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Identification and functional annotation of the Src-regulated kinome, part 2

ABSTRACT: Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. Herein, we describe a novel, integrated approach that addresses this knowledge gap. This utilizes mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotates the regulated kinases. As an example, we identified approximately one hundred protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. Screening with corresponding siRNAs identified nine kinases, including SGK1, as being essential for Src-induced transformation. In contrast, MAP4K5 suppressed transformation in a manner enhanced by S335 phosphorylation. In triple negative breast cancer cells, Src positively regulated SGK1 expression and combined inhibition of Src and SGK1 was more effective at inhibiting cell proliferation than either treatment alone. Therefore, this approach not only provides major mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Breast Cancer

SUBMITTER: Hugh Ma

LAB HEAD: Roger Daly

PROVIDER: PXD011146 | Pride | 2018-11-21

REPOSITORIES: Pride

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			Action	DRS
	20180802_MM231_AZD_TiO2_gel_01.raw	Raw
	20180802_MM231_AZD_TiO2_gel_01_reinject.raw	Raw
	20180802_MM231_AZD_TiO2_gel_01_reinject_2.raw	Raw
	20180802_MM231_AZD_TiO2_gel_02.raw	Raw
	20180802_MM231_AZD_TiO2_gel_02_reinject.raw	Raw

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Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation

Project description:Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. Herein, we describe a novel, integrated approach that addresses this knowledge gap. This utilizes mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotates the regulated kinases. As an example, we identified approximately seventy protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. An integrated siRNA screen identified nine kinases, including SGK1, as being essential for Src-induced transformation. In triple negative breast cancer cells, which exhibit a prominent signalling network governed by src family kinases, Src positively regulated SGK1 expression and combined inhibition of Src and SGK1 was more effective at inhibiting colony formation in vitro, and xenograft growth in vivo, than either treatment alone. Therefore, this approach not only provides major mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies.

2019-01-14 | PXD010687 | Pride

Global phosphoproteomic mapping of early mitotic exit in human cells identifies novel substrate dephosphorylation motifs

Project description:Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3,300 unique proteins during early mitotic exit, providing up to 8-fold greater resolution than previous studies. Only a small fraction (~10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical transition.

2015-06-25 | PXD001559 | Pride

Src modulates its oncogenic signaling via regulation of miRNA-129-3p [microRNA]

Project description:To address the molecular mechanisms underlying c-Src-induced cell transformation, we previously developed a model system using Csk-deficient fibroblasts that can be transformed by wild-type c-Src. In this study, we applied this system for the analysis of the potential contribution of mRNA and miRNA to c-Src-induced cell transformation. We found miR-129-3p was downregulated in c-Src-induced cell transformation in a Dox-inducible expression system via c-Src, c-Yes, and Fer.

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Src modulates its oncogenic signaling via regulation of miRNA-129-3p [mRNA]

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Gene profiling of v-Src transformed primary chicken cells

Project description:Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. To describe these changes, investigators have relied extensively on the study of immortalized rodent cell lines or heterogeneous tumor samples that limit the identification of differentially expressed genes or may not represent the full spectrum of biological processes regulated during transformation. In this study, we took advantage of transformation-deficient and temperature sensitive mutants of the Rous sarcoma virus to characterize the patterns of gene expression in two types of primary cells, namely chicken embryo fibroblasts (CEF) and chicken neuro-retinal (CNR) cells. Keywords: viral transformation of primary cells, transformation, transformation deficient mutant, temperature sensitive mutant, v-Src Chicken embryo fibroblasts (CEF) were infected with the wild-type strain Schmidt-Ruppin A RSV or non-transforming strain NY315 RSV or the non-transforming control virus RCASBP(A) to assess genes involved in v-Src-dependent transformation of CEF. Chicken embryo fibroblasts (CEF) were infected with the temperature sensitve strain NY72-4 RSV and cultured either at non-permissive temperature (41.5M-KM-^ZC) or permissive temperature (37M-KM-^ZC) to assess genes involved in v-Src-dependent transformation of CEF. Chicken neuroretina cells (CNR) were infected with the temperature sensitve strain NY72-4 RSV and cultured either at non-permissive temperature (41.5M-KM-^ZC) or permissive temperature (37M-KM-^ZC) to assess genes involved in v-Src-dependent transformation of CNR and compared to CEF.

2010-02-21 | E-GEOD-14489 | biostudies-arrayexpress

Stable expression of intracellular Notch suppresses v-Src-induced transformation in avian neural cells

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2010-12-01 | GSE22054 | GEO

Stable expression of intracellular Notch suppresses v-Src-induced transformation in avian neural cells

2010-12-01 | E-GEOD-22054 | biostudies-arrayexpress

STAT3 knockdown during transformation

Project description:siSTAT3 knockdown of a tamoxifen initiated, transformation inducible, breast cancer model system (MCF10A-ER-Src), with associated controls of EtOH and siNEG treatments. A description of MCF10A-ER-Src cells can be found here: www.encodeproject.org

2015-06-01 | GSE64536 | GEO

Gene profiling of v-Src transformed primary chicken cells

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Gene profiling of v-Src transformed primary chicken embryo fibroblasts (CEFs) under the condition of AP-1 repression

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