2D-DIGE analysis of phospho-enriched fractions from dasatinib-treated melanoma cell lines

Eustace, Alex J.; Dowling, Paul; Henry, Michael; Doolan, Padraig; Clynes, Martin; Crown, John; O’Donovan, Norma

doi:10.1016/j.jprot.2010.12.011

Cited by 14 publications

(9 citation statements)

References 35 publications

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“…6C). This finding is reminiscent of a previous study showing that dasatinib could inhibit the growth and reduce the migration and invasion of WM-115 cells, but not WM-266 -4 cells (27).…”

Section: Quantitative Profiling Of the Global Kinomes Of Wm-115 And Wsupporting

confidence: 84%

“…These two cell lines are particularly interesting because dasatinib, a tyrosine kinase inhibitor, was found to inhibit the growth and reduce the migration and invasion of WM-115, but not WM-266 -4 cells (27). However, conventional immunoblot assay for a limited number of kinases did not reveal the target kinase(s) conferring the distinct sensitivities of these two cell lines (27). We reason that quantitative global kinome profiling of these two cell lines may uncover the target kinases of dasatinib in WM-115 cells.…”

Section: Quantitative Profiling Of the Global Kinomes Of Wm-115 And Wmentioning

confidence: 99%

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A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues

Xiao

Guo

Wang

2014

Molecular & Cellular Proteomics

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Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anticancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy. Molecular

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“…6C). This finding is reminiscent of a previous study showing that dasatinib could inhibit the growth and reduce the migration and invasion of WM-115 cells, but not WM-266 -4 cells (27).…”

Section: Quantitative Profiling Of the Global Kinomes Of Wm-115 And Wsupporting

confidence: 84%

Section: Quantitative Profiling Of the Global Kinomes Of Wm-115 And Wmentioning

confidence: 99%

A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues

Xiao

Guo

Wang

2014

Molecular & Cellular Proteomics

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“…This is correlated with markedly elevated sensitivity of WM‐115 cells toward dasatinib, a potent inhibitor for EphA2, EphB3, and EphB4 (Karaman et al, ), than WM‐266‐4 cells. This finding is reminiscent of a previous study showing that dasatinib could suppress the growth and reduce the migration and invasion of WM‐115 cells, but not WM‐266‐4 cells (Eustace et al, ). Therefore, this facile and accurate kinome profiling assay, in combination with the kinome‐inhibitor interaction map (Fabian et al, ; Karaman et al, ; Davis et al, ), may provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer therapy.…”

Section: Global Kinome Enrichment/detection Platforms and Their Applisupporting

confidence: 79%

Global discovery of protein kinases and other nucleotide‐binding proteins by mass spectrometry

Xiao

Wang

2014

Mass Spectrometry Reviews

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Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein–nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein–nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed.

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“…A decrease in p-MAPK was also observed indicating that numerous alterations in protein phosphorylation/kinase signaling pathways are occurring simultaneously in SKBR3-L cells. Thus, we compared the global phosphorylation pattern of SKBR3-L and SKBR3-par cells using 2D-gel electrophoresis coupled with MALDI-Tof/Tof mass spectrometry analysis, a technique which has been successfully applied to the study of altered protein phosphorylation [ 28 ]. eEF2 showed the largest decrease in phosphorylation levels in SKBR3-L compared to the SKBR3 parental cells and was selected for further analysis.…”

Section: Discussionmentioning

confidence: 99%

PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

et al. 2014

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BackgroundHER2 targeted therapies including trastuzumab and more recently lapatinib have significantly improved the prognosis for HER2 positive breast cancer patients. However, resistance to these agents is a significant clinical problem. Although several mechanisms have been proposed for resistance to trastuzumab, the mechanisms of lapatinib resistance remain largely unknown. In this study we generated new models of acquired resistance to HER2 targeted therapy and investigated mechanisms of resistance using phospho-proteomic profiling.ResultsLong-term continuous exposure of SKBR3 cells to low dose lapatinib established a cell line, SKBR3-L, which is resistant to both lapatinib and trastuzumab. Phospho-proteomic profiling and immunoblotting revealed significant alterations in phospho-proteins involved in key signaling pathways and molecular events. In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells. SKBR3-L cells exhibited significantly increased activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF2. SKBR3-L cells showed increased sensitivity to PP2A inhibition, with okadaic acid, compared to SKBR3 cells. PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells. Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib. The alteration in eEF2 phosphorylation, PP2A activity and sensitivity to okadaic acid were also observed in a second HER2 positive cell line model of acquired lapatinib resistance, HCC1954-L.ConclusionsOur data suggests that decreased eEF2 phosphorylation, mediated by increased PP2A activity, contributes to resistance to HER2 inhibition and may provide novel targets for therapeutic intervention in HER2 positive breast cancer which is resistant to HER2 targeted therapies.

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2D-DIGE analysis of phospho-enriched fractions from dasatinib-treated melanoma cell lines

Cited by 14 publications

References 35 publications

A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues

A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues

Global discovery of protein kinases and other nucleotide‐binding proteins by mass spectrometry

PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

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