Qinqin Ji

Cytochrome c is tyrosine 97 phosphorylated by neuroprotective insulin treatment

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway

The kinase ZAP-70 has both positive and negative functions in T cell signaling. T cell signaling gets ZAPped Binding of peptide antigen to the T cell receptor (TCR) complex results in the sequential activation of the tyrosine kinases Lck and ZAP-70. Phosphorylation of immunoreceptor tyrosine–based activation motifs (ITAMs) in TCR complex subunits by these kinases results in the recruitment of effectors required for T cell activation. By comparing the phosphoproteomic profiles of unstimulated and TCR-stimulated cells in which the catalytic activity of ZAP-70 was or was not inhibited, Sjölin-Goodfellow et al. found that ZAP-70 activated feedback inhibition that dampened both TCR-dependent and TCR-independent activation of Lck. Biochemical and computational analyses suggested that the signaling network was organized such that ZAP-70 was both downstream and upstream of Lck to both promote T cell activation and prevent inappropriate T cell activation. T cell activation by antigens binding to the T cell receptor (TCR) must be properly regulated to ensure normal T cell development and effective immune responses to pathogens and transformed cells while avoiding autoimmunity. The Src family kinase Lck and the Syk family kinase ZAP-70 (ζ chain–associated protein kinase of 70 kD) are sequentially activated in response to TCR engagement and serve as critical components of the TCR signaling machinery that leads to T cell activation. We performed a mass spectrometry–based phosphoproteomic study comparing the quantitative differences in the temporal dynamics of phosphorylation in stimulated and unstimulated T cells with or without inhibition of ZAP-70 catalytic activity. The data indicated that the kinase activity of ZAP-70 stimulates negative feedback pathways that target Lck and thereby modulate the phosphorylation patterns of the immunoreceptor tyrosine–based activation motifs (ITAMs) of the CD3 and ζ chain components of the TCR and of signaling molecules downstream of Lck, including ZAP-70. We developed a computational model that provides a mechanistic explanation for the experimental findings on ITAM phosphorylation in wild-type cells, ZAP-70–deficient cells, and cells with inhibited ZAP-70 catalytic activity. This model incorporated negative feedback regulation of Lck activity by the kinase activity of ZAP-70 and predicted the order in which tyrosines in the ITAMs of TCR ζ chains must be phosphorylated to be consistent with the experimental data.

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling.

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor–mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr112, Tyr128, and Tyr145, in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr192 of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr440 of Fyn, Tyr702 of PLCγ1, Tyr204, Tyr397, and Tyr69 of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues.

Quantitative Phosphoproteomic Analysis Identifies Activation of the RET and IGF-1R/IR Signaling Pathways in Neuroblastoma

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: Implications for AMP Kinase

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation.

The activation of T lymphocytes through antigen-mediated T cell receptor (TCR) clustering is vital in regulating the adaptive immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperatures have initiated some of the hallmarks of TCR signaling, such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum, as well as coalesced the T cell membrane microdomains. The precise mechanism of the TCR signaling initiation due to temperature change remains obscure. One critical question is whether the signaling initiated by the cold treatment of T cells differs from the signaling initiated by the cross-linking of the T cell receptor. To address this uncertainty, we performed a wide-scale, quantitative mass-spectrometry-based phosphoproteomic analysis on T cells stimulated either by temperature shifts or through the cross-linking of the TCR. Careful statistical comparisons between the two stimulations revealed a striking level of identity among the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, in unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns that were nearly identical to those of soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells.

Abstract B042: Regulation of Lck activity by a cryptic phosphosite

Src family kinases (SFKs) are initiators of receptor-mediated signaling events, enabling cells to perceive and interact with their environment. For immune cells SFKs are especially crucial because they couple antigen recognition to intracellular signaling pathways. In T cells, the SFK Lck phosphorylates the T cell antigen receptor (TCR) complex upon antigen encounter. Because Lck phosphorylates the TCR complex, its activity sets a critical threshold for T cell activation; yet how Lck activity is regulated in resting or stimulated cells is poorly understood. Through quantitative mass spectrometry (SILAC), we recently identified a cryptic Lck phosphosite (Y192) as sensitive to inhibition of the effector kinase Zap70. Because Zap70 is an Lck substrate, and dramatic reduction in phosphorylation of Y192 occurred upon Zap70 inhibition, we reasoned this site might function to regulate Lck in response to changes in Zap70 activity. To test this hypothesis we reconstituted Lck-deficient Jurkat T cells with Lck Y192 variants. Mutation of Y192 reveals that this site has a profound impact on Lck activity. Cells reconstituted with Y192E or Y192A show a marked reduction in Lck activity, characterized by hyperphosphorylation on Lck9s inhibitory C-terminus (Y505) and a reduction in Lck autophosphorylation within the activation loop (Y394). Consistent with a defect in Lck activity, cells that express Lck Y192E/A variants, when assessed for hallmarks of T cell activation, reveal striking defects in TCR signaling. Interestingly, these defects are reminiscent of CD45-deficient T cells. Preliminary findings suggest that mutation of Y192 disrupts the ability of the phosphatase CD45 to dephosphorylate Lck9s inhibitory C-terminus (Y505). Because of the profound effect that Y192 modification exerts on Lck activity, and therefore TCR signaling, we believe that phosphorylation of Y192 comprises a Zap70-dependent negative feedback mechanism that tunes Lck activity. Such a mechanism predicts that increases in Zap70 activity cause phosphorylation of Y192 within Lck9s SH2 domain, favouring Lck inactivation by preventing CD45-mediated dephosphorylation of the inhibitory C-terminus. Citation Format: Adam H. Courtney, Hanna E. Sjolin-Goodfellow, Qinqin Ji, Theresa A. Kadlecek, Arthur R. Salomon, Arthur Weiss. Regulation of Lck activity by a cryptic phosphosite. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B042.