A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1.

Protein-Cadmium Interactions in Crowded Biomolecular Environments Probed by In-cell and Lysate NMR Spectroscopy.

One of the mechanisms by which toxic metal ions interfere with cellular functions is ionic mimicry, where they bind to protein sites in lieu of native metals Ca and Zn . The influence of crowded intracellular environments on these interactions is not well understood. Here, we demonstrate the application of and lysate NMR spectroscopy to obtain atomic-level information on how a potent environmental toxin cadmium interacts with its protein targets.

A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated Protein Kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1.

Mechanisms by which small molecules of diverse chemotypes arrest Sec14 lipid transfer activity.

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood.

Regulation of eukaryotic protein kinases by Pin1, a peptidyl-prolyl isomerase.

The peptidyl-prolyl isomerase Pin1 cooperates with proline-directed kinases and phosphatases to regulate multiple oncogenic pathways. Pin1 specifically recognizes phosphorylated Ser/Thr-Pro motifs in proteins and catalyzes their cis-trans isomerization. The Pin1-catalyzed conformational changes determine the stability, activity, and subcellular localization of numerous protein substrates.

Structural anatomy of Protein Kinase C C1 domain interactions with diacylglycerol 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. Effector proteins translocate to available DAG pools in the membranes by 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.

Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy.

Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd as a probe was prompted by previous findings that xenobiotic metal ions modulate PKC activity.

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1.

Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain.

Structural insights into C1-ligand interactions: Filling the gaps by in silico methods.

Protein Kinase C isoenzymes (PKCs) are the key mediators of the phosphoinositide signaling pathway, which involves regulated hydrolysis of phosphatidylinositol (4,5)-bisphosphate to diacylglycerol (DAG) and inositol-1,4,5-trisphosphate. Dysregulation of PKCs is implicated in many human diseases making this class of enzymes an important therapeutic target. Specifically, the DAG-sensing cysteine-rich conserved homology-1 (C1) domains of PKCs have emerged as promising targets for pharmaceutical modulation.

Deletion of the Trichoderma virens NRPS, Tex7, induces accumulation of the anti-cancer compound heptelidic acid.

The anticancer antibiotic heptelidic acid is a sesquiterpene lactone produced by the beneficial plant fungus Trichoderma virens. This species has been separated into two strains, referred to as P and Q, based on its biosynthesis of secondary metabolites; notably, only P-strains were reported to produce heptelidic acid. While characterizing a Q-strain of T.

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions.

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb, a structural and functional surrogate of Ca, to generate the protein states with well-defined protein-metal ion stoichiometry.

Interference of pH buffer with Pb-peripheral domain interactions: obstacle or opportunity?

Pb is a xenobiotic metal ion that competes for Ca-binding sites in proteins. Using the peripheral Ca-sensing domains of Syt1, we show that the chelating pH buffer Bis-Tris enables identification and functional characterization of high-affinity Pb sites that are likely to be targeted by bioavailable Pb.

Cd(II)- and Pb(II)-Induced Self-Assembly of Peripheral Membrane Domains from Protein Kinase C.

Cd and Pb are xenobiotic heavy metal ions that use ionic mimicry to interfere with the cellular function of biomacromolecules. Using a combination of SAXS, electron microscopy, FRET, and solution NMR spectroscopy, we demonstrate that treatment with Cd and Pb causes self-assembly of protein kinase C regulatory domains that peripherally associate with membranes. The self-assembly process successfully competes with ionic mimicry and is mediated by conserved protein regions that are distinct from the canonical Ca-binding motifs of protein kinase C.

High affinity interactions of Pb with synaptotagmin I.

Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca2+-evoked neurotransmitter release, has two high-affinity Pb2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb2+-complexed C2 domains revealed that protein-bound Pb2+ ions have holodirected coordination geometries and all-oxygen coordination spheres.

Structural Basis of Protein Kinase Cα Regulation by the C-Terminal Tail.

Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca-dependent manner. In these cases, membrane association is triggered by Ca binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd, in lieu of Ca to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding.

Toggling of Diacylglycerol Affinity Correlates with Conformational Plasticity in C1 Domains.

Conserved homology-1 (C1) domains are peripheral membrane domains that target their host proteins to diacylglycerol (DAG)-containing membranes. It has been previously shown that a conservative aromatic mutation of a single residue in the C1 domain has a profound effect on DAG affinity. We report that the "DAG-toggling" mutation changes the conformational dynamics of the loop region that forms the binding site for the C1 activators.

Dynamic Response of the C2 Domain of Protein Kinase Cα to Ca Binding.

Ca-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy.

Dynamics and Membrane Interactions of Protein Kinase C.

Protein kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. Significant research efforts have been directed at understanding the structure, function, and regulatory modes of the enzyme since its discovery and identification as the first receptor for tumor-promoting phorbol esters. The activation of PKC involves a transition from the cytosolic autoinhibited latent form to the membrane-associated active form.

Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis.

Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it.

Expression and purification of the N-terminal regulatory domain of Protein Kinase C for biophysical studies.

We report the protocol for heterologous expression and purification of the N-terminal regulatory region of two Protein Kinase C (PKC)(1) isozymes, one conventional and one novel. Previous studies of these domains relied almost exclusively on the fusion constructs with high-molecular-weight solubility fusion partners such as GST and MBP. We developed experimental procedures that enabled us to overcome challenges associated with the amphiphilic character of the regulatory domain and generate sufficient quantities of fusion partner-free proteins for biophysical work.

Interfacial partitioning of a loop hinge residue contributes to diacylglycerol affinity of conserved region 1 domains.

Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant.

Cd2+ as a Ca2+ surrogate in protein-membrane interactions: isostructural but not isofunctional.

Due to its favorable spectroscopic properties, Cd(2+) is frequently used as a probe of Ca(2+) sites in proteins. We investigate the ability of Cd(2+) to act as a structural and functional surrogate of Ca(2+) in protein-membrane interactions. C2 domain from protein kinase Cα (C2α) was chosen as a paradigm for the Ca(2+)-dependent phosphatidylserine-binding peripheral membrane domains.

The C-terminal V5 domain of Protein Kinase Cα is intrinsically disordered, with propensity to associate with a membrane mimetic.

The C-terminal V5 domain is one of the most variable domains in Protein Kinase C isoforms (PKCs). V5 confers isoform specificity on its parent enzyme through interactions with isoform-specific adaptor proteins and possibly through specific intra-molecular interactions with other PKC domains. The structural information about V5 domains in solution is sparse.