Proteomic and Functional Genomic Landscape of Receptor Tyrosine Kinase and Ras to Extracellular Signal–Regulated Kinase Signaling

Friedman, Adam; Tucker, George; Singh, Rohit; Dong, Yan; Vinayagam, Arunachalam; Hu, Yanhui; Binari, Richard; Hong, Pengyu; Sun, Xiaoyun; Porto, Maura; Pacifico, Svetlana; Murali, Thilakam; Finley, Russell L.; Asara, John M.; Berger, Bonnie; Perrimon, Norbert

doi:10.1126/scisignal.2002029

Cited by 87 publications

(86 citation statements)

References 60 publications

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“…To determine whether the control of resting phospho-ERK levels by PDE8A activity occurs at the organismal level, we studied the phospho-ERK levels in wild-type fly strains that are known to exhibit canonical ERK signaling (35) and in a PDE8-knockout (PDE8 −/− ) Drosophila mutant (Fig. 6D).…”

Section: Resultsmentioning

confidence: 99%

Phosphodiesterase-8A binds to and regulates Raf-1 kinase

Brown¹,

Day²,

Huston³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A −/− ) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

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Section: Resultsmentioning

confidence: 99%

Phosphodiesterase-8A binds to and regulates Raf-1 kinase

Brown¹,

Day²,

Huston³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, Akt expression is reported to be regulated by phosphoinositide 3-kinase (PI3K), mTOR, and phosphate and tensin homolog (PTEN) deleted from chromosome 10 (28-31). Erk expression is also reported to be regulated by several receptors protein tyrosine kinases and the mitogenactivated protein kinase (MAPK) pathway (32)(33)(34)(35). One possible explanation for the lack of significant effect of let7c transfection on Akt and Erk could be that Akt and Erk pathways are regulated mainly by signals other than IL-6 whereas STAT3 is regulated by IL-6 expression in esophageal cancer cells.…”

Section: Discussionmentioning

confidence: 99%

Let-7 Expression Is a Significant Determinant of Response to Chemotherapy through the Regulation of IL-6/STAT3 Pathway in Esophageal Squamous Cell Carcinoma

Sugimura

Miyata

Tanaka

et al. 2012

Clinical Cancer Research

146

114

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Purpose: Cisplatin-based chemotherapy is widely used for esophageal cancer, sometimes in combination with surgery/radiotherapy, but poor response to chemotherapy is not uncommon. The aim of this study was to examine whether miRNA expression is useful to predict the response to chemotherapy in patients with esophageal cancer.Experimental Design: Using pretreatment biopsy samples from 98 patients with esophageal cancer who received preoperative chemotherapy, we measured the expression level of several miRNAs whose expression was altered in cisplatin-resistant esophageal cancer cell lines compared with those parent cell lines and examined the relationship between the miRNA expression and response to chemotherapy. In vitro assays were conducted to clarify the mechanism of miRNA-induced changes in chemosensitivity.Results: The expression levels of 15 miRNAs were altered in cisplatin-resistant cells. Of these, low expression of let-7b and let-7c in before-treatment biopsies from 74 patients of the training set correlated significantly with poor response to chemotherapy, both clinically and histopathologically. Low expression of let-7c also correlated with poor prognosis (P ¼ 0.032). The relationship between let-7b and let-7c expression and response to chemotherapy was confirmed in the other 24 patients of the validation set. In in vitro assay, transfection of let-7c restored sensitivity to cisplatin and increased rate of apoptosis after exposure to cisplatin. Let-7c directly repressed cisplatin-activated interleukin (IL)-6/STAT3 prosurvival pathway.Conclusions: Let-7 expression in esophageal cancer can be potentially used to predict the response to cisplatin-based chemotherapy. Let-7 modulates the chemosensitivity to cisplatin through the regulation of IL-6/STAT3 pathway in esophageal cancer.

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“…This can be achieved by performing an immunoprecipitation (IP) with a bait protein and analyzing the prey proteins using microcapillary-tandem mass spectrometry (LC-MS/MS) (6)(7)(8)(9)(10)(11)(12)(13). These experiments have included various types of affinity tags and have been overlaid with RNAi genetic screens (14,15). Undoubtedly, PPI networks provide a valuable framework for a better understanding of the functional organization of the proteome (16).…”

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confidence: 99%

Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS)

Breitkopf

Yuan

Pihán

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.targeted therapies | personalized medicine | protein-protein interactions P roteomics studies over the last decade have provided significant advancements in our understanding of the functional landscape of proteins, their posttranslational modifications and the protein-protein interactions (PPI) (1-3). This is especially important for diseases such as cancer because proteins and their transient posttranslational modifications (PTMs) reflect the changes cells undergo during pathogenic development. Modern high-resolution mass spectrometers are well suited for these analyses and have led the efforts in this field of proteomics, allowing researchers to analyze dynamic changes in cell lines or tissue and to distinguish between normal and unhealthy states (4, 5). PPI networks and phosphoproteomics are important for understanding the function and regulation of pathways leading to cell growth and proliferation in diseases such as cancer. The advantage of analyzing purified protein complexes is the ability to identify specific interacting proteins and posttranslational modifications that may otherwise go undetected in large-scale global analyses. This can be achieved by performing an immunoprecipitation (IP) with a bait protein and analyzing the prey proteins using microcapillary-tandem mass spectrometry (LC-MS/MS) (6-1...

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Proteomic and Functional Genomic Landscape of Receptor Tyrosine Kinase and Ras to Extracellular Signal–Regulated Kinase Signaling

Cited by 87 publications

References 60 publications

Phosphodiesterase-8A binds to and regulates Raf-1 kinase

Phosphodiesterase-8A binds to and regulates Raf-1 kinase

Let-7 Expression Is a Significant Determinant of Response to Chemotherapy through the Regulation of IL-6/STAT3 Pathway in Esophageal Squamous Cell Carcinoma

Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS)

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