Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death.

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis

JUN dependency in distinct early and late BRAF inhibition adaptation states of melanoma.

A prominent mechanism of acquired resistance to BRAF inhibitors in BRAF (V600) -mutant melanoma is associated with the upregulation of receptor tyrosine kinases. Evidences suggested that this resistance mechanism is part of a more complex cellular adaptation process. Using an integrative strategy, we found this mechanism to invoke extensive transcriptomic, (phospho-) proteomic and phenotypic alterations that accompany a cellular transition to a de-differentiated, mesenchymal and invasive state. Even short-term BRAF-inhibitor exposure leads to an early adaptive, differentiation state change-characterized by a slow-cycling, persistent state. The early persistent state is distinct from the late proliferative, resistant state. However, both differentiation states share common signaling alterations including JUN upregulation. Motivated by the similarities, we found that co-targeting of BRAF and JUN is synergistic in killing fully resistant cells; and when used up-front, co-targeting substantially impairs the formation of the persistent subpopulation. We confirmed that JUN upregulation is a common response to BRAF inhibitor treatment in clinically treated patient tumors. Our findings demonstrate that events shared between early- and late-adaptation states provide candidate up-front co-treatment targets

Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin.

Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36

Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

<div><p>Specific oxidized phospholipids (oxPC_CD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPC_CD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPC_CD36 as well as by the strong physiological agonist thrombin. oxPC_CD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPC_CD36. Subsequent <i>ex vivo</i> studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPC_CD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPC_CD36.</p></div

Phosphoproteome network regulating integrin in platelets activated by thrombin.

<p>Proteins and their sites of phosphorylation identified by mass spectrometry were mapped onto an integrin protein- interaction network (reference 32) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084488#pone.0084488.s003" target="_blank">Materials and Methods S1</a>. Graphical representation was done with Cytoscape. Proteins are colored based on the changes in phosphorylation (yellow circle for at least one phosphorylation event induced by thrombin; white circle for no measurable change by thrombin). Phosphorylation sites are colored based on the up-regulation (red diamond), down-regulation (green diamond), or absence of modification (white diamond) by thrombin.</p

KODA-PC activates the sequential phosphorylation of SFK, SYK, and PLCγ2 through CD36.

<p><b>(A)</b> Human platelets isolated by gel filtration were incubated with buffer alone (–), CD36 blocking antibody clone FA6-152 (CD36), or a negative control antibody (IgG); then, 50 µM PLPC or 50 µM KODA-PC were added to the platelets for 7 minutes. Equal amount of protein were separated by gel electrophoresis and probed with antibodies raised against phosphorylated SYK (pTyr323) and PLCγ2 (pTyr1217). The blots were reprobed with SYK and PLCγ2 antibodies for normalization. <b>(B)</b> Human platelets isolated by gel filtration were incubated with DMSO, 10 µM SFK inhibitor (PP2), or 0.2 µM SYK inhibitor (BAY61-3606) for 30 min; then, 50 µM PLPC or 50 µM KODA-PC were added to the platelets for 7 minutes. Equal amount of protein were separated by gel electrophoresis and probed with antibodies raised against phosphorylated SYK (pTyr525/526) and PLCγ2 (pTyr759 and pTyr1217). The blots were reprobed with SYK and PLCγ2 antibodies for normalization.</p

Phosphorylation dependent responses in integrin β3 adaptor proteins observed in platelets activated by thrombin.

<p>¹ Phosphorylation at tyrosine, serine, or threonine is indicated by “pY”, “pS”, and “pT” respectively.</p><p>² Calculated as the peak area ratio of the tryptic peptide between Thrombin and Resting samples. The largest fold change is shown for peptides with the same sequence but different charge as shown in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084488#pone.0084488.s002" target="_blank">File S2</a>.</p

KODA-PC induces platelet P-selectin expression in a SFK- and SYK-dependent manner.

<p><b>(A-B)</b> Human platelets isolated by gel filtration were incubated with 10 µM SFK inhibitor (PP2), 0.2 µM SYK inhibitor (BAY 61-3606), or controls (DMSO and 10 µM PP3) for 30 min. Then, 30 µM PLPC, 30 µM KODA-PC, 10 µM ADP, or 0.05 U/mL thrombin were added for 30 min. Platelet P-selectin expression was determined by flow cytometry using PE-conjugated antibody to P-selectin. <b>(A)</b> Flow cytometry histograms from representative experiments are shown. <b>(B)</b> Quantitation of flow cytometry data presented as mean ± SD of at least 3 independent experiments. * P<0.05.</p