This cell-based assay is designed to monitor the phosphorylation of SHP1 on Tyr564, 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 the activation of SHP1 and/or SHP2 by small molecule inhibitors is believed to contribute to restoring the immune response against tumors.
Phospho-SHP1 (Tyr564) assay principle
The Phospho-SHP1 (Tyr564) assay measures SHP1 when phosphorylated at Tyr564. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-SHP1 (Tyr564) assay uses 2 labeled antibodies, one with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, which brings the donor fluorophore into close proximity to the acceptor, thus 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-SHP1 (Tyr564) 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 the addition of Phospho-SHP1 (Tyr564) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-SHP1 (Tyr564) one-plate assay protocol
Detection of Phosphorylated SHP1 (Tyr564) 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, SHP1 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-SHP1 (Tyr564) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
In Jurkat cells, SHP1 is transiently phosphorylated, thus making its detection difficult. By inhibiting phosphatase activity, pervanadate prevents the rapid dephosphorylation of SHP1 and enables a clear detection of phosphorylated SHP1.
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 30min with Pervanadate (30µM), 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-SHP1 (Tyr564) or total SHP1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
As described elsewhere, a dose dependent inhibition of SHP1 Tyr564 phosphorylation was obtained following treatment with Saracatinib, whereas the SHP1 expression level remained stable under the same experimental conditions.
HTRF phospho-SHP1 cellular assays 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 16µL of each dilution were transferred into a low volume white microplate before the addition of 4µL of HTRF phospho-SHP1 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
Using the HTRF Phospho-SHP1 Y564 assay, 1,250 cells/well were sufficient to detect a signal, while 5,000 cells were needed for Western Blot, relying on ECL detection. These results demonstrate that the HTRF phospho-SHP1 assay is 4 times more sensitive than the Western Blot.
Function and regulation of SHP1
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.
