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

Thompson CM, Bloom LR, Ogiue-Ikeda M, Machida K - BMC Biotechnol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, 06030, Farmington, CT, USA. thompsc1@mskcc.org.

ABSTRACT

Background: There 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.

Results: We 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.

Conclusions: We 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.

No MeSH data available.


Related in: MedlinePlus

In-solution SH2 domain binding assay using proximity ligation and real-time PCR. a, Schematic Illustration of SH2-PLA. A pair of PLA probes is used to detect the interaction of tyrosine phosphorylated EGFR and a GST-SH2 domain. The 3′ SH2-PLA probe consists of an anti-EGFR antibody conjugated with the 3′ proximity oligonucleotide (3′ Prox-Oligo). The 5′ SH2-PLA probes consists of an anti-GST antibody conjugated with the 5′ Prox-Oligo and a GST-SH2 domain. When the GST-SH2 domain binds to tyrosine phosphorylation sites of EGFR, the 5′ and 3′ PLA probes are brought in close proximity, allowing ligation of the two Prox-Oligos which is detectable by real-time PCR. b, Experimental workflow of SH2-PLA Method 1. Lysates are prepared with or without EGF stimulation. Biotinylated anti-GST and anti-EGFR antibodies are conjugated with the 5′ and 3′ Prox-Oligos, respectively, and stored at −20 °C. The 5′ SH2-PLA probe is mixed with purified GST-SH2, and the 3′ SH2-PLA probe is mixed with cell lysates allowing the antibodies to bind their respective epitopes. Subsequently, the 5′ and 3′ PLA probe solutions are combined to induce interaction between the SH2 and pEGFR. Then, the amount of the complex is quantified by proximity ligation and real-time PCR. An alternative method is also possible (Additional file 1: Figure S2). Estimated assay runtime including sample-handling steps for each procedure is noted on the right
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Fig1: In-solution SH2 domain binding assay using proximity ligation and real-time PCR. a, Schematic Illustration of SH2-PLA. A pair of PLA probes is used to detect the interaction of tyrosine phosphorylated EGFR and a GST-SH2 domain. The 3′ SH2-PLA probe consists of an anti-EGFR antibody conjugated with the 3′ proximity oligonucleotide (3′ Prox-Oligo). The 5′ SH2-PLA probes consists of an anti-GST antibody conjugated with the 5′ Prox-Oligo and a GST-SH2 domain. When the GST-SH2 domain binds to tyrosine phosphorylation sites of EGFR, the 5′ and 3′ PLA probes are brought in close proximity, allowing ligation of the two Prox-Oligos which is detectable by real-time PCR. b, Experimental workflow of SH2-PLA Method 1. Lysates are prepared with or without EGF stimulation. Biotinylated anti-GST and anti-EGFR antibodies are conjugated with the 5′ and 3′ Prox-Oligos, respectively, and stored at −20 °C. The 5′ SH2-PLA probe is mixed with purified GST-SH2, and the 3′ SH2-PLA probe is mixed with cell lysates allowing the antibodies to bind their respective epitopes. Subsequently, the 5′ and 3′ PLA probe solutions are combined to induce interaction between the SH2 and pEGFR. Then, the amount of the complex is quantified by proximity ligation and real-time PCR. An alternative method is also possible (Additional file 1: Figure S2). Estimated assay runtime including sample-handling steps for each procedure is noted on the right

Mentions: We chose the epidermoid carcinoma cell line A431, which overexpresses wild type epidermal growth factor receptor (EGFR), as a developmental platform. The premise of the SH2-PLA assay is that 1) EGF stimulation induces tyrosine phosphorylation of the intracellular domain of EGFR, which creates specific binding sites for SH2 domains such as Grb2, Src, PLCγ1, Vav2, etc.; 2) GST-SH2 domain coupled to anti-GST 5′ Prox-Oligo binds to these phosphotyrosines in cell lysate; 3) This interaction brings the anti-GST 5′ Prox-Oligo and anti-EGFR 3′ Prox-Oligo probes together in close proximity, thus allowing for the proximity ligation reaction to be subsequently quantified by real-time PCR (Fig. 1). In other words, detection of a specific assay signal requires the creation of the quaternary complex: anti-EGFR 3′Prox-Oligo probe:phosphorylated EGFR:GST-SH2 protein:anti-GST 5′ Prox-Oligo probe.Fig. 1


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

Thompson CM, Bloom LR, Ogiue-Ikeda M, Machida K - BMC Biotechnol. (2015)

In-solution SH2 domain binding assay using proximity ligation and real-time PCR. a, Schematic Illustration of SH2-PLA. A pair of PLA probes is used to detect the interaction of tyrosine phosphorylated EGFR and a GST-SH2 domain. The 3′ SH2-PLA probe consists of an anti-EGFR antibody conjugated with the 3′ proximity oligonucleotide (3′ Prox-Oligo). The 5′ SH2-PLA probes consists of an anti-GST antibody conjugated with the 5′ Prox-Oligo and a GST-SH2 domain. When the GST-SH2 domain binds to tyrosine phosphorylation sites of EGFR, the 5′ and 3′ PLA probes are brought in close proximity, allowing ligation of the two Prox-Oligos which is detectable by real-time PCR. b, Experimental workflow of SH2-PLA Method 1. Lysates are prepared with or without EGF stimulation. Biotinylated anti-GST and anti-EGFR antibodies are conjugated with the 5′ and 3′ Prox-Oligos, respectively, and stored at −20 °C. The 5′ SH2-PLA probe is mixed with purified GST-SH2, and the 3′ SH2-PLA probe is mixed with cell lysates allowing the antibodies to bind their respective epitopes. Subsequently, the 5′ and 3′ PLA probe solutions are combined to induce interaction between the SH2 and pEGFR. Then, the amount of the complex is quantified by proximity ligation and real-time PCR. An alternative method is also possible (Additional file 1: Figure S2). Estimated assay runtime including sample-handling steps for each procedure is noted on the right
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482279&req=5

Fig1: In-solution SH2 domain binding assay using proximity ligation and real-time PCR. a, Schematic Illustration of SH2-PLA. A pair of PLA probes is used to detect the interaction of tyrosine phosphorylated EGFR and a GST-SH2 domain. The 3′ SH2-PLA probe consists of an anti-EGFR antibody conjugated with the 3′ proximity oligonucleotide (3′ Prox-Oligo). The 5′ SH2-PLA probes consists of an anti-GST antibody conjugated with the 5′ Prox-Oligo and a GST-SH2 domain. When the GST-SH2 domain binds to tyrosine phosphorylation sites of EGFR, the 5′ and 3′ PLA probes are brought in close proximity, allowing ligation of the two Prox-Oligos which is detectable by real-time PCR. b, Experimental workflow of SH2-PLA Method 1. Lysates are prepared with or without EGF stimulation. Biotinylated anti-GST and anti-EGFR antibodies are conjugated with the 5′ and 3′ Prox-Oligos, respectively, and stored at −20 °C. The 5′ SH2-PLA probe is mixed with purified GST-SH2, and the 3′ SH2-PLA probe is mixed with cell lysates allowing the antibodies to bind their respective epitopes. Subsequently, the 5′ and 3′ PLA probe solutions are combined to induce interaction between the SH2 and pEGFR. Then, the amount of the complex is quantified by proximity ligation and real-time PCR. An alternative method is also possible (Additional file 1: Figure S2). Estimated assay runtime including sample-handling steps for each procedure is noted on the right
Mentions: We chose the epidermoid carcinoma cell line A431, which overexpresses wild type epidermal growth factor receptor (EGFR), as a developmental platform. The premise of the SH2-PLA assay is that 1) EGF stimulation induces tyrosine phosphorylation of the intracellular domain of EGFR, which creates specific binding sites for SH2 domains such as Grb2, Src, PLCγ1, Vav2, etc.; 2) GST-SH2 domain coupled to anti-GST 5′ Prox-Oligo binds to these phosphotyrosines in cell lysate; 3) This interaction brings the anti-GST 5′ Prox-Oligo and anti-EGFR 3′ Prox-Oligo probes together in close proximity, thus allowing for the proximity ligation reaction to be subsequently quantified by real-time PCR (Fig. 1). In other words, detection of a specific assay signal requires the creation of the quaternary complex: anti-EGFR 3′Prox-Oligo probe:phosphorylated EGFR:GST-SH2 protein:anti-GST 5′ Prox-Oligo probe.Fig. 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, 06030, Farmington, CT, USA. thompsc1@mskcc.org.

ABSTRACT

Background: There 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.

Results: We 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.

Conclusions: We 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.

No MeSH data available.


Related in: MedlinePlus