER and FRET experiments to measure the binding of multiple CDK inhibitors and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. We reveal that the allosteric coupling between these biochemical effectors is responsible for the differential recognition of Cdk2 and Cdk4 inhibitors. Finally, we used sequence analysis, DEER, FRET and activity assays to identify and measure the effects of mutating an allosteric hub that has diverged between Cdk2 and Cdk4. We demonstrate that this hub controls the strength of allosteric coupling, and that the altered architecture and allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).
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    Development of Allosteric Inhibitors against Cyclin-dependent Kinase 2 (CDK2)
    (2021-05) Faber, Erik
    Cyclin-dependent kinase 2 (CDK2) is a validated therapeutic target for male nonhormonal contraception as well as various anticancer indications. Despite this, selectively targeting CDK2 has been challenging, largely due to the structural homology of the active site among kinases that most developed therapeutics bind. Our group previously discovered an unexplored allosteric site in CDK2 that binds the commercial dye 8-anilino-1-naphthalene sulfonic acid (ANS) with moderate affinity. In this work, I explored the cooperative effects of ANS with other ATP-site (i.e. orthosteric) ligands to enhance the affinity of ANS. Next, I developed various synthetic methods, including an Ullmann coupling method, to make ANS derivatives whereas this had been previously challenging. Using the fluorescent properties of ANS, we discovered a new chemical scaffold that binds this allosteric site. From there, we conducted a structure-activity relationship (SAR) campaign to improve the affinity of this compound ~5,000 fold from the original screening hit, leading to compounds with single-digit nanomolar affinity. We observed a negatively cooperative relationship with this series and cyclin binding, representing an underexplored mechanism of CDK2 inhibition.
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    Positive And Negative Regulation Of Defense Responses Against Pseudomonas Syringae In Arabidopsis
    (2014-03) Sreekanta, Suma
    Immune signaling in plants involves both positive and negative regulators. Maintaining a balance between growth and defense responses is important because there is a fitness cost to the plants if immune responses are left unchecked. Suppression of immune responses in the absence of pathogens as well as after the threat has passed is critical in maintaining such a balance between growth and defense responses. Upon pathogen perception, the positive regulators counter the immune repression to induce defense responses. We investigated the roles of two genes, CBP60a and PCRK1 in the regulation of defense responses against Pseudomonas syringae pathogen in the model system Arabidopsis thaliana . CBP60a is a negative regulator of immune responses. We showed that CBP60a is a CaM binding protein and that CaM binding is important for its function in transducing defense signals. Mutants of CBP60a were more resistant to Pseudomonas syringae infection suggesting that CBP60a was a negative regulator of defense responses. We found that CBP60a functions in repressing immune signaling under conditions where the plants are not challenged by a pathogen. We also investigated the role of a putative kinase, PCRK1, in immune signaling. We showed that pcrk1 mutants are more susceptible to Pseudomonas syringae than wild type plants suggesting that PCRK1 has a positive role in immune responses. We also showed that PCRK1 is important for immunity triggered by some of the conserved Microbe Associated Molecular Patterns (MAMP) as well endogenous signals generated as a result of pathogen activity called Damage Associated Molecular Patterns (DAMP).
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    Targeting Cellular Retinoic Acid Binding Protein 1 to Modulate Non-Canonical Retinoic Acid Signaling
    (2023-09) Nhieu, Jennifer
    All-trans-retinoic acid (atRA) is the principle active metabolite of vitamin A and is essential for almost all biological functions. Canonically, atRA exerts its actions through retinoic acid receptors (RARs) located in the nucleus to regulate gene transcription. atRA also possesses “non-canonical activities” that modulate cell signaling, and is defined by (1) RAR-independence, (2) a rapid time-scale and (3) cytosolic localization. The primary mediator of this non-canonical activity is the highly conserved cellular retinoic acid binding protein 1 (CRABP1). CRABP1 was previously thought to only function in the binding and sequestration of atRA to regulate cellular bio-availability. However, studies of two non-canonical pathways have established CRABP1 as a mediator of non-canonical atRA activity. The first is CRABP1-mediated regulation of the mitogen activated protein kinase (MAPK) pathway with physiological and disease relevance in stem cell proliferation, cancer, immune regulation, and obesity. The second is CRABP1-mediated regulation of calcium (Ca2+)-calmodulin dependent kinase II (CaMKII) activation with physiological and disease relevance in cardiac dysfunction and motor neuron degenerative diseases such as amyotrophic lateral sclerosis (ALS). Nuclear Magnetic Resonance (NMR) spectroscopy and molecular studies determined the structural and molecular mechanism underlying CRABP1-mediated regulation of CaMKII activation. Mechanistically, CRABP1 preferentially complexes with the inactive form of CaMKII to ultimately dampen CaMKII activation. Alanine mutagenesis studies have determined that CRABP1 residues within a proposed CaMKII interaction surface and an allosteric site maintain this preference. Mutation of these residues can shift CRABP1 preference towards the active form of CaMKII. Two novel CRABP1 ligands (C4 and C32) were also characterized as potential therapeutic agents that may be developed to target the CRABP1-CaMKII pathway in motor neuron (MN) diseases. In a reconstituted MN culture model, C4 and C32 can dampen CaMKII activation in a CRABP1-dependent manner. In an immortalized MN cell line (MN1) C4 and C32 can protect against excitotoxic-mediated MN death induced by ionomycin treatment. The primary sequence of CRABP1 is extremely conserved among animal species, with only one substitution observed at amino acid position 86, suggesting important functional constraints placed upon CRABP1 sequence during evolution. Data mining of reported human studies was performed to determine the relevance of CRABP1 in human health and disease. Associations of CRABP1 with various human diseases were identified, including altered human CRABP1 gene expression and the presence of variants in cancers, ALS, and several rare diseases. The studies within this dissertation elucidate the structural and molecular mechanism of CRABP1-mediated regulation of cell signaling, specifically in CaMKII activation. The results suggest a potential therapeutic approach in targeting the CRABP1-CaMKII pathway with CRABP1-selective ligands to manage MN diseases. These results expand our understanding of CRABP1 in mediating the non-canonical activity of atRA hormone, particularly in modulating various cell signaling pathways to maintain health. The results also uncover complex mechanisms through which CRABP1-selective, atRA-like compounds may be further developed in therapeutic applications.