Identifying Transmembrane Interactions in Receptor Protein Tyrosine Phosphatase Homodimerization and Heterodimerization.

Receptor protein tyrosine phosphatases (RPTPs) are one of the key regulators of receptor tyrosine kinases (RTKs) and therefore play a critical role in modulating signal transduction. While the structure-function relationship of RTKs has been widely studied, the mechanisms modulating the activity of RPTPs still need to be fully understood. On the other hand, homodimerization has been shown to antagonize RPTP catalytic activity and appears to be a general feature of the entire family. Conversely, their documented ability to physically interact with RTKs is integral to their negative regulation of RTKs, but there is a yet-to-be proposed common model. However, specific transmembrane (TM) domain interactions and residues have been shown to be essential in regulating RPTP homodimerization, interactions with RTK substrates, and activity. Therefore, elucidating the contribution of the TM domains in RPTP regulation can provide significant insights into how these receptors function, interact, and eventually be modulated. This chapter describes the dominant-negative AraC-based transcriptional reporter (DN-AraTM) assay to identify specific TM interactions essential to homodimerization and heteroassociation with other membrane receptors, such as RTKs.

System-Level Analysis of the Effects of RPTPs on Cellular Signaling Networks.

Tyrosine phosphorylation regulates signaling network activity downstream of receptor tyrosine kinase (RTK) activation. Receptor protein tyrosine phosphatases (RPTPs) serve to dephosphorylate RTKs and their proximal adaptor proteins, thus serving to modulate RTK activity. While the general function of RPTPs is well understood, the direct and indirect substrates for each RPTP are poorly characterized. Here we describe a method, quantitative phosphotyrosine phosphoproteomics, that enables the identification of specific phosphorylation sites whose phosphorylation levels are altered by the expression and activity of a given RPTP. In a proof-of-concept application, we use this method to highlight several direct or indirect substrate phosphorylation sites for PTPRJ, also known as DEP1, and show their quantitative phosphorylation in the context of wild-type PTPRJ compared to a mutant form of PTPRJ with increased activity, in EGF-stimulated cells. This method is generally applicable to define the signaling network effects of each RPTP in cells or tissues under different physiological conditions.

Promoting the activity of a receptor tyrosine phosphatase with a novel pH-responsive transmembrane agonist inhibits cancer-associated phenotypes.

Cell signaling by receptor protein tyrosine kinases (RTKs) is tightly controlled by the counterbalancing actions of receptor protein tyrosine phosphatases (RPTPs). Due to their role in attenuating the signal-initiating potency of RTKs, RPTPs have long been viewed as therapeutic targets. However, the development of activators of RPTPs has remained limited. We previously reported that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ) is regulated by specific transmembrane (TM) residues. Disrupting this interaction by single point mutations promotes PTPRJ access to its RTK substrates (e.g., EGFR and FLT3), reduces RTK's phosphorylation and downstream signaling, and ultimately antagonizes RTK-driven cell phenotypes. Here, we designed and tested a series of first-in-class pH-responsive TM peptide agonists of PTPRJ that are soluble in aqueous solution but insert as a helical TM domain in lipid membranes when the pH is lowered to match that of the acidic microenvironment of tumors. The most promising peptide reduced EGFR's phosphorylation and inhibited cancer cell EGFR-driven migration and proliferation, similar to the PTPRJ's TM point mutations. Developing tumor-selective and TM-targeting peptide binders of critical RPTPs could afford a potentially transformative approach to studying RPTP's selectivity mechanism without requiring less specific inhibitors and represent a novel class of therapeutics against RTK-driven cancers.

Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD.

The receptor protein tyrosine phosphatase (RPTP) PTPRJ (also known as DEP-1) has been identified as a negative regulator of the receptor tyrosine kinase FLT3 signalling in vitro. The inactivation of the PTPRJ gene in mice expressing the constitutively active, oncogenic receptor tyrosine kinase FLT3 ITD aggravated known features of leukaemogenesis, revealing PTPRJ's antagonistic role. FLT3 ITD mutations resulting in constitutively kinase activity and cell transformation frequently occur in patients with acute myeloid leukaemia (AML). Thus, in situ activation of PTPRJ could be used to abrogate oncogenic FLT3 signalling. The activity of PTPRJ is suppressed by homodimerization, which is mediated by transmembrane domain (TMD) interactions. Specific Glycine-to-Leucine mutations in the TMD disrupt oligomerization and inhibit the Epidermal Growth Factor Receptor (EGFR) and EGFR-driven cancer cell phenotypes. To study the effects of PTPRJ TMD mutant proteins on FLT3 ITD activity in cell lines, endogenous PTPRJ was inactivated and replaced by stable expression of PTPRJ TMD mutants. Autophosphorylation of wild-type and ITD-mutated FLT3 was diminished in AML cell lines expressing the PTPRJ TMD mutants compared to wild-type-expressing cells. This was accompanied by reduced FLT3-mediated global protein tyrosine phosphorylation and downstream signalling. Further, PTPRJ TMD mutant proteins impaired the proliferation and in vitro transformation of leukemic cells. Although PTPRJ's TMD mutant proteins showed impaired self-association, the specific phosphatase activity of immunoprecipitated proteins remained unchanged. In conclusion, this study demonstrates that the destabilization of PTPRJ TMD-mediated self-association increases the activity of PTPRJ in situ and impairs FLT3 activity and FLT3-driven cell phenotypes of AML cells. Thus, disrupting the oligomerization of PTPRJ in situ could prove a valuable therapeutic strategy to restrict oncogenic FLT3 activity in leukemic cells.