Epidermal growth factor-induced vacuolar (H+)-atpase assembly: a role in signaling via mTORC1 activation.

Using proteomics and immunofluorescence, we demonstrated epidermal growth factor (EGF) induced recruitment of extrinsic V(1) subunits of the vacuolar (H(+))-ATPase (V-ATPase) to rat liver endosomes. This was accompanied by reduced vacuolar pH. Bafilomycin, an inhibitor of V-ATPase, inhibited EGF-stimulated DNA synthesis and mammalian target of rapamycin complex 1 (mTORC1) activation as indicated by a decrease in eukaryotic initiation factor 4E-binding 1 (4E-BP1) phosphorylation and p70 ribosomal S6 protein kinase (p70S6K) phosphorylation and kinase activity. There was no corresponding inhibition of EGF-induced Akt and extracellular signal-regulated kinase (Erk) activation. Chloroquine, a neutralizer of vacuolar pH, mimicked bafilomycin effects. Bafilomycin did not inhibit the association of mTORC1 with Raptor nor did it affect AMP-activated protein kinase activity. Rather, the intracellular concentrations of essential but not non-essential amino acids were decreased by bafilomycin in EGF-treated primary rat hepatocytes. Cycloheximide, a translation elongation inhibitor known to augment intracellular amino acid levels, prevented the effect of bafilomycin on amino acids levels and completely reversed its inhibition of EGF-induced mTORC1 activation. In vivo administration of EGF stimulated the recruitment of Ras homologue enriched in brain (Rheb) but not mammalian target of rapamycin (mTOR) to endosomes and lysosomes. This was inhibited by chloroquine treatment. Our results suggest a role for vacuolar acidification in EGF signaling to mTORC1.

Global analysis of protein phosphorylation networks in insulin signaling by sequential enrichment of phosphoproteins and phosphopeptides.

Although the important role of protein phosphorylation in insulin signaling networks is well recognized, its analysis in vivo has not been pursued in a systematic fashion through proteome-wide studies. Here we undertake a global analysis of insulin-induced changes in the rat liver cytoplasmic and endosomal phosphoproteome by sequential enrichment of phosphoproteins and phosphopeptides. After subcellular fractionation proteins were denatured and loaded onto iminodiacetic acid-modified Sepharose with immobilized Al³âº ions (IMAC-Al resin). Retained phosphoproteins were eluted with 50 mM phosphate and proteolytically digested. The digest was then loaded onto an IMAC-Al resin and phosphopeptides were eluted with 50 mM phosphate, and resolved by 2-dimensional liquid chromatography, which combined offline weak anion exchange and online reverse phase separations. The peptides were identified by tandem mass spectrometry, which also detected the phosphorylation sites. Non-phosphorylated peptides found in the flow-through of the IMAC-Al columns were also analyzed providing complementary information for protein identification. In this study we enriched phosphopeptides to ~85% purity and identified 1456 phosphopeptides from 604 liver phosphoproteins. Eighty-nine phosphosites including 45 novel ones in 83 proteins involved in vesicular transport, metabolism, cell motility and structure, gene expression and various signaling pathways were changed in response to insulin treatment. Together these findings could provide potential new markers for evaluating insulin action and resistance in obesity and diabetes.

Stromal niche cells protect early leukemic FLT3-ITD+ progenitor cells against first-generation FLT3 tyrosine kinase inhibitors.

Targeting constitutively activated FMS-like tyrosine kinase 3 [(FLT3); FLT3-ITD] with tyrosine kinase inhibitor (TKI) in acute myeloid leukemia (AML) leads to clearance of blasts in the periphery but not in the bone marrow, suggesting a protective effect of the marrow niche on leukemic stem cells. In this study, we examined the effect of stromal niche cells on CD34(+) progenitors from patients with FLT3-ITD(+) or wild-type FLT3 (FLT3-WT) AML treated with the TKIs SU5614 or sorafenib. TKIs effectively and specifically inhibited FLT3 and increased the fraction of undivided progenitors in both FLT3-ITD(+) and FLT3-WT samples. Treatment with SU5614 and sorafenib also reduced the number of mature leukemic progenitors, whereas contact with stroma protected against this cell loss. In contrast, primitive long-term progenitors from both FLT3-ITD(+) and FLT3-WT AML were resistant to TKIs. Additional contact with niche cells significantly expanded long-term FLT3-ITD(+) but not FLT3-WT progenitors in the presence of SU5614 but not that of sorafenib. Thus, TKIs with first-generation inhibitors fail to eradicate early leukemic stem/progenitor cells in FLT3-ITD(+) AML. Further, we defined a specific interaction between FLT3-ITD(+) progenitors and niche cells that enables the maintenance of leukemic progenitors in the presence of TKI. Collectively, our findings suggest that molecular therapy may have unpredicted effects on leukemic progenitors, underscoring the necessity of developing strategies to selectively eliminate the malignant stem cell clone.

Oncostatin M-mediated regulation of KIT-ligand-induced extracellular signal-regulated kinase signaling maintains hematopoietic repopulating activity of Lin-CD34+CD133+ cord blood cells.

We investigated whether KIT signaling was sufficient to maintain human hematopoietic stem cells in culture or whether, as with murine stem cells, signaling through glycoprotein 130 (gp130) is additionally required. Sorted CD34(+)CD133(+)(CD33/CD38/CD71)(-) cells from human umbilical cord blood (UCB) were cultured in the presence of combinations of KIT-ligand (KL) and the gp130 stimulating molecule oncostatin M (OSM). We found that OSM increased KL-induced proliferation, which was accompanied by an expansion in numbers of mature progenitors colony-forming cells (CFC, CAFCw2). More primitive progenitors, CAFCw6 and long-term culture-CFC, were not maintained by KL as a single factor. Although addition of OSM did not improve survival, the KL/OSM combination showed improved maintenance of immature progenitors as well as higher CD34 expression. Similarly, both KL and OSM were required to maintain NOD/SCID-repopulating activity. In experiments to investigate the underlying mechanism, we found that extracellular signal-regulated kinase (ERK) and its downstream target p90 ribosomal S6 kinase were activated by KL and downregulated by the inclusion of OSM during stimulation. The p38 mitogen-activated protein kinase (p38 MAPK) was not modulated by either KL or OSM. Indeed, many of the effects of OSM (increased cell division, maintenance of CFC, and maintenance of high CD34 expression) could be mimicked by using the mitogen-activated protein kinase kinase inhibitor U0126. More importantly, NOD/SCID-repopulating activity was preserved in the KL/U0126-stimulated cells, but not in cells stimulated with a combination of KL and the p38 MAPK inhibitor SB203580. Our results show that the loss of repopulating activity during KL stimulation is counteracted by OSM through the downregulation of ERK pathway signaling. Disclosure of potential conflicts of interest is found at the end of this article.

Quantitative analysis of a proteome by N-terminal stable-isotope labelling of tryptic peptides.

Covalent modification of peptides and proteins with compounds containing stable isotopes (isotope tagging) has become an essential tool to detect dynamic changes in the proteome following external or internal influence; however, using terminal amino groups for global isotope labelling of tryptic peptides is challenged by the similar reactivity of the amino groups of lysine residues. We describe a new quantitative method based on selective tagging of the terminal amino groups of tryptic peptides with pentafluorophenyl esters containing stable isotopes. The labelled peptides were resolved by two-dimensional nanoflow liquid chromatography on weak anion-exchange and reversed-phase columns and then identified and quantified by tandem mass spectrometry. The method was applied to compare the proteomes of plasma membranes from proliferating and differentiated human colorectal adenocarcinoma (Caco-2) cells and endosomes purified from the livers of rats stimulated with insulin and epidermal growth factor. The comparison of the results obtained by isotope tagging and biochemical assays demonstrate that global isotope tagging with pentafluorophenyl esters allows accurate quantification of complex protein samples.

Compartmentalization of signaling-competent epidermal growth factor receptors in endosomes.

In this study, the preparation of detergent-resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in raft markers were used to investigate the effect of physiological doses of epidermal growth factor (EGF) in vivo on the compartmentalization and activation of EGF receptor (EGFR) in rat liver endosomes. Both of these techniques show that after EGF administration, a distinctive population of intracellular EGFR, which was characterized by a high level of tyrosine phosphorylation, accumulated in endosomes. EGFR recruited to early endosomes were more tyrosine phosphorylated than those from late endosomes. However, the level of tyrosine phosphorylation of EGFR in DRMs isolated from early and late endosomes was comparable, suggesting that EGFR in endosomal DRMs are more resistant to tyrosine dephosphorylation. In accordance with the higher level of Tyr phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc to endosomal DRMs compared with whole endosomes. Furthermore, a proteomic analysis identified a selective increase of many alpha-subunits of heterotrimeric G proteins in endosomal DRMs in response to EGF. These observations suggest that a distinctive pool of endocytic EGFR, potentially competent for signaling, is actively trafficking through intracellular compartments with the characteristic of lipid rafts.