Selective activation of protein kinase C-δ and -ɛ by 6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U)

Abstract

6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U) is a diterpene compound isolated from Plectranthus grandidentatus with an antiproliferative effect on several human cancer cell lines. Herein, we studied the modulatory activity of coleon U on individual isoforms of the three protein kinase C (PKC) subfamilies, classical (cPKC- and -I), novel (nPKC- and -) and atypical (aPKC-), using a yeast PKC assay. The results showed that, whereas the PKC activator phorbol-12-myristate-13-acetate (PMA) activated every PKC tested except aPKC coleon U had no effect on aPKC and cPKCs. Besides, the effect of coleon U on nPKCs was higher than that of PMA. This revealed that coleon U was a potent and selective activator of nPKCs The isoform-selectivity of coleon U for nPKC- and - was confirmed using an in vitro PKC assay. Most importantly, while PMA activated nPKCs inducing an isoform translocation from the cytosol to the plasma membrane and a G2/M cell cycle arrest, coleon U induced nPKCs translocation to the nucleus and a metacaspase-and mitochondrial-dependent apoptosis. This work therefore reconstitutes in yeast distinct subcellular translocations of a PKC isoform and the subsequent distinct cellular responses reported for mammalian cells. Together, our study identifies a new isoform-selective PKC activator with promising pharmacological applications. Indeed, since coleon U has no effect on cPKCs and aPKC recognized as anti-apoptotic proteins, and selectively induces an apoptotic pathway dependent on nPKC- and - activation, it represents a promising compound for evaluation as an anti-cancer drug.

Differential regulation of p53 function by protein kinase C isoforms revealed by a yeast cell system

“…1 A and B). Instead, and as previously reported by us [8,9], in our experimental conditions expression of a mammalian PKC isoform did not significantly interfere with yeast growth (Fig. 1A and B).…”

“…(A) Cells with DNA fragmentation (TUNEL+) and necrotic cells (PI+) obtained from yeast expressing WT p53 for 35, 45 and 60 h incubation; (B) TUNEL and PI positive cells, (C) Yca1p activation and (D) ROS accumulation, obtained from yeast expressing WT p53 and/or a PKC isoform for 45 h incubation. In B, C and D, yeasts expressing PKC‐ε treated with 1 μM coleon U (see [8]) were used as positive control (C+). Data represent means ± S.E.M.…”

“…Since the yeast PKC (Pkc1p in Saccharomyces cerevisiae ) is a structural but not a functional homologue of mammalian PKC isoforms, yeast was considered a well‐suited organism to study individual mammalian PKC isoforms [7]. Indeed, the yeast PKC assay has been used by us to search for isoform‐selective PKC modulators [8] and to study the regulation of human apoptotic proteins by PKC isoforms [9]. Similarly, though no orthologues of human p53 have been identified in yeast, S. cerevisiae has been extensively used to score several functional properties of human p53 [10].…”

New insights into cancer‐related proteins provided by the yeast model

“…The applicability of this small molecule as an analytical probe for studying nPKCδ and ε cellular signalling pathways has been confirmed in mammalian cells [80]. Furthermore, the stimulation of an apoptotic pathway independent of anti‐apoptotic PKC isoforms (α, βI and ζ), as revealed by a study in yeast [79], also supports the promising application of coleon U as an anticancer drug. Interestingly, it was also shown in yeast that, although 4β‐phorbol l2‐myristate 13‐acetate activated nPKCδ and ε, inducing their translocation from the cytosol to the plasma membrane and G2/M cell cycle arrest, coleon U induced the translocation of these isoforms to the nucleus and metacaspase‐ and mitochondria‐dependent apoptosis [79].…”

“…The yeast PKC phenotypic assay was also extensively used in the characterization of the potency and selectivity of known PKC activators [73] and inhibitors [74,75], as well as in the screening for new potent and selective PKC pharmacological modulators of individual mammalian PKC isoforms [74,76–78]. More recently, using yeast cells expressing PKCα, βI, δ, ε or ζ, a new potent and selective activator of nPKCδ and ε was discovered [79]. The applicability of this small molecule as an analytical probe for studying nPKCδ and ε cellular signalling pathways has been confirmed in mammalian cells [80].…”