Deciphering Phosphotyrosine-Dependent Signaling Networks in Cancer by SH2 Profiling

Machida, Kazuya; Khenkhar, Malik; Nollau, Peter

doi:10.1177/1947601912459048

Cited by 14 publications

(8 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We observed that the cell line deficient in Rptpζ proliferated at higher rate, which was also confirmed by BrdU incorporation assays ( Fig 5A ). We further assessed differences in the state of tyrosine phosphorylation between the Trp53 +/- /Ptprz1 +/- and Trp53 +/- /Ptprz1 -/- OS cell lines using far-Western Blotting with different SH2-domains [ 46 , 47 ]. Here we observed that several proteins were specifically detected in Trp53 +/- /Ptprz1 -/- cells with the most obvious differences in the case of SH2-domains of ABL2, CRK, NCK2, Pi3KN and SRC ( Fig 5B , S2 Fig ).…”

Section: Resultsmentioning

confidence: 99%

“…After incubation on ice for 30 min cellular lysates were cleared by centrifugation at 4°C for 10 min and supernatants were stored at -80°C. SH2 profiling based on far-Western blotting was essentially performed as described previously [ 46 , 47 ]. Briefly, 15 μg of OS lysates were separated by SDS-PAGE (4–12% acrylamide gels) and transferred to PVDF membranes.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice

et al. 2015

View full text Add to dashboard Cite

Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice

et al. 2015

View full text Add to dashboard Cite

show abstract

“…17 In contrast to MCL19-expressing and untransduced Ramos cells, we observed a substantial increase of phosphorylation in MCL11-expressing cells, further demonstrating the activation of BCR downstream signaling by SpA (Online Supplementary Figure S5). These findings proved that SpA is able to crosslink the BCR and activate its downstream signaling cascade.…”

mentioning

confidence: 78%

Complementarity determining region-independent recognition of a superantigen by B-cell antigen receptors of mantle cell lymphoma

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…SH2 profiling is a unique proteomic method in which interactions between an array of SH2 domains and protein samples are quantitatively analyzed, thereby defining the functional output of tyrosine phosphorylation. There are three assay platforms for SH2 profiling: quantitative far-Western, rosette, and oligonucleotide-tagged multiplex (OTM) assays [ 10 ]. In far-Western, protein samples are separated by electrophoresis and replicate blots are separately probed with labeled SH2 domains [ 6 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

“…Signal is detected either by chemiluminescence (far-Western and rosette) or by PCR (OTM). Quantified values are used to classify samples, such as different cancer tissues, based on SH2 binding preferences (SH2 profiles) [ 5 , 6 , 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR

et al. 2015

View full text Add to dashboard Cite

BackgroundThere is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required.ResultsWe have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1–2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment.ConclusionsWe showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain–ligand interactions which could have wide-ranging applications in both basic and translational cancer research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0169-1) contains supplementary material, which is available to authorized users.

show abstract

Deciphering Phosphotyrosine-Dependent Signaling Networks in Cancer by SH2 Profiling

Cited by 14 publications

References 48 publications

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice

Complementarity determining region-independent recognition of a superantigen by B-cell antigen receptors of mantle cell lymphoma

SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR

Contact Info

Product

Resources

About