Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.     

Ligand Entry and Exit Pathways in the β2-Adrenergic Receptor

The recently determined crystal structure of the human β₂-adrenergic (β₂AR) G-protein-coupled receptor provides an excellent structural basis for exploring β₂AR-ligand binding and dissociation process. Based on this crystal structure, we simulated ligand exit from the β₂AR receptor by applying the random acceleration molecular dynamics (RAMD) simulation method. The simulation results showed that the extracellular opening on the receptor surface was the most frequently observed egress point (referred to as pathway A), and a few other pathways through interhelical clefts were also observed with significantly lower frequencies. In the egress trajectories along pathway A, the D192-K305 salt bridge between the extracellular loop 2 (ECL2) and the apex of the transmembrane helix 7 (TM7) was exclusively broken. The spatial occupancy maps of the ligand computed from the 100 RAMD simulation trajectories indicated that the receptor-ligand interactions that restrained the ligand in the binding pocket were the major resistance encountered by the ligand during exit and no second barrier was notable. We next performed RAMD simulations by using a putative ligand-free conformation of the receptor as input structure. This conformation was obtained in a standard molecular dynamics simulation in the absence of the ligand and it differed from the ligand-bound conformation in a hydrophobic patch bridging ECL2 and TM7 due to the rotation of F193 of ECL2. Results from the RAMD simulations with this putative ligand-free conformation suggest that the cleft formed by the hydrophobic bridge, TM2, TM3, and TM7 on the extracellular surface likely serves as a more specific ligand-entry site and the ECL2-TM7 hydrophobic junction can be partially interrupted upon the entry of ligand that pushes F193 to rotate, resulting in a conformation as observed in the ligand-bound crystal structure. These results may help in the design of β₂AR-targeting drugs with improved efficacy, as well as in understanding the receptor subtype selectivity of ligand binding in the β family of the adrenergic receptors that share almost identical ligand-binding pockets, but show notable amino acid sequence divergence in the putative ligand-entry site, including ECL2 and the extracellular end of TM7.     

Cell adaptation to activated FGFR3 includes Sprouty4 up regulation to inhibit the receptor-mediated ERKs activation from the endoplasmic reticulum

The kinase activity of the thanatophoric dysplasia type II-fibroblast growth factor receptor 3 mutant (TDII-FGFR3) hampers its maturation. As a consequence, the immature receptor activates extracellular regulated kinases (ERKs) from the endoplasmic reticulum (ER), which leads to apoptosis. On the other hand, in stable TDII-FGFR3 cells receptor biosynthesis is restored and ERKs are activated from the cell surface. To identify potential mediators of cell adaptation to the activated receptor we investigated gene products that are differently regulated in TDII and wild-type FGFR3 cells. cDNA representational difference analysis reveals Sprouty4 up regulation in the TDII-FGFR3 cells. Interestingly, Sprouty4 inhibits the TDII-FGFR3-mediated ERKs activation from the ER, but fails to suppress ERKs activation from cell surface. We conclude that cell adaptation to activated FGFR3 include Sprouty4 activity, which silences the premature receptor signaling and suppress apoptosis.     

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.     

Characterization of a biofilm-like extracellular matrix in FLO1-expressing Saccharomyces cerevisiae cells

Like bacteria, fungi growing in biofilms are often embedded in a so-called extracellular matrix (ECM), a complex and species-specific mixture of compounds secreted by cells in the biofilm. The precise physiological role of this ECM and its importance for the stress and drug resistance that is so characteristic of biofilms remain vague. Here, we describe the discovery of an ECM produced by flocculating Saccharomyces cerevisiae cells. Although S. cerevisiae has long been believed not to produce an ECM, our results indicate that flocculating cells secrete a mixture of glucose and mannose polysaccharides that surrounds flocculating cells. This matrix impedes the penetration of large molecules into the floc, but does not seem to play a role in the resistance of flocculating cultures to drugs and ethanol. Together, our results provide a new model system to study the formation and biological role of microbial extracellular matrices.     

Distribution, levels and phosphorylation of Raf-1 in Alzheimer's disease

Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase pathway, has been increasingly implicated in the pathogenesis of Alzheimer's disease due to its critical role in brain function. While we previously demonstrated that ERK is activated in Alzheimer's disease, the upstream cascade leading to its activation had not been fully examined. In this study, we focused on Raf-1, one of the physiological activators of the ERK pathway. Raf-1 is activated by phosphorylation at Ser338 and Tyr340/341 and inhibited by phosphorylation at Ser259. Interestingly, phosphorylation at all three sites on Raf-1 was increased as evidenced by both immunocytochemistry and immunoblot analysis in Alzheimer's disease brains compared to age-matched controls. Both phospho-Raf-1 (Ser259) and phospho-Raf-1 (Ser338) were localized to intracytoplasmic granular structures, whereas phospho-Raf-1 (Tyr340/341) was localized to neurofibrillary tangles and granules in pyramidal neurons in Alzheimer's disease hippocampus. There is extensive overlap between phospho-Raf-1 (Ser338) and phospho-Mek1/2, the downstream effector of Raf-1, suggestive of a mechanistic link. Additionally, increased levels of Raf-1 are associated with Ras and MEK1 in Alzheimer's disease as evidenced by its coimmunoprecipitation with Ras and Mek1, respectively. Based on these findings, we speculate that Raf-1 is activated to effectively mediate Ras-dependent signals in Alzheimer's disease.