This cell-based assay is designed to monitor the phosphorylation of SHP2 on Tyr542, which represents a hallmark of its activation.
Many cancer cells overexpress checkpoint inhibitor ligands such as PD-L1. PD-L1 binds its counterpart checkpoint inhibitor receptor PD1, present at the surface of T lymphocytes. In turn, the PD1-PDL1 complex recruits and activates inhibitory effectors, such as SHP1 or SHP2. These two phosphatases, which are phosphorylated on Tyr564 and Tyr542 respectively by the kinase Lck, trigger the dephosphorylation of signaling proteins such as ZAP-70 or SLP-76, involved in the T cell activation pathway. Finally, activated SHP1 and SHP2 participate in T cell inactivation.
Preventing activation of SHP1 and/or SHP2 by small molecule inhibitors is believed to contribute to restoring the immune response against tumors.
Phospho-SHP2 (Tyr542) assay principle
The Phospho-SHP2 (Tyr542) assay measures SHP2 when phosphorylated at Tyr542. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-SHP2 (Tyr542) assay uses 2 labeled antibodies, one with a donor fluorophore, and the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.
Phospho-SHP2 (Tyr542) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before adding Phospho-SHP2 (Tyr542) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-SHP2 (Tyr542) one-plate assay protocol
Detection of Phosphorylated SHP2 (Tyr542) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
In Jurkat cells, SHP2 phosphorylation is detected in the presence of pervanadate
Human Jurkat suspension cells were plated at 100,000 cells/well in a 96 well half area plate and incubated for 30 min in the presence or absence of Pervanadate at 30µM. Following this incubation, cells were lysed with 10µL of 4X supplemented lysis buffer for 30min at RT under gentle shaking. Then 16 µL of lysate were transferred into a 384-well low volume white microplate, and 4µL of the HTRF phospho-SHP2 (Tyr542) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
In Jurkat cells, SHP2 is transiently phosphorylated, thus making its detection difficult. By inhibiting phosphatases activity, pervanadate prevents the rapid dephosphorylation of SHP2 and enables a clear detection of phosphorylated SHP2.
Pharmacological validation using the Lck inhibitor, saracatinib, in Jurkat T-cells
Human Jurkat suspension cells were plated at 100,000 cells/ well in a 96 well half area plate, and incubated for 24h at 37°C, 5% CO2, with increasing concentrations of Saracatinib. Before lysis, Jurkat cells were incubated for 30 min with Pervanadate 30 µM followed by the addition of 10µL of supplemented lysis buffer 4X. After 30min lysis at RT under gentle shaking, 16µL of lysate were transferred into a 384-well low volume white microplate, and 4µL of the HTRF phospho-SHP2 (Tyr542) or total SHP2 detection reagents were added. The HTRF signal was recorded after a 4 hour incubation.
As described elsewhere, a dose dependent inhibition of SHP2 Tyr542 phosphorylation was obtained following treatment with Saracatinib, whereas the SHP2 expression level remained stable under the same experimental conditions.
Pharmacological validation on NIH-3T3 mouse cells stimulated with PDGF
Mouse NIH 3T3 adherent cells were seeded at 100,000 cells/ well in a 96-well plate and incubated at 37°C, 5% CO2. After an overnight serum deprivation, cells were stimulated with increasing concentrations of PDGF for 20 min. Cell culture medium was harvested, and cells were lysed with 50 µL supplemented lysis buffer. After 30min lysis at RT under gentle shaking, 16µL of lysate were transferred into a 384-well low volume white microplate, and 4µL of the HTRF phospho-SHP2 (Tyr542) or total SHP2 detection reagents were added. The HTRF signal was recorded after a 4 hour incubation.
As shown here, PDGF stimulation induced a strong SHP2 Tyr542 phosphorylation in NIH3T3 cells, whereas SHP2 expression remained stable under the same experimental conditions.
HTRF assay compared to Western Blot
The human Jurkat cell line was seeded in a T175 flask, and incubated at 37 °C, 5% CO2. The cells were then treated with Pervanadate (30 µM) for 30 min before lysis.
Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-SHP2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
Using the HTRF Phospho-SHP2 Y542 assay, 620 cells/well were sufficient to detect a signal, while 10,000 cells were needed using Western Blot relying on ECL detection. These results demonstrate that the HTRF phospho-SHP2 assay is 16 times more sensitive than the Western Blot.
Function and regulation of SHP2
SHP1 (also known as tyrosine-protein phosphatase non-receptor type 6, PTPN6) is a tyrosine phosphatase mainly expressed in hematopoietic cells, activated by Lck and recruited by cellular surface receptors. SHP2 (also known as tyrosine-protein phosphatase non-receptor type 11) is ubiquitously expressed in hematopoietic or non-hematopoietic cells. Although SHP2 negatively regulates T cell activation, SHP2 is positively involved in ERK activation in response to growth factors such as PDGF or FGF.
In T lymphocyte cells, SHP1 and 2 are recruited by immune checkpoint inhibitors, thereby participating in the suppression of the TCR signaling pathway. SHP1 and 2 interact with PD1 ITIM domains and are phosphorylated and activated by the Lck kinase. Activated SHP1 and 2 phosphatases lead to the dephosphorylation of key TCR signaling effectors such as ZAP70 or SLP76, which are required for T-cell proliferation and function.
