Functional proteomics to identify critical proteins in signal transduction pathways

Reversible protein phosphorylation plays a crucial role in the regulation of signaling pathways that control various biological responses, such as cell growth, differentiation, invasion, metastasis and apoptosis. Proteomics is a powerful research approach for fully monitoring global molecular responses to the activation of signal transduction pathways. Identification of different phosphoproteins and their phosphorylation sites by functional proteomics provides informational insights into signaling pathways triggered by all kinds of factors. This review summarizes how functional proteomics can be used to answer specific questions related to signal transduction systems of interest. By examining our own example on identifying the novel phosphoproteins in signaling pathways activated by EB virus-encoded latent membrane protein 1 (LMP1), we demonstrated a functional proteomic strategy to elucidate the molecular activity of phosphorylated annexin A2 in LMP1 signaling pathway. Functional profiling of signaling pathways is promising for the identification of novel targets for drug discovery and for the understanding of disease pathogenesis.     

Identifying responsive functional modules from protein-protein interaction network

Proteins interact with each other within a cell, and those interactions give rise to the biological function and dynamical behavior of cellular systems. Generally, the protein interactions are temporal, spatial, or condition dependent in a specific cell, where only a small part of interactions usually take place under certain conditions. Recently, although a large amount of protein interaction data have been collected by high-throughput technologies, the interactions are recorded or summarized under various or different conditions and therefore cannot be directly used to identify signaling pathways or active networks, which are believed to work in specific cells under specific conditions. However, protein interactions activated under specific conditions may give hints to the biological process underlying corresponding phenotypes. In particular, responsive functional modules consist of protein interactions activated under specific conditions can provide insight into the mechanism underlying biological systems, e.g. protein interaction subnetworks found for certain diseases rather than normal conditions may help to discover potential biomarkers. From computational viewpoint, identifying responsive functional modules can be formulated as an optimization problem. Therefore, efficient computational methods for extracting responsive functional modules are strongly demanded due to the NP-hard nature of such a combinatorial problem. In this review, we first report recent advances in development of computational methods for extracting responsive functional modules or active pathways from protein interaction network and microarray data. Then from computational aspect, we discuss remaining obstacles and perspectives for this attractive and challenging topic in the area of systems biology.     

Phosphoproteomic Analysis of Neurotrophin Receptor TrkB Signaling Pathways in Mouse Brain

SUMMARY 1. The signaling pathways activated by trkB neurotrophin receptor have been studied in detail in cultured neurons, but little is known about the pathways activated by trkB in intact brain. TrkB is a tyrosine kinase and protein phosphorylation is a key regulatory process in the neuronal signal transduction pathways.2. We have investigated trkB signaling in the transgenic mice overexpressing trkB in postnatal neurons (trkB.TK) using phosphoproteomics.3. We found that several proteins are overphosphorylated on tyrosine residues in the brain of trkB.TK mice and identified some of these proteins.4. We demonstrate that the well characterized signaling molecules mitogen-activated protein kinase (MAPK) and cyclic AMP responsive element binding protein (CREB) were phosphorylated at a higher level in the brain of trkB.TK mice when compared to the wild type littermates. Furthermore, we found that β-actin was tyrosine phosphorylated in the brain of the transgenic mice.5. Our results demonstrate that phosphoproteomics is a sensitive approach to investigate signaling pathways activated in mouse brain.     

Global expression profiling and pathway analysis of mouse mammary tumor reveals strain and stage specific dysregulated pathways in breast cancer progression

It is believed that the alteration of tissue microenvironment would affect cancer initiation and progression. However, little is known in terms of the underlying molecular mechanisms that would affect the initiation and progression of breast cancer. In the present study, we use two murine mammary tumor models with different speeds of tumor initiation and progression for whole genome expression profiling to reveal the involved genes and signaling pathways. The pathways regulating PI3K-Akt signaling and Ras signaling were activated in Fvb mice and promoted tumor progression. Contrastingly, the pathways regulating apoptosis and cellular senescence were activated in Fvb.B6 mice and suppressed tumor progression. We identified distinct patterns of oncogenic pathways activation at different stages of breast cancer, and uncovered five oncogenic pathways that were activated in both human and mouse breast cancers. The genes and pathways discovered in our study would be useful information for other researchers and drug development.     

Making sense of cross-talk between steroid hormone receptors and intracellular signaling pathways: who will have the last word?

In classical models of nuclear steroid hormone receptor function, ligand binds receptor, heat shock proteins dissociate, and receptor dimers enter or are withheld in the nucleus and interact with coregulatory molecules to mediate changes in gene expression. The footnotes, "receptors become phosphorylated" and "dynamic nucleo-cytoplasmic shuttling occurs" describe well-accepted, but less well-understood aspects of receptor action. Recently, the idea that several protein kinases are activated in response to steroid hormone binding to cognate cytoplasmic or membrane-associated receptors has become fashionable. However, the precise role of steroid hormone receptor phosphorylation and our understanding of which cytoplasmic kinases are activated and their functional significance remain elusive. This review provides an overview of the primary ways in which steroid hormone receptor and growth factor cross-talk occurs, using the human progesterone receptor (PR) as a model. The functional consequences of PR phosphorylation by protein kinases classically activated in response to peptide growth factors and novel extranuclear or nongenomic functions of PR as potential independent initiators of signal transduction pathways are discussed. Intracellular protein kinases are emerging as key mediators of steroid hormone receptor action. Cross-talk between steroid receptor- and growth factor-initiated signaling events may explain how gene subsets are coordinately regulated by mitogenic stimuli in hormonally responsive normal tissues, and is suspected to play a role in their cancer biology.     

幽门螺杆菌通过激活磷脂酰肌醇3-激酶信号来调节细胞迁移

目的幽门螺杆菌被认为是诱发胃癌的最强的风险因素。幽门螺旋杆菌的毒性成分是可以增加癌症危险的cag分泌系统,它可以使cagA和肽聚糖易位进入宿主细胞,进而激活信号转导通路。AKT是磷脂酰肌醇3。激酶（PI3K）的目的蛋白,并在胃癌中被激活,但PI3K-AKT和具有潜在致癌性的幽门螺旋杆菌诱导的细胞反应之间的关系尚不清楚。方法我们揭示了介导幽门螺旋杆菌刺激的AKT活化和胃上皮细胞的这些生物学结果之间的分子通路。结果幽门螺旋杆菌以Scr和表皮生长因子受体依赖性方式增加PI3K-AKT的信号,是幽门螺旋杆菌诱导的细胞迁移不可或缺的。结论这些结果表明,PI3K-AKT信号调节幽门螺旋杆菌诱发的病理生理反应,从而降低癌变门槛。     

Expression profiling of MAP kinase-mediated meiotic progression in Caenorhabditis elegans

The LET-60 (Ras)/LIN-45 (Raf)/MPK-1 (MAP kinase) signaling pathway plays a key role in the development of multiple tissues in Caenorhabditis elegans. For the most part, the identities of the downstream genes that act as the ultimate effectors of MPK-1 signaling have remained elusive. A unique allele of mpk-1, ga111, displays a reversible, temperature-sensitive, tissue-specific defect in progression through meiotic prophase I. We performed gene expression profiling on mpk-1(ga111) animals to identify candidate downstream effectors of MPK-1 signaling in the germ line. This analysis delineated a cohort of genes whose expression requires MPK-1 signaling in germ cells in the pachytene stage of meiosis I. RNA in situ hybridization analysis shows that these genes are expressed in the germ line in an MPK-1-dependent manner and have a spatial expression pattern consistent with the location of activated MPK-1. We found that one MPK-1 signaling-responsive gene encoding a C2H2 zinc finger protein plays a role in meiotic chromosome segregation downstream of MPK-1. Additionally, discovery of genes responsive to MPK-1 signaling permitted us to order MPK-1 signaling relative to several events occurring in pachytene, including EFL-1/DPL-1 gene regulation and X chromosome reactivation. This study highlights the utility of applying global gene expression methods to investigate genes downstream of commonly used signaling pathways in vivo.     

Kinase activity profiling reveals active signal transduction pathways in pediatric acute lymphoblastic leukemia: A new approach for target discovery

Still about 20% of patients with acute lymphoblastic leukemia (ALL) struggle with relapse, despite intensive chemotherapy. We and others have shown that kinase activity profiling is able to give more insights in active signal transduction pathways and point out interesting signaling hubs as well as new potential druggable targets. With this technique the gap between newly designed drugs and ALL may be bridged. The aim of this study was to perform kinome profiling on 20 pediatric ALL samples (14 BCP‐ALL and six T‐ALL) to identify signaling proteins relevant to ALL. We defined 250 peptides commonly activated in both BCP‐ALL and T‐ALL representing major signal transduction pathways including MAPK, PI3K/Akt, and regulators of the cell cycle/p53 pathway. For 27 peptides, differentially phosphorylation between BCP‐ALL and T‐ALL was observed. Among these, ten peptides were more highly phosphorylated in BCP‐ALL while 17 peptides showed increased phosphorylation in T‐ALL. Furthermore we selected one lead of the list of commonly activated peptides (HGFR_Y1235) in order to test its efficacy as a potential target and provide proof of principle for this approach. In conclusion kinome profiling is an elegant approach to study active signaling and identify interesting potential druggable targets.     

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.     

Identification of novel therapeutic targets in microdissected clear cell ovarian cancers

Clear cell ovarian cancer is an epithelial ovarian cancer histotype that is less responsive to chemotherapy and carries poorer prognosis than serous and endometrioid histotypes. Despite this, patients with these tumors are treated in a similar fashion as all other ovarian cancers. Previous genomic analysis has suggested that clear cell cancers represent a unique tumor subtype. Here we generated the first whole genomic expression profiling using epithelial component of clear cell ovarian cancers and normal ovarian surface specimens isolated by laser capture microdissection. All the arrays were analyzed using BRB ArrayTools and PathwayStudio software to identify the signaling pathways. Identified pathways validated using serous, clear cell cancer cell lines and RNAi technology. In vivo validations carried out using an orthotopic mouse model and liposomal encapsulated siRNA. Patient-derived clear cell and serous ovarian tumors were grafted under the renal capsule of NOD-SCID mice to evaluate the therapeutic potential of the identified pathway. We identified major activated pathways in clear cells involving in hypoxic cell growth, angiogenesis, and glucose metabolism not seen in other histotypes. Knockdown of key genes in these pathways sensitized clear cell ovarian cancer cell lines to hypoxia/glucose deprivation. In vivo experiments using patient derived tumors demonstrate that clear cell tumors are exquisitely sensitive to antiangiogenesis therapy (i.e. sunitinib) compared with serous tumors. We generated a histotype specific, gene signature associated with clear cell ovarian cancer which identifies important activated pathways critical for their clinicopathologic characteristics. These results provide a rational basis for a radically different treatment for ovarian clear cell patients.