The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.     

Medea in full self-control

Medea, a loving but betrayed character from Greek mythology who murdered her own children, inspired the naming of an Arabidopsis regulatory protein involved in the control of endosperm development in the maturing seed. In contrast to the Medea of antiquity who, according to Euripides, cannot be called a model of self-control, three recent publications demonstrate that MEDEA in plants is in command of its own action by autoregulation.     

DRBC agar: a new tool for <Emphasis Type="Italic">Candida dubliniensis</Emphasis> identification

Candida dubliniensis pathogenic species, which shares many phenotypic features with C. albicans, may be misidentified in the microbiology laboratory. The growth on DRBC agar at 25 °C was shown to be a new tool for differentiation between C. dubliniensis and C. albicans. All 27 isolates of C. dubliniensis showed in this medium rough colonies (peripheral hyphal fringes) and abundant chlamydospore production, while all 103 isolates of C. albicans showed smooth colonies without fringes or chlamydospores. DRBC agar allowed the differentiation of C. albicans from C. dubliniensis with 100 % sensitivity and specificity.     

Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad.     

Under siege: virus control in plant meristems and progeny

In the arms race between plants and viruses, two frontiers have been utilized for decades to combat viral infections in agriculture. First, many pathogenic viruses are excluded from plant meristems, which allows the regeneration of virus-free plant material by tissue culture. Second, vertical transmission of viruses to the host progeny is often inefficient, thereby reducing the danger of viral transmission through seeds. Numerous reports point to the existence of tightly linked meristematic and transgenerational antiviral barriers that remain poorly understood. In this review, we summarize the current understanding of the molecular mechanisms that exclude viruses from plant stem cells and progeny. We also discuss the evidence connecting viral invasion of meristematic cells and the ability of plants to recover from acute infections. Research spanning decades performed on a variety of virus/host combinations has made clear that, beside morphological barriers, RNA interference (RNAi) plays a crucial role in preventing—or allowing—meristem invasion and vertical transmission. How a virus interacts with plant RNAi pathways in the meristem has profound effects on its symptomatology, persistence, replication rates, and, ultimately, entry into the host progeny.

We review what is known about the biological mechanisms regulating virus exclusion from—or invasion of—plant host meristems and progeny, including possible consequences and implications of these phenomena.     

In vitro activity of essential oils extracted from plants used as spices against fluconazole-resistant and fluconazole-susceptible Candida spp

In the present study, the antifungal activity of selected essential oils obtained from plants used as spices was evaluated against both fluconazole-resistant and fluconazole-susceptible Candida spp. The Candida species studied were Candida albicans, Candida dubliniensis, Candida tropicalis, Candida glabrata, and Candida krusei. For comparison purposes, they were arranged in groups as C. albicans, C. dubliniensis, and Candida non-albicans. The essential oils were obtained from Cinnamomum zeylanicum Breyn, Lippia graveolens HBK, Ocimum basilicum L., Origanum vulgare L., Rosmarinus officinalis L., Salvia officinalis L., Thymus vulgaris L., and Zingiber officinale. The susceptibility tests were based on the M27-A2 methodology. The chemical composition of the essential oils was obtained by gas chromatography-mass spectroscopy and by retention indices. The results showed that cinnamon, Mexican oregano, oregano, thyme, and ginger essential oils have different levels of antifungal activity. Oregano and ginger essential oils were found to be the most and the least efficient, respectively. The main finding was that the susceptibilities of fluconazole-resistant C. albicans, C. dubliniensis, and Candida non-albicans to Mexican oregano, oregano, thyme, and ginger essential oils were higher than those of the fluconazole-susceptible yeasts (P<0.05). In contrast, fluconazole-resistant C. albicans and Candida non-albicans were less susceptible to cinnamon essential oil than their fluconazole-susceptible counterparts (P<0.05). A relationship between the yeasts' susceptibilities and the chemical composition of the essential oils studied was apparent when these 2 parameters were compared. Finally, basil, rosemary, and sage essential oils did not show antifungal activity against Candida isolates at the tested concentrations.     

Serine 396 of PDK1 is required for maximal PKB activation

Protein kinase B (PKB; also known as Akt) is important for mediating survival and proliferation signals. Following activation, PKB shuttles to various compartments of the cell, including the nucleus, where it phosphorylates an array of targets. PKB is phosphorylated at T308 by its activator PDK1. PDK1 is normally excluded from the nucleus via a nuclear exclusion sequence (NES), and our previous work suggested that nuclear exclusion can be attenuated by IGF-1-induced phosphorylation of S396 proximal to the NES. No studies have been done to test the significance of S396 phosphorylation or the impact of nuclear accumulation of PDK1 on PKB activation. To address these questions, we created isogenic embryonic stem cell (ESC) lines expressing various alleles of PDK1 within a PDK1-/- background. Disruption of the NES domain of PDK1 correlated with elevated PKB phosphorylation at both T308 and S473. In contrast, mutation of S396 to alanine reduced PDK1 nuclear localization and reduced PKB phosphorylation and activation. The loss of phosphorylation of PKB by S396A mutation was rescued by forcing nuclear PDK1 or by conversion of S396 to an aspartic acid. The phosphorylation of the PKB substrate FOXO3alpha was reduced in S396A PDK1 ESC. Other known and suspected PKB substrates, including GSK3 and Raf1, were unaffected. This study therefore reveals that S396 plays a role in the activation of PKB leading to the regulated phosphorylation of some PKB substrates including FOXO3alpha.