Concept: Protein tyrosine phosphatase
PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.
Consequences of expression of the protein tyrosine phosphatase nonreceptor 22 (PTPN22) gain-of-function variant were evaluated in leukocytes from patients with anti-neutrophil cytoplasmic autoantibody (ANCA) disease. The frequency of the gain-of-function allele within the Caucasian patient cohort was 22% (OR 1.45), compared to general American Caucasian population (16.5%, p = 0.03). Examination of the basal phosphatase activity of PTPN22 gain-of-function protein indicated persistently elevated activity in un-stimulated peripheral leukocytes, while basal activity was undetectable in leukocytes from patients without the gain-of-function variant. To examine consequences of persistently high PTPN22 activity, the activation status of ERK and p38 MAPK were analyzed. While moderate levels of activated ERK were observed in controls, it was undetectable in leukocytes expressing PTPN22 gain-of-function protein and instead p38MAPK was up-regulated. IL-10 transcription, reliant on the ERK pathway, was negatively affected. Over the course of disease, patients expressing variant PTPN22 did not show a spike in IL-10 transcription as they entered remission in contrast to controls, implying that environmentally triggered signals were blunted. Sustained activity of PTPN22, due to the gain-of-function mutation, acts as a dominant negative regulator of ERK activity leading to blunted cellular responsiveness to environmental stimuli and expression of protective cytokines.
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 7 years ago
A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC(50) values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC(50) of 27 ± 0.83 μM.
Recent advances in genome-wide association studies (GWAS) across autoimmune and immune-mediated diseases have augmented our understanding of pathogenic mechanisms underlying these diseases. This has further highlighted their heterogeneous nature, both within and between diseases. Furthermore, varying responses to therapy have also served to underline the importance of this heterogeneity in the manner in which these diseases are diagnosed and treated. Here we discuss our current understanding of the shared pathways of autoimmunity, including the tumor necrosis factor (TNF), major histocompatibility complex (MHC), interleukin 23 receptor (IL23R) and protein tyrosine phosphatase non-receptor type 22 (PTPN22) pathways. In addition, we summarize effective specific therapies tested across major autoimmune diseases, highlighting the insight they have provided into disease mechanisms and their implications for potential future improvements.
As a part of our ongoing effort to identify anti-diabetic constituents from natural sources, we examined the inhibitory activity of the methanol extracts of 12 species of the genus Artemisia, against α-glucosidase and protein tyrosine phosphatase 1B (PTP1B). The methanol extracts of different species exhibited promising α-glucosidase and PTP1B inhibitory activities. Since the methanol extract of Artemisia capillaris exhibited the highest α-glucosidase inhibitory activity together with significant PTP1B inhibitory activity, it was selected for further investigation. Repeated column chromatography based on bioactivity guided fractionation yielded 10 coumarins (esculetin, esculin, scopolin, isoscopolin, daphnetin, umbelliferone, 7-methoxy coumarin, scoparone, scopoletin, 6-methoxy artemicapin C), 8 flavonoids (hyperoside, quercetin, isorhamnetin, cirsilineol, arcapillin, isorhamnetin 3-robinobioside, linarin, isorhamnetin 3-glucoiside), 6 phenolic compounds (1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid methyl ester, 4,5-dicaffeoylquinic acid, 3-caffeoylquinic acid), and one chromone (capillarisin). Among these compounds, esculetin, scopoletin, quercetin, hyperoside, isorhamnetin, 3,5-dicaffeoylquinic acid methyl ester, 3,4-dicaffeoylquinic acid, and 1,5-dicaffeoylquinic acid exhibited potent α-glucosidase inhibitory activity when compared to the positive control acarbose. In addition, esculetin and 6-methoxy artemicapin C displayed PTP1B inhibitory activity. Interestingly, all isolated dicaffeoylquinic acids showed significant PTP1B inhibitory activity. Therefore, the results of the present study clearly demonstrate the potential of the A. capillaris extract to inhibit α-glucosidase and PTP1B. These inhibitory properties can be largely attributed to a combination of different chemical structures, including coumarins, flavonoids, and dicaffeoylquinic acids, which could be further explored to develop therapeutic or preventive agents for the treatment of diabetes.
OBJECTIVES: Although c-Src (Src) has emerged as a potential pancreatic cancer target in preclinical studies, Src inhibitors have not demonstrated a significant therapeutic benefit in clinical trials. The objective of these studies was to examine the effects of combining Src inhibition with inhibition of the protein tyrosine phosphatase SHP-2 in pancreatic cancer cells in vitro and in vivo. METHODS: SHP-2 and Src functions were inhibited by siRNA or small molecule inhibitors. The effects of dual Src/SHP-2 functional inhibition were evaluated by Western blot analysis of downstream signaling pathways; cell biology assays to examine caspase activity, viability, adhesion, migration, and invasion in vitro; and an orthotopic nude mouse model to observe pancreatic tumor formation in vivo. RESULTS: Dual targeting of Src and SHP-2 induces an additive or supra-additive loss of phosphorylation of Akt and ERK-½ and corresponding increases in expression of apoptotic markers, relative to targeting either protein individually. Combinatorial inhibition of Src and SHP-2 significantly reduces viability, adhesion, migration, and invasion of pancreatic cancer cells in vitro and tumor formation in vivo, relative to individual Src/SHP-2 inhibition. CONCLUSIONS: These data suggest that the antitumor effects of Src inhibition in pancreatic cancer may be enhanced through simultaneous inhibition of SHP-2.
In this study, we identified water-soluble C60 and C70 fullerene derivatives as a novel class of protein tyrosine phosphatase inhibitors. The evaluated compounds were found to inhibit CD45, PTP1B, TC-PTP, SHP2, and PTPβ with IC50 values in the low micromolar to high nanomolar range. These results demonstrate a new strategy for designing effective nanoscale protein tyrosine phosphatase inhibitors.
Leptin signaling in the hypothalamus plays a crucial role in the regulation of body weight. Leptin resistance, in which leptin signaling is disrupted, is a major obstacle to the improvement of obesity. We herein demonstrated that protein tyrosine phosphatase receptor type J (Ptprj) is expressed in hypothalamic neurons together with leptin receptors, and that PTPRJ negatively regulates leptin signaling by inhibiting the activation of JAK2, the primary tyrosine kinase in leptin signaling, through the dephosphorylation of Y813 and Y868 in JAK2 autophosphorylation sites. Leptin signaling is enhanced in Ptprj-deficient mice, and they exhibit lower weight gain than wild-type mice because of a reduced food intake. Diet-induced obesity and the leptin treatment up-regulated PTPRJ expression in the hypothalamus, while the overexpression of PTPRJ induced leptin resistance. Thus, the induction of PTPRJ is a factor contributing to the development of leptin resistance, and the inhibition of PTPRJ may be a potential strategy for improving obesity.
We used a loss-of-function screen to investigate the role of classical Protein Tyrosine Phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling. The study revealed a novel role for PTPD2 as a positive regulator of ERBB2 signaling. Suppression of PTPD2 attenuated the ERBB2-induced multiacinar phenotype in three-dimensional cultures specifically by inhibiting ERBB2-mediated loss of polarity and lumen filling. In contrast, overexpression of PTPD2 enhanced the ERBB2 phenotype. We also found that a lipid second messenger, phosphatidic acid, bound PTPD2 in vitro and enhanced its catalytic activity. Small-molecule inhibitors of Phospholipase D (PLD), an enzyme that produces phosphatidic acid in cells, also attenuated the ERBB2 phenotype. Exogenously added phosphatidic acid rescued the PLD-inhibition phenotype, but only when PTPD2 was present. These findings illustrate a novel pathway involving PTPD2 and the lipid second messenger phosphatidic acid that promotes ERBB2 function.
Formation of a functional neuronal network requires not only precise target recognition, but also stabilization of axonal contacts within their appropriate synaptic layers. Little is known about the molecular mechanisms underlying the stabilization of axonal connections after reaching their specifically targeted layers. Here, we show that two receptor protein tyrosine phosphatases (RPTPs), LAR and Ptp69D, act redundantly in photoreceptor afferents to stabilize axonal connections to the specific layers of the Drosophila visual system. Surprisingly, by combining loss-of-function and genetic rescue experiments, we found that the depth of the final layer of stable termination relied primarily on the cumulative amount of LAR and Ptp69D cytoplasmic activity, while specific features of their ectodomains contribute to the choice between two synaptic layers, M3 and M6, in the medulla. These data demonstrate how the combination of overlapping downstream but diversified upstream properties of two RPTPs can shape layer specific wiring.