DigesTip: A new device for a rapid and efficient in-solution protein digestion
DigesTip is a new device for in-solution protein digestion, based on a patent pending technology, able to immobilize enzymes (trypsin, in this case) on a solid surface, keeping their activity preserved. DigesTip is a standard pipette tip, usable both by human and by robots. Its main performances are: very short digestion time (1 min) and usability with low protein sample concentrations (5 µg/mL). DigesTip obtains a clear signal in MS measurements and its usage rules out several preparative steps.
Covalent Binding of Flavins to RnfG and RnfD in the Rnf Complex from Vibrio cholerae
Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na+-NQR complexes.
Akt–RSK–S6 Kinase Signaling Networks Activated by Oncogenic Receptor Tyrosine Kinases
Receptor tyrosine kinases (RTKs) activate pathways mediated by serine-threonine kinases, such as the PI3K (phosphatidylinositol 3-kinase)–Akt pathway, the Ras–MAPK (mitogen-activated protein kinase)–RSK (ribosomal S6 kinase) pathway, and the mTOR (mammalian target of rapamycin)–p70 S6 pathway, that control important aspects of cell growth, proliferation, and survival. The Akt, RSK, and p70 S6 family of protein kinases transmits signals by phosphorylating substrates on an RxRxxS/T motif (R, arginine; S, serine; T, threonine; and x, any amino acid). We developed a large-scale proteomic approach to identify more than 300 substrates of this kinase family in cancer cell lines driven by the c-Met, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor (PDGFR) RTKs. We identified a subset of proteins with RxRxxS/T sites for which phosphorylation was decreased by RTK inhibitors (RTKIs), as well as by inhibitors of the PI3K, mTOR, and MAPK pathways, and we determined the effects of small interfering RNA directed against these substrates on cell viability. Phosphorylation of the protein chaperone SGTA (small glutamine-rich tetratricopeptide repeat–containing protein ) at serine-305 was essential for PDGFR stabilization and cell survival in PDGFR-dependent cancer cells. Our approach provides a new view of RTK and Akt–RSK–S6 kinase signaling, revealing previously unidentified Akt–RSK–S6 kinase substrates that merit further consideration as targets for combination therapy with RTKIs.