Sap-1/PTPRH activity is regulated by reversible dimerization.

Sap-1/PTPRH, a receptor protein tyrosine phosphatase (RPTP), is a ubiquitously expressed enzyme that is upregulated in human gastrointestinal cancers. Using both chemical cross-linkers and co-immunoprecipitation we show that overexpressed full-length Sap-1 is present as a stable homodimer. Unlike a number of adhesion RPTPs which have tandem catalytic domains that are involved in dimerization, Sap-1 has a single catalytic domain, and we show that this domain is not required for Sap-1 dimerization, which is mediated instead by the large extracellular and transmembrane domains.

Applying the SPOT peptide synthesis procedure to the study of protein tyrosine phosphatase substrate specificity: probing for the heavenly match in vitro.

This section provides detailed protocols for peptide synthesis on membrane (SPOT) and describes the application of this technology to protein tyrosine phosphatase (PTPs) substrate selectivity studies. Applications include PTP binding and dephosphorylation assays on phosphotyrosine peptides derived from known substrates, such as the insulin receptor (IR) autophosphorylation site, and on peptides from focused or random SPOT peptide libraries, to discover consensus binding motifs. Weak or transient interactions that cannot be revealed by regular SPOT binding can be uncovered using SPOT double synthesis (SPOT-DS), whereby two different peptides are synthesized on the same spot.

Probing protein-tyrosine phosphatase substrate specificity using a phosphotyrosine-containing phage library.

Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality.

Identification of protein tyrosine phosphatases with specificity for the ligand-activated growth hormone receptor.

Protein tyrosine phosphatases (PTPs) play key roles in switching off tyrosine phosphorylation cascades, such as initiated by cytokine receptors. We have used substrate-trapping mutants of a large set of PTPs to identify members of the PTP family that have substrate specificity for the phosphorylated human GH receptor (GHR) intracellular domain. Among 31 PTPs tested, T cell (TC)-PTP, PTP-beta, PTP1B, stomach cancer-associated PTP 1 (SAP-1), Pyst-2, Meg-2, and PTP-H1 showed specificity for phosphorylated GHR that had been produced by coexpression with a kinase in bacteria.

Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides.

Protein-protein recognition usually involves multiple interactions among different motifs that are scattered over protein surfaces. To identify such weak interactions, we have developed a novel double peptide synthesis (DS) method. This method allows us to map protein-protein interactions that involve two linear dis- continuous components from a polypeptide by the use of spatially addressable synergistic pairs of synthetic peptides.

The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities.

The activity of protein tyrosine phosphatases (PTPs) is restricted by their substrate specificities. The analysis of PTP specificity was greatly helped by the discovery that "substrate-trapping" PTP mutants, such as PTP-1B D181A, stably and specifically bind their substrates. We have set up a PTP substrate specificity assay based on the SPOT technique, which involves the microsynthesis of (phospho)peptides on membranes.