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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

Validation of the SH2-PLA assay. a, Representative PCR amplification plot for SH2-PLA experiments. Increased binding between SH2 and pEGFR upon EGF stimulation is expressed as a reduced threshold cycle value (Ct). Here, ∆Ct is defined as [Ctcontrol – CtEGF stimulated].b, Specificity of SH2-PLA. SH2-PLA (top panel) and far-Western (middle panel) results for EGF-stimulated and control A431 cell samples are shown. Results for GST control probe are shown in lanes 1–4; Grb2 SH2 probe in lanes 5–8; and PLCγ1 tandem SH2 probe in lanes 9–12. Lanes 3, 4, 7, 8, 11, and 12 show the assay result in the presence of pY1068 blocking peptide, which contains the Grb2 SH2 consensus binding site of EGFR. c, SH2-PLA assay performance. The SH2-PLA assay was performed three times using a two fold dilution series of EGF-stimulated and control A431 cell lysates. Average Ct values, normalized to non protein control (NPC), are shown in the upper panel. The intra-assay variation for Ct values was 0.07-2.36 (mean 0.60) and the inter-assay %CV was 0.32-3.08 (mean 1.28). Since EGF-stimulated samples always showed a greater signal (lower Ct) than the unstimulated control throughout the dilution series, the range of assay detection is estimated to be at least 1.1–1100 μg/ml of lysate concentration, and the lower limit of detection is approximately 2 ng of protein per assay. The lower panel shows the approximately linear region of the mean Ct plot against log input lysate concentrations, and the ∆Ct (unstimulated – stimulated) of about three cycles. The log2 fold change between EGF-stimulated and control samples was estimated to be 6.0 - 6.4 using the ProteinAssist software tool (Additional file 1: Figure S3). d, Adoption of other phosphotyrosine recognizing domains. The SH2-PLA methodology was applied to protein tyrosine phosphatase (PTP) and phosphotyrosine binding (PTB) domains. ShcA PTB domain and the substrate-trapping mutant of PTP1B PTP domain displayed activity comparable to Grb2 SH2 (lanes 1–4 and 7–8). Signal was undetectable for the wild type (wt) PTP1B PTP domain, likely due to the intrinsic phosphatase activity (lanes 5–6)
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Fig2: Validation of the SH2-PLA assay. a, Representative PCR amplification plot for SH2-PLA experiments. Increased binding between SH2 and pEGFR upon EGF stimulation is expressed as a reduced threshold cycle value (Ct). Here, ∆Ct is defined as [Ctcontrol – CtEGF stimulated].b, Specificity of SH2-PLA. SH2-PLA (top panel) and far-Western (middle panel) results for EGF-stimulated and control A431 cell samples are shown. Results for GST control probe are shown in lanes 1–4; Grb2 SH2 probe in lanes 5–8; and PLCγ1 tandem SH2 probe in lanes 9–12. Lanes 3, 4, 7, 8, 11, and 12 show the assay result in the presence of pY1068 blocking peptide, which contains the Grb2 SH2 consensus binding site of EGFR. c, SH2-PLA assay performance. The SH2-PLA assay was performed three times using a two fold dilution series of EGF-stimulated and control A431 cell lysates. Average Ct values, normalized to non protein control (NPC), are shown in the upper panel. The intra-assay variation for Ct values was 0.07-2.36 (mean 0.60) and the inter-assay %CV was 0.32-3.08 (mean 1.28). Since EGF-stimulated samples always showed a greater signal (lower Ct) than the unstimulated control throughout the dilution series, the range of assay detection is estimated to be at least 1.1–1100 μg/ml of lysate concentration, and the lower limit of detection is approximately 2 ng of protein per assay. The lower panel shows the approximately linear region of the mean Ct plot against log input lysate concentrations, and the ∆Ct (unstimulated – stimulated) of about three cycles. The log2 fold change between EGF-stimulated and control samples was estimated to be 6.0 - 6.4 using the ProteinAssist software tool (Additional file 1: Figure S3). d, Adoption of other phosphotyrosine recognizing domains. The SH2-PLA methodology was applied to protein tyrosine phosphatase (PTP) and phosphotyrosine binding (PTB) domains. ShcA PTB domain and the substrate-trapping mutant of PTP1B PTP domain displayed activity comparable to Grb2 SH2 (lanes 1–4 and 7–8). Signal was undetectable for the wild type (wt) PTP1B PTP domain, likely due to the intrinsic phosphatase activity (lanes 5–6)

Mentions: A431 cell lysates were prepared in the presence or absence of EGF stimulation and the SH2-PLA assay was performed as outlined in Fig. 1b. We employed several SH2 domain containing proteins for validation that are known to be physiological ligands of EGFR such as Grb2, Vav2, and PLCγ1 [26–28]. Figure 2a shows a representative real-time PCR amplification plot of the SH2-PLA assay using the Grb2 SH2 domain probe and A431 cell samples. PCR product in the EGF-stimulated A431 sample was amplified more rapidly than in the unstimulated sample resulting in a lower threshold cycle (Ct) value. The difference in Ct values between the two samples (∆Ct) is an indicator of enhanced binding by the Grb2 SH2 domain probe to tyrosine phosphorylated EGFR (pEGFR). To validate the specificity of the assay, we compared signal from a GST-SH2 domain probe and GST control. The Ct value for the GST control was unchanged with EGF stimulation (lanes 1 vs. 2, Fig. 2b upper panel). On the other hand, the SH2 domains of Grb2 and PLCγ1 showed a marked reduction in their Ct values upon stimulation (lanes 5 vs. 6 and 9 vs. 10). Using the same set of samples and SH2 domains, far-Western blotting was performed as a reference (Fig. 2b middle panel). In far-Western, proteins were separated on polyacrylamide and transferred to a nitrocellulose membrane which was then probed with HRP-labeled GST-SH2 domains [23]. The identity of a major band at approximately 180 KDa in the SH2-far-Western blotting has previously been confirmed to be EGFR by anti-EGFR immunodepletion (data not shown). As shown in the middle panel of Fig. 2b, the signal profiles of the SH2-PLA and far-Western are similar despite their use of distinctive assay readouts (Ct values vs. bands). SH2 domains are known to have both unique and overlapping ligand binding characteristics [7, 29–32]. To determine if the SH2 binding is tyrosine site dependent, a synthesized phosphopeptide corresponding to EGFR tyrosine 1068, containing the Grb2 SH2 consensus binding site, was added as a blocker. In both assays, Grb2 SH2 binding was significantly reduced in the presence of the blocker, while the blocking effect on PLCγ SH2 domain binding was relatively modest (lanes 6 vs. 8 and 10 vs. 12). Taken together, these results indicate that, like far-Western, the SH2-PLA assay performed with the anti-GST 5′ Prox-Oligo antibody and anti-EGFR 3′ Prox-Oligo antibody probe pair is specific enough to distinguish between EGF-stimulated and control cell samples.Fig. 2


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)

Validation of the SH2-PLA assay. a, Representative PCR amplification plot for SH2-PLA experiments. Increased binding between SH2 and pEGFR upon EGF stimulation is expressed as a reduced threshold cycle value (Ct). Here, ∆Ct is defined as [Ctcontrol – CtEGF stimulated].b, Specificity of SH2-PLA. SH2-PLA (top panel) and far-Western (middle panel) results for EGF-stimulated and control A431 cell samples are shown. Results for GST control probe are shown in lanes 1–4; Grb2 SH2 probe in lanes 5–8; and PLCγ1 tandem SH2 probe in lanes 9–12. Lanes 3, 4, 7, 8, 11, and 12 show the assay result in the presence of pY1068 blocking peptide, which contains the Grb2 SH2 consensus binding site of EGFR. c, SH2-PLA assay performance. The SH2-PLA assay was performed three times using a two fold dilution series of EGF-stimulated and control A431 cell lysates. Average Ct values, normalized to non protein control (NPC), are shown in the upper panel. The intra-assay variation for Ct values was 0.07-2.36 (mean 0.60) and the inter-assay %CV was 0.32-3.08 (mean 1.28). Since EGF-stimulated samples always showed a greater signal (lower Ct) than the unstimulated control throughout the dilution series, the range of assay detection is estimated to be at least 1.1–1100 μg/ml of lysate concentration, and the lower limit of detection is approximately 2 ng of protein per assay. The lower panel shows the approximately linear region of the mean Ct plot against log input lysate concentrations, and the ∆Ct (unstimulated – stimulated) of about three cycles. The log2 fold change between EGF-stimulated and control samples was estimated to be 6.0 - 6.4 using the ProteinAssist software tool (Additional file 1: Figure S3). d, Adoption of other phosphotyrosine recognizing domains. The SH2-PLA methodology was applied to protein tyrosine phosphatase (PTP) and phosphotyrosine binding (PTB) domains. ShcA PTB domain and the substrate-trapping mutant of PTP1B PTP domain displayed activity comparable to Grb2 SH2 (lanes 1–4 and 7–8). Signal was undetectable for the wild type (wt) PTP1B PTP domain, likely due to the intrinsic phosphatase activity (lanes 5–6)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Validation of the SH2-PLA assay. a, Representative PCR amplification plot for SH2-PLA experiments. Increased binding between SH2 and pEGFR upon EGF stimulation is expressed as a reduced threshold cycle value (Ct). Here, ∆Ct is defined as [Ctcontrol – CtEGF stimulated].b, Specificity of SH2-PLA. SH2-PLA (top panel) and far-Western (middle panel) results for EGF-stimulated and control A431 cell samples are shown. Results for GST control probe are shown in lanes 1–4; Grb2 SH2 probe in lanes 5–8; and PLCγ1 tandem SH2 probe in lanes 9–12. Lanes 3, 4, 7, 8, 11, and 12 show the assay result in the presence of pY1068 blocking peptide, which contains the Grb2 SH2 consensus binding site of EGFR. c, SH2-PLA assay performance. The SH2-PLA assay was performed three times using a two fold dilution series of EGF-stimulated and control A431 cell lysates. Average Ct values, normalized to non protein control (NPC), are shown in the upper panel. The intra-assay variation for Ct values was 0.07-2.36 (mean 0.60) and the inter-assay %CV was 0.32-3.08 (mean 1.28). Since EGF-stimulated samples always showed a greater signal (lower Ct) than the unstimulated control throughout the dilution series, the range of assay detection is estimated to be at least 1.1–1100 μg/ml of lysate concentration, and the lower limit of detection is approximately 2 ng of protein per assay. The lower panel shows the approximately linear region of the mean Ct plot against log input lysate concentrations, and the ∆Ct (unstimulated – stimulated) of about three cycles. The log2 fold change between EGF-stimulated and control samples was estimated to be 6.0 - 6.4 using the ProteinAssist software tool (Additional file 1: Figure S3). d, Adoption of other phosphotyrosine recognizing domains. The SH2-PLA methodology was applied to protein tyrosine phosphatase (PTP) and phosphotyrosine binding (PTB) domains. ShcA PTB domain and the substrate-trapping mutant of PTP1B PTP domain displayed activity comparable to Grb2 SH2 (lanes 1–4 and 7–8). Signal was undetectable for the wild type (wt) PTP1B PTP domain, likely due to the intrinsic phosphatase activity (lanes 5–6)
Mentions: A431 cell lysates were prepared in the presence or absence of EGF stimulation and the SH2-PLA assay was performed as outlined in Fig. 1b. We employed several SH2 domain containing proteins for validation that are known to be physiological ligands of EGFR such as Grb2, Vav2, and PLCγ1 [26–28]. Figure 2a shows a representative real-time PCR amplification plot of the SH2-PLA assay using the Grb2 SH2 domain probe and A431 cell samples. PCR product in the EGF-stimulated A431 sample was amplified more rapidly than in the unstimulated sample resulting in a lower threshold cycle (Ct) value. The difference in Ct values between the two samples (∆Ct) is an indicator of enhanced binding by the Grb2 SH2 domain probe to tyrosine phosphorylated EGFR (pEGFR). To validate the specificity of the assay, we compared signal from a GST-SH2 domain probe and GST control. The Ct value for the GST control was unchanged with EGF stimulation (lanes 1 vs. 2, Fig. 2b upper panel). On the other hand, the SH2 domains of Grb2 and PLCγ1 showed a marked reduction in their Ct values upon stimulation (lanes 5 vs. 6 and 9 vs. 10). Using the same set of samples and SH2 domains, far-Western blotting was performed as a reference (Fig. 2b middle panel). In far-Western, proteins were separated on polyacrylamide and transferred to a nitrocellulose membrane which was then probed with HRP-labeled GST-SH2 domains [23]. The identity of a major band at approximately 180 KDa in the SH2-far-Western blotting has previously been confirmed to be EGFR by anti-EGFR immunodepletion (data not shown). As shown in the middle panel of Fig. 2b, the signal profiles of the SH2-PLA and far-Western are similar despite their use of distinctive assay readouts (Ct values vs. bands). SH2 domains are known to have both unique and overlapping ligand binding characteristics [7, 29–32]. To determine if the SH2 binding is tyrosine site dependent, a synthesized phosphopeptide corresponding to EGFR tyrosine 1068, containing the Grb2 SH2 consensus binding site, was added as a blocker. In both assays, Grb2 SH2 binding was significantly reduced in the presence of the blocker, while the blocking effect on PLCγ SH2 domain binding was relatively modest (lanes 6 vs. 8 and 10 vs. 12). Taken together, these results indicate that, like far-Western, the SH2-PLA assay performed with the anti-GST 5′ Prox-Oligo antibody and anti-EGFR 3′ Prox-Oligo antibody probe pair is specific enough to distinguish between EGF-stimulated and control cell samples.Fig. 2

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