Browsing by Subject "PKC"

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    Differential functional regulation of protein kinase C (PKC) orthologs in fission yeast.
    (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2017-07) Madrid, M.; Vázquez-Marín, B.; Soto, T.; Franco, A.; Gómez-Gil, E.; Vicente-Soler, J.; Gacto, M.; Pérez, P.; Cansado Vizoso, José; Genética y Microbiología
    The two PKC orthologs Pck1 and Pck2 in the fission yeast Schizosaccharomyces pombe operate in a redundant fashion to control essential functions, including morphogenesis and cell wall biosynthesis, as well as the activity of the cell integrity pathway (CIP) and its core element the MAPK Pmk1. We show here that despite the strong structural similarity and functional redundancy of these two enzymes, the mechanisms regulating their maturation, activation, and stabilization have a remarkably distinct biological impact on both kinases. We found that in contrast to Pck2, putative in vivo phosphorylation of Pck1 within the conserved activation loop, turn and hydrophobic motifs is essential for Pck1 stability and biological functions. Constitutive Pck activation promoted dephosphorylation and destabilization of Pck2, while it enhanced Pck1 levels to interfere with proper downstream signaling to the CIP via Pck2. Importantly, whereas catalytic activity was essential for Pck1 function, Pck2 remained partially functional independently of its catalytic activity. Our findings suggest that early divergence from a common ancestor in fission yeast involved important changes in the mechanisms regulating catalytic activation and stability of PKC family members to allow for flexible and dynamic control of downstream functions, including MAPK signaling.
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    PKCε controls the fusion of secretory vesicles in mast cells in a phosphatidic acid-dependent mode
    (ELSEVIER, 2021-06-18) Serrano-Lopez, Emilio M.; Lopez-Martínez, David; Gomez-Fernandez, Juan C.; Corbalán García, Senena; Bioquímica y Biología Molecular A
    PKCε is highly expressed in mast cells and plays a fundamental role in the antigen-triggered activation of the allergic reaction. Although its regulation by diacylglycerols has been described, its regulation by acidic phospholipids and how this regulation leads to the control of downstream vesicle secretion is barely known. Here, we used structural and evolutionary studies to find the molecular mechanism that explains the selectivity of the C1B domain of PKCε by Phosphatidic Acid (PA). This resided in a collection of Arg residues that form a specific rim on the outer surface of the C1B domain, around the diacylglycerol binding cleft. In RBL-2H3 cells, this basic rim allowed the kinase to respond specifically to phosphatidic acid signals that induced its translocation to the plasma membrane and subsequent activation. Further experiments in cells that overexpress PKCε and a mutant of the PA binding site, showed that PA-dependent PKCε activation increased vesicle degranulation in RBL-2H3 cells, and this correlated with increased SNAP23 phosphorylation. Over-expression of PKCε in these cells also induced an increase in the number of docked vesicles containing SNAP23, when stimulated with PA. This accumulation could be attributed to the stabilizing effect of phosphorylation on the formation of the SNARE complex, which ultimately led to increased release of content in the presence of Ca2+ during the fusion process. Therefore, these findings reinforce the importance of PA signaling in the activation of PKCε, which could be an important target to inhibit the exacerbated responses of these cells in the allergic reaction.