Dimerization In Vivo and Inhibition of the Nonreceptor Form of Protein Tyrosine Phosphatase Epsilon

Abstract

cyt-PTP is a naturally occurring nonreceptor form of the receptor-type protein tyrosine phosphatase (PTP) epsilon. As such, cyt-PTP enables analysis of phosphatase regulation in the absence of extracellular domains, which participate in dimerization and inactivation of the receptor-type phosphatases receptor-type protein tyrosine phosphatase alpha (RPTP␣) and CD45. Using immunoprecipitation and gel filtration, we show that cyt-PTP forms dimers and higher-order associations in vivo, the first such demonstration among nonreceptor phosphatases. Although cyt-PTP readily dimerizes in the absence of exogenous stabilization, dimerization is increased by oxidative stress. Epidermal growth factor receptor stimulation can affect cyt-PTP dimerization and tyrosine phosphorylation in either direction, suggesting that cell surface receptors can relay extracellular signals to cyt-PTP, which lacks extracellular domains of its own. The inactive, membrane-distal (D2) phosphatase domain of cyt-PTP is a major contributor to intermolecular binding and strongly interacts in a homotypic manner; the presence of D2 and the interactions that it mediates inhibit cyt-PTP activity. Intermolecular binding is inhibited by the extreme C and N termini of D2. cyt-PTP lacking these regions constitutively dimerizes, and its activities in vitro towards para-nitrophenylphosphate and in vivo towards the Kv2.1 potassium channel are markedly reduced. We conclude that physiological signals can regulate dimerization and phosphorylation of cyt-PTP in the absence of direct interaction between the PTP and extracellular molecules. Furthermore, dimerization can be mediated by the D2 domain and does not strictly require the presence of PTP extracellular domains.Phosphorylation of tyrosine residues in proteins is a central mechanism for protein regulation and is well established as a master regulator of physiological processes. Tyrosine phosphorylation is reversible and is controlled by the generically opposing activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs) (25). Receptor-type PTPs (RPTPs), which are a major structural subfamily of the PTP superfamily, are integral membrane proteins which possess extracellular domains of various lengths and typically two cytosolic phosphatase domains (2,12,49). Although their importance in regulating biological processes is well established, relatively little is known about how activities of RPTPs are regulated.Extracellular molecules have been suggested to bind RPTPs and to influence PTP activity or function, much as binding by pleiotrophin inhibits activity of PTP␤/ (36). However, little is known about how such binding affects signal transduction across cell membranes or RPTP activity (5, 41, 42, 53). Structural studies have suggested that ligand-induced dimerization inhibits activity of RPTPs by stabilizing homodimeric structures, in which the helix-turn-helix "wedge" domain of one molecule prevents access of substrates to the catalytic domain of its dimerization partner (6,34,51). Dimerizati...

Neu-mediated phosphorylation of protein tyrosine phosphatase epsilon is critical for activation of Src in mammary tumor cells

“…Following the immune precipitation of FLAG-tagged molecules, presence of dimerized, coprecipitating HAtagged molecules was determined by blotting the precipitate with anti-HA antibodies. In agreement with previous studies (Toledano-Katchalski et al, 2003), RPTPe dimers were detected readily among both nonglycosylated and fully-glycosylated RPTPe molecules (Figure 6a). Expression of NeuNT significantly reduced dimerization of the fully glycosylated, membrane-associated RPTPe molecules without affecting the nonglycosylated molecules ( Figure 6a).…”

“…Since 12-25% of PTPe molecules are dimerized (Toledano-Katchalski et al, 2003) and since phosphorylation reduces but does not eliminate RPTPe dimerization (Figure 6a), phosphorylation may increase the fraction of monomeric RPTPe molecules by a relatively small extent that is below the sensitivity of the in vivo method used here (Figure 6b). In all, our results are broadly consistent with phosphorylated RPTPe activating c-Src mainly via the phosphotyrosine displacement mechanism, that is, by RPTPe specifically targeting c-Src and related molecules.…”

“…A similar system was used previously to show that increased dimerization of cyt-PTPe reduces its ability to dephosphorylate Kv2.1 in cells (Toledano-Katchalski et al, 2003). cyt-PTPe dephosphorylates Kv2.1 predominantly at Y124, thereby down-regulating channel activity and countering phosphorylation of this residue by c-Src.…”

Epidermal Growth Factor Receptor (EGFR)-mediated Positive Feedback of Protein-tyrosine Phosphatase ϵ (PTPϵ) on ERK1/2 and AKT Protein Pathways Is Required for Survival of Human Breast Cancer Cells

“…RPTP⑀ is located at the plasma membrane and is highly glycosylated in the extracellular region, while the cytPTP⑀, p67 and p65 are mainly localized in the cytosol (8,11,13,14). Both RPTP⑀ and cytPTP⑀ can form dimers, making the phosphatase inactive, and removing of the 22 N-terminal residues causes enzyme inactivation by constitutive dimerization of the protein (15). In mammary tumor cells, as well as in HEK293 cells, RPTP⑀ can be phosphorylated at its C-terminal Tyr-695 residue by HER2, which reduces dimerization and augments RPTP⑀ activity to dephosphorylate and activate Src (16).…”

PTPε Represses LPS-Mediated TNF-α Induction in RAW264.7 Cells by Inducing Dephosphorylation of p38

“…In addition, ERK phosphorylation is increased in mouse fibroblasts and in mammary tumor cells lacking PTPε. 11,12 Tumor necrosis factor-α (TNF-α) is a major cytokine that induces inflammation and becomes a therapeutic target in inflammation diseases. 13 The regulation site for TNF-α expression is an adenosine/uridine-rich element (ARE) residing in the 3' untranslated region of the TNF-α mRNA that represses TNF-α expression post-transcriptionally.…”