Marı́a J. Mazón

Incorporation of 32P into yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) was observed after addition of glucose to a cell suspension incubated with (32P)orthophosphoric acid. The 32P counts were coincident with the enzyme band when immunoprecipitates were subjected to sodium dodecyl sulfate disc gel electrophoresis. The incorporation of phosphate was associated with a decrease in enzyme activity. Approximately 1 mol of phosphate was incorporated/mol of enzyme. The phosphate is bound to the enzyme in a phosphoester linkage with a serine residue. Release of 32P accompanying enzyme reactivation was observed both in vivo and in cell-free extracts.

Addition of glucose to yeast cells causes a phosphorylation and an inactivation of the gluconeogenic enzyme fructose-bisphosphatase [Mazón, M.J., Gancedo, J.M., and Gancedo, C. (1982) J. Biol. Chem. 257, 1128-1130]. We report here that the addition of the proton ionophores 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone to yeast cells produces the same effect as that of glucose. Both glucose and ionophores produced: (a) phosphorylation and inactivation of fructose-bisphosphatase, (b) an immediate rise in the intracellular concentration of cAMP, (c) an instant inhibition of the transport of amino acids driven by the membrane potential. It is proposed that the effect of glucose on fructose-bisphosphatase involves as a first step the depolarization of the plasma membrane resulting in an increase of the intracellular concentration of cAMP. This in turn would stimulate phosphorylation of fructose-bisphosphatase.

Epithelial-to-Mesenchymal Transition (EMT) is a key process contributing to the aggressiveness of cancer cells. EMT is triggered by activation of different transcription factors collectively known as EMT-TFs. Different cellular cues and cell signalling networks activate EMT at transcriptional and posttranscriptional level in different biological and pathological situations. Among them, overexpression of LOXL2 (lysyl oxidase-like 2) induces EMT independent of its catalytic activity. Remarkably, perinuclear/cytoplasmic accumulation of LOXL2 is a poor prognosis marker of squamous cell carcinomas and is associated to basal breast cancer metastasis by mechanisms no yet fully understood. Here, we report that overexpression of LOXL2 promotes its accumulation in the Endoplasmic Reticulum where it interacts with HSPA5 leading to activation of the IRE1-XBP1 signalling pathway of the ER-stress response. LOXL2-dependent IRE1-XBP1 activation induces the expression of several EMT-TFs: SNAI1, SNAI2, ZEB2 and TCF3 that are direct transcriptional targets of XBP1. Remarkably, inhibition of IRE1 blocks LOXL2-dependent upregulation of EMT-TFs thus hindering EMT induction.

An increasing number of plasma membrane proteins have been shown to be attached to the membrane via a glycosylphosphatidylinositol (GPI) moiety. All eukaryotes share a highly conserved GPI-core structure EthN-P-Man3-GlcN-PI, where EthN is ethanolamine. We have identified a protein encoded by the yeast open reading frame YGL142C that shares 33% identity with the human Pig-B protein. Deletion of this essential gene leads to a block in GPI anchor biosynthesis. We therefore named the gene GPI10. Gpi10p and Pig-B are functional homologues and the lethal deletion of GPI10 can be rescued by expression of the PIG-B cDNA. As found for PIG-B mutant cells, gpi10 deletant cells cannot attach the third mannose in an alpha-1,2 linkage to the GPI core-structure intermediate. Overexpression of GPI10 gives partial resistance to the GPI-synthesis inhibitor YW3548, suggesting that this gene product may affect the target of the inhibitor.