Researchers at Cold Spring Harbor Laboratory have developed a cysteinyl-binding assay to help them identify protein tyrosine phosphatases that are oxidized in vivo, according to a recent paper.
The assay uses the unique chemistry of the invariant catalytic Cys residue in labeling the PTP active site with biotinylated small molecules under mildly acidic conditions.
“What we are actually trying to do here is to define the signaling function of critical PTPs,” said Nicholas Tonks, a professor at CSHL.
By understanding how these phosphatases are oxidized and inactivated, the researchers can then determine the signaling function of the PTPs in a particular signaling pathway to define the substrate specificity of the phosphatases. Tonks said researchers can do this by processes such as RNAi or by using substrate-trapping mutants.
The CSHL assay allows the investigators to look at a wide variety of signaling pathways involving things such as growth factors, hormones, cytokines, GPCRs, and cell stressors, and identify which phosphatases are oxidized and inactivated in response to that stimulus.
It also allows them to look at the substrate specificity of the phosphatases and exactly what signaling pathways are regulated by those phosphatases downstream of the stimuli; and from that, “get a better picture of the physiological function of the PTPs themselves, and be able to identify critical drug targets from signaling pathways that are regulated,” Tonks told CBA News this week.
Scientists can expand their repertoire of candidate therapeutic targets by extending their knowledge of important signaling pathways based on their response to certain stimuli. Researchers now know a lot about the function of PTPs. Several PTPs have been linked to human disease.
“Where we are getting to with this assay is that this could predict that activity of PTPs must be tightly controlled in vivo.”
For example, PTP1B is an outstanding therapeutic target for the treatment of diabetes and obesity, said Tonks, who was corresponding author on the paper. It plays a major role in insulin and leptin receptor signaling.
It is also an excellent target for the treatment of breast cancer. “It would appear that in insulin signaling, [PTP1B] plays an inhibitory role and down-regulates insulin or leptin receptor signaling. However, in the context of Her2-positive breast cancer, it plays a positive role and promotes Her2 signaling, suggesting that an inhibitor of PTP1B could be a good adjunct therapy with Herceptin in treating Her2-positive breast cancers,” Tonks said.
This example illustrates that PTPs not only antagonize PTK function, but that they can also, in some cases, function positively and coordinate with PTKs to promote signaling function.
“Where we are getting to with this assay is that this could predict that activity of PTPs must be tightly controlled in vivo,” Tonks said. “You do not want PTPs aberrantly regulating tyrosine phosphatase-dependent signaling.”
The scientists used the assay to study platelet-derived growth factor receptor signaling in an angiomyolipoma cell model. The cells were lysed, and a buffer exchange was performed. The lysates were then allowed to incubate for 30 minutes on a shaker at room temperature. During this phase, the sulfenamide and sulenic acid forms of the active site Cys residues, which were protected from alkylation in the previous step, reverted to their thiolate states.
The lysates were incubated with biotinylated BPP or IAP probes for one hour on a shaker at room temperature. Biotinylated proteins were enriched by using streptavidin-Sepharose beads for 16 hours at 4°C on a rotating wheel with sequential rounds of centrifugation. The beads were resuspended in 4x Laermmli sample buffer and heated to 90°C for 1 minute.
The researchers observed the reversible oxidation of PTP SHP2 and the tumor suppressor phosphatase PTEN in response to PDGF stimulation, a finding they previously saw in other cell systems. They also detected the reversible oxidation of the receptor PTP LAR and the dual-specificity MKP1.
The researchers concluded that these data demonstrate the broad selectivity of this assay.
A Better Way
The method described in the PNAS paper overcomes several technical challenges associated with existing methods.
For example, an in-gel phosphatase assay requires a denaturation/renaturation cycle to restore the activity of those PTPs in which the active site Cys has not been subjected to stimulus-dependent oxidation, said Tonks. However, those PTPs that were not oxidized in response to the initial stimulus and were irreversibly alkylated in a lysis step remain inactive and are not registered in the assay.
By replacing the in-gel assay with biotinylated probes in the new assay, the investigators removed the required cycles and activity-based detection and as a result, can now detect the reversible oxidation of receptor PTPs, Tonks said.
The use of biotinylated, active site-directed probes also permits detection on a blot with steptavidin-HRP, and promotes enrichment of low-abundance PTPs.
“What you get is a read-out on a blot that gives you an estimate of the molecular weight of the phosphatase, and then you can test for identity using antibodies,” Tonks said.
In addition, by performing the assay under mildly acidic conditions, side reactions with noncatalytic cysteines and other amino acids that can occur at a higher pH appear to be minimized.
“What we are trying to do now is a mass spec-based identification,” said Tonks. He said that the next step in this work is linking these approaches to mass spectometry, so that we can “get a more comprehensive analysis of what is being oxidized and inactivated in response to these stimuli, and getting a more comprehensive understanding of the signaling function of these enzymes.”