A high-throughput assay for phosphoprotein-specific phosphatase activity in cellular extracts.

Protein phosphatases undo the post-translational modifications of kinase-signaling networks, but phosphatase activation in cells is difficult to measure and interpret. Here, we report the design of a quantitative and high-throughput assay platform for monitoring cellular phosphatase activity toward specific phosphoprotein targets. Protein substrates of interest are purified recombinantly, phosphorylated in vitro using the upstream kinase, and adsorbed to 96-well plates. Total phosphatase extracts from cells are then added to trigger a solid-phase dephosphorylation reaction. After stopping the reaction, phosphoprotein levels are quantified by ELISA with a phospho-specific antibody, and the loss of phospho-specific immunoreactivity is used as the readout of phosphatase activity. We illustrate the generality of the method by developing specific phosphatase-activity assays for the three canonical mitogen-activated protein phospho-kinases: ERK, JNK, and p38. The assays capture changes in activity with a dynamic range of 25-100-fold and are sensitive to a limit of detection below 25,000 cells. When applied to cytokine-induced signaling, the assays revealed complex and dynamic regulation of phosphatases suggesting cross-communication and a means for cellular memory. Our assay platform should be beneficial for phosphoproteomic surveys and computational-systems models of signaling, where phosphatases are known to be important but their activities are rarely measured.

Unbiased identification of substrates of protein tyrosine phosphatase ptp-3 in C. elegans.

The leukocyte antigen related (LAR) family of receptor-like protein tyrosine phosphatases has three members in humans - PTPRF, PTPRD and PTPRS - that have been implicated in diverse processes including embryonic development, inhibition of cell growth and axonal guidance. Mutations in the LAR family are associated with developmental defects such as cleft palate as well as various cancers including breast, neck, lung, colon and brain. Although this family of tyrosine phosphatases is important for many developmental processes, little is known of their substrates. This is partially due to functional redundancy within the LAR family, as deletion of a single gene in the LAR family does not have an appreciable phenotype, but a dual knockout is embryonically lethal in mouse models. To circumvent the inability to knockout multiple members of the LAR family in mouse models, we used a knockout of ptp-3, which is the only known ortholog of the LAR family in Caenorhabditis elegans and allows for the study of the LAR family at the organismal level. Using SILAC-based quantitative phosphoproteomics, we identified 255 putative substrates of ptp-3, which included four of the nine known annotated substrates of the LAR family. A motif analysis of the identified phosphopeptides allowed for the determination of sequences that appear to be preferentially dephosphorylated. Finally, we discovered that kinases were overrepresented in the list of identified putative substrates and tyrosine residues whose phosphorylation is known to increase kinase activity were dephosphorylated by ptp-3. These data are suggestive of ptp-3 as a potential negative regulator of several kinase families, such as the mitogen activated kinases (MAPKs), and multiple tyrosine kinases including FER, MET, and NTRK2.