Structural anatomy of C1 domain interactions with DAG and other agonists

Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. DAG effector proteins compete for available lipid using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the first high-resolution crystal structures of a C1 domain (C1B from PKCdelta) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.

Protein Kinase C gamma Mutations Drive Spinocerebellar Ataxia Type 14 by Impairing Autoinhibition

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+-regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. The majority of the approximately 50 mutations identified in PKCγ cluster to the DG-sensing C1 domains. Here, we use a FRET-based activity reporter to show that ataxia-associated PKCγ mutations enhance basal activity by compromising autoinhibition. Although impaired autoinhibition generally leads to PKC degradation, the C1 domain mutations protect PKCγ from phorbol ester-induced downregulation. Furthermore, it is the degree of disrupted autoinhibition, not changes in the amplitude of agonist-stimulated activity, that correlate with disease severity. Specifically, a SCA14 mutation in which phenylalanine 48 in the C1A domain is deleted had high basal activity both in cells and in vitro, yet was unresponsive to agonist stimulation. Validating that the pathology arises from disrupted autoinhibition, we show that the degree of impaired autoinhibition correlates inversely with age of disease onset in patients: mutations that cause high basal activity are associated with early onset, whereas those that only modestly increase basal activity, including a previously undescribed mutation D115Y, are associated with later onset. Molecular modeling indicates that almost all SCA14 mutations that are not in the C1 domains are at interfaces with the C1B domain, and bioinformatics analysis reveals that mutations in the C1B domain are under-represented in cancer. Thus, clustering of SCA14 mutations to the C1B domain provides a unique mechanism to enhance PKCγ basal activity while protecting the enzyme from downregulation.

Design, Synthesis and Characterization of Novel sn-1 Heterocyclic DAGlactones as PKCe Activators

<p>In this study we describe the synthesis and characterization of novel diacylglycerol (DAG)-lactones that bind to protein kinase C (PKC). DAG-lactones proved to be useful templates for the design of potent and selective C1 domain ligands. The ester moiety at <i>sn-1</i> position, a common feature in this template, is relevant for interaction with the PKC C1 domains, although it represents a labile group susceptible to endogenous esterases. Our studies identified the DAG-lactone 10B12 with an isozazole ring as a nanomolar affinity PKC ligand. This compound shows preferential selectivity for PKCepsilon, and strongly activates actin cytoskeleton reorganization into peripheral ruffles in cancer cells, an effect mediated by PKCepsilon. Therefore, introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC selectivity.</p><div><br></div>

Design, Synthesis and Characterization of Novel sn-1 Heterocyclic DAGlactones as PKCe Activators

<p>In this study we describe the synthesis and characterization of novel diacylglycerol (DAG)-lactones that bind to protein kinase C (PKC). DAG-lactones proved to be useful templates for the design of potent and selective C1 domain ligands. The ester moiety at <i>sn-1</i> position, a common feature in this template, is relevant for interaction with the PKC C1 domains, although it represents a labile group susceptible to endogenous esterases. Our studies identified the DAG-lactone 10B12 with an isozazole ring as a nanomolar affinity PKC ligand. This compound shows preferential selectivity for PKCepsilon, and strongly activates actin cytoskeleton reorganization into peripheral ruffles in cancer cells, an effect mediated by PKCepsilon. Therefore, introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC selectivity.</p><div><br></div>

Elucidating the interaction of γ-hydroxymethyl-γ-butyrolactone substituents with model membranes and protein kinase C–C1 domains

Investigation of γ-hydroxymethyl-γ-butyrolactone substituents as protein kinase C ligands, in an effort to develop small molecule-based regulators with higher specificity for C1 domain than the endogenous ligand, diacylglycerols.