The mammalian target of rapamycin (mTOR) regulates cell growth and survival via two different multiprotein complexes, mTORC1 and mTORC2. The assembly of these serine-threonine kinase multiprotein complexes occurs via poorly understood molecular mechanisms. Here, we demonstrate that GRp58/ERp57 regulates the existence and activity of mTORC1. Endogenous mTOR interacts with GRp58/ERp57 in different mammalian cells. In vitro, recombinant GRp58/ERp57 preferentially interacts with mTORC1. GRp58/ERp57 knockdown reduces mTORC1 levels and phosphorylation of 4E-BP1 and p70 S6K in response to insulin. In contrast, GRp58/ERp57 overexpression increases mTORC1 levels and activity. A redox-sensitive mechanism that depends on GRp58/ERp57 expression activates mTORC1. Although GRp58/ERp57 is known as an endoplasmic reticulum (ER) resident, we demonstrate its presence at the cytosol, together with mTOR, Raptor, and Rictor as well as a pool of these proteins associated to the ER. In addition, the presence of GRp58/ERp57 at the ER decreases in response to insulin or leucine. Interestingly, a fraction of p70 S6K, but not 4E-BP1, is associated to the ER and phosphorylated in response to serum, insulin, or leucine. Altogether, our results suggest that GRp58/ERp57 is involved in the assembly of mTORC1 and positively regulates mTORC1 signaling at the cytosol and the cytosolic side of the ER.

A distinct 5¢ flanking var gene region regulates Plasmodium falciparum variant erythrocyte surface antigen expression in placental malaria opposite sense when compared with the usual orientation of telomere-adjacent var genes. This unique arrangement might explain why the varCSA gene is relatively conserved in genetically distinct parasites despite being located in a highly recombinogenic chromosome compartment. The 5¢ untranslated region (UTR) of the varCSA-type sequence is also transcribed in placental isolates that bind to CSA, illustrating an important role for the unique 5¢ varCSA-type sequence in the regulation of var genes involved in malaria pathogenesis in pregnant women. However, this promoter is not always found to be transcribing var genes selected for expression of products that bind to CSA in vitro. Our work identifies a sequence tag for the identification of varCSA genes in placental isolates for the first time. IntroductionPlasmodium falciparum is responsible for nearly all malaria-specific mortality in endemic areas. Both morbidity and mortality associated with malaria have been attributed, in part, to the unique ability of P. falciparum-infected red blood cells (iRBCs) to adhere to small vascular endothelial cells and to uninfected erythrocytes (Wahlgren et al., 1999). Adhesion to host cells seems to be essential for parasite survival, as it prevents destruction of the iRBCs in the spleen. In holoendemic areas, novel parasite variants emerge in pregnant women that seem to be able to evade pre-existing immunity (Fried and Duffy, 1996). Falciparum malaria during the first pregnancy is a major cause of maternal anaemia and low birthweight (Steketee et al., 1996). Antibodies that develop after multiple pregnancies are associated with lower levels of iRBCs in the placenta, presumably by reducing sequestration (Fried et al., 1998). P. falciparum-infected erythrocytes adhere to syncytiothrophoblast cells in the placenta via chondroitin sulphate A (CSA). A direct correlation between CSA parasite sequestration and gestational malaria has been established. Cytoadherence of iRBCs is mediated by receptor-ligand interactions between parasite ligands on the infected erythrocyte membranes and cellular adhesion molecules on the surface of vascular endothelial cells such as CD36, ICAM-1, VCAM-1, Eselectin and CSA. Adhesion to different receptors is medi- ated by the parasite-derived antigen P. falciparum erythrocyte membrane protein 1 (PfEMP-1) encoded by ª 50 var genes per haploid genome (reviewed by Craig and Scherf, 2001). Most var genes are subtelomeric; however, clusters of var genes have also been found in internal chromosome positions. Members of the var gene family are subject to clonal antigenic variation, but only a single antigen variant is expressed on the iRBC surface in a mutually exclusive manner (Chen et al., 1998;Scherf et al., 1998). In situ activation of silent var genes appears to occur at the transcriptional level, as demonstrated by nuclear run-on assays in trophozoites (Scherf et al., 199...

Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation was studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appears to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor- α1B-adrenergic receptor crosstalk.

Proteasome pathway regulates TGF-b signaling; degradation of activated Smad2/3 and receptors turns TGF-b signal off, while degradation of negative modulators such as Ski and SnoN maintains the signal. We have found that anisomycin is able to downregulate Ski and SnoN via proteasome as TGF-b does, but through a mechanism independent of Smad activation. The mechanism used by anisomycin to downregulate Ski and SnoN is also independent of MAPK activation and protein synthesis inhibition. TGF-b signal was the only pathway described causing Ski and SnoN degradation, thus this new effect of anisomycin on endogenous Ski and SnoN proteins suggests alternative processes to downregulate these negative modulators of TGF-b signaling.