Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase Cα

The C2 domain of protein kinase Cα (PKCα) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCα isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-L-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCα-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-L-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCα crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-L-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 Å and at 2.0 Å, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-L-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCα is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCα activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed. © 2002 Elsevier Science Ltd. All rights reserved.This research was supported by grants PB98-0389 to the Universidad de Murcia, and BIO099-0865 to the IBMB and by 1FD97-1558 from DGESIC (Spain) to a collaborative project between the Universidad de Murcia and the IBMB. Data were collected at the EMBL protein crystallography beamlines at ESRF (Grenoble) within a Block Allocation Group (BAG Barcelona), as at ESRF BM14. This work was supported financially by the ESRF and by grant HPRI-CT-1999-00022 of the European Union.Peer Reviewe

Structural study of the catalytic domain of PKCfusinginfrared spectroscopy and two-dimensional infraredcorrelation spectroscopy

The secondary structure of the catalytic domain from protein kinase Cfwas studied using IR spectroscopy. In the presence of the substrateMgATP, there was a significant change in the secondary structure. Afterheating to 80°C, a 14% decrease in thea-helix component was observed,accompanied by a 6% decrease in theb-pleated sheet; no change wasobserved in the large loops or in 310-helix plus associated loops. The maxi-mum increase with heating was observed in the aggregatedb-sheet compo-nent, with an increase of 14%. In the presence of MgATP, and comparedwith the sample heated in its absence, there was a substantial decrease inthe 310-helix plus associated loops and an increase ina-helix. Synchronous2D-IR correlation showed that the main changes occurred at 1617 cm)1,which was assigned to changes in the intermolecular aggregatedb-sheet ofthe denaturated protein. This increase was mainly correlated with thechange ina-helix. In the presence of MgATP, the main correlation wasbetween aggregatedb-sheet and the large loops component. The asynchro-nous 2D-correlation spectrum indicated that a number of components aretransformed in intermolecularly aggregatedb-sheet, especially thea-helixandb-sheet components. It is interesting that changes in 310-helix plusassociated loops and ina-helix preceded changes in large loops, which sug-gests that the open loops structure exists as an intermediate state duringdenaturation. In summary, IR spectroscopy revealed an important effect ofMgATP on the secondary structure and on the thermal unfolding processwhen this was induced, whereas 2D-IR correlation spectroscopy allowed usto show the establishment of the denaturation pathway of this proteinMedicin

Deciphering the Role and Signaling Pathways of PKCα in Luminal A Breast Cancer Cells.

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor. Here, we analyzed the importance of PKCα in cellular processes such as proliferation, migration, or apoptosis by inhibiting its gene expression in a luminal A breast cancer cell line (MCF-7). Differential expression analysis and phospho-kinase arrays of PKCα-KD vs. PKCα-WT MCF-7 cells identified an essential set of proteins and oncogenic kinases of the JAK/STAT and PI3K/AKT pathways that were down-regulated, whereas IGF1R, ERK1/2, and p53 were up-regulated. In addition, unexpected genes related to the interferon pathway appeared down-regulated, while PLC, ERBB4, or PDGFA displayed up-regulated. The integration of this information clearly showed us the usefulness of inhibiting a multifunctional kinase-like PKCα in the first step to control the tumor phenotype. Then allowing us to design a possible selection of specific inhibitors for the unexpected up-regulated pathways to further provide a second step of treatment to inhibit the proliferation and migration of MCF-7 cells. The results of this study suggest that PKCα plays an oncogenic role in this type of breast cancer model. In addition, it reveals the signaling mode of PKCα at both gene expression and kinase activation. In this way, a wide range of proteins can implement a new strategy to fine-tune the control of crucial functions in these cells and pave the way for designing targeted cancer therapies.Work in Murcia was supported by grants BFU2017-87222-P (MICINN, Spain-FEDER) to S.C.-G. and J.C.G.-F. and Fundación Séneca Region de Murcia 20885/PI/18 to S.C.-G.S

Contribución al estudio de la estructura y función de la Ca2+-ATPasa del retículo sarcoplásmico / María Senena Corbalán García ; directores Juan Carmelo Gómez Fernández, José Antonio Teruel Puche.

Tesis-Universidad de Murcia.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. T.M.-1136.Consulte la tesis en: BCA. GENERAL. Fac. Veterinaria. Sala de estudio. Tesis-V 71

Correction: Crystal structure of the C-terminal four-helix bundle of the potassium channel KCa3.1.

[This corrects the article DOI: 10.1371/journal.pone.0199942.]

Mecanismo de Doble Diana de las Proteínas Periféricas de Membrana

Gran cantidad de funciones celulares dependen de la interacción de proteínas con la superficie interna de la membrana plasmática o de otras membranas intracelulares. Entre otras funciones destacan el tráfico celular, las rutas de señalización y el mantenimiento de la propia estructura celular. (Lemmon, 2008; Moravcevic, Oxley, & Lemmon, 2012). Entre un 30-40% de las proteínas celulares existentes interaccionan con algún tipo de membrana, manifestando así la importancia de las funciones que desempeñan (Arora & Tamm, 2001)

Deer Island Treatment Plant Performance (2007-09)

KCa3.1 (also known as SK4 or IK1) is a mammalian intermediate-conductance potassium channel that plays a critical role in the activation of T cells, B cells, and mast cells, effluxing potassium ions to maintain a negative membrane potential for influxing calcium ions. KCa3.1 shares primary sequence similarity with three other (low-conductance) potassium channels: KCa2.1, KCa2.2, and KCa2.3 (also known as SK1-3). These four homotetrameric channels bind calmodulin (CaM) in the cytoplasmic region, and calcium binding to CaM triggers channel activation. Unique to KCa3.1, activation also requires phosphorylation of a single histidine residue, His358, in the cytoplasmic region, which relieves copper-mediated inhibition of the channel. Near the cytoplasmic C-terminus of KCa3.1 (and KCa2.1-2.3), secondary-structure analysis predicts the presence of a coiled-coil/heptad repeat. Here, we report the crystal structure of the C-terminal coiled-coil region of KCa3.1, which forms a parallel four-helix bundle, consistent with the tetrameric nature of the channel. Interestingly, the four copies of a histidine residue, His389, in an 'a' position within the heptad repeat, are observed to bind a copper ion along the four-fold axis of the bundle. These results suggest that His358, the inhibitory histidine in KCa3.1, might coordinate a copper ion through a similar binding mode