Tyrosine Dephosphorylation Research Articles

Protein Tyrosine Phosphatases in Neuroblastoma: Emerging Roles as Biomarkers and Therapeutic Targets.

Neuroblastoma is a type of cancer intimately related with early development and differentiation of neuroendocrine cells, and constitutes one of the pediatric cancers with higher incidence and mortality. Protein tyrosine phosphatases (PTPs) are key regulators of cell growth and differentiation by their direct effect on tyrosine dephosphorylation of specific protein substrates, exerting major functions in the modulation of intracellular signaling during neuron development in response to external cues driving cell proliferation, survival, and differentiation. We review here the current knowledge on the role of PTPs in neuroblastoma cell growth, survival, and differentiation. The potential of PTPs as biomarkers and molecular targets for inhibition in neuroblastoma therapies is discussed.

Recent advances in the development of allosteric protein tyrosine phosphatase inhibitors for drug discovery.

Protein tyrosine phosphatases (PTPs) superfamily catalyzes tyrosine de-phosphorylation which affects a myriad of cellular processes. Imbalance in signal pathways mediated by PTPs has been associated with development of many human diseases including cancer, metabolic, and immunological diseases. Several compelling evidence suggest that many members of PTP family are novel therapeutic targets. However, the clinical development of conventional PTP-based active-site inhibitors originally was hampered by the poor selectivity and pharmacokinetic properties. In this regard, PTPs has been widely dismissed as "undruggable." Nonetheless, allosteric modulation has become increasingly an influential and alternative approach that can be exploited for drug development against PTPs. Unlike active-site inhibitors, allosteric inhibitors exhibit a remarkable target-selectivity, drug-likeness, potency, and in vivo activity. Intriguingly, there has been a high interest in novel allosteric PTPs inhibitors within the last years. In this review, we focus on the recent advances of allosteric inhibitors that have been explored in drug discovery and have shown an excellent result in the development of PTPs-based therapeutics. A special emphasis is placed on the structure-activity relationship and molecular mechanistic studies illustrating applications in chemical biology and medicinal chemistry.

Helicobacter pylori CagA Induces Cortactin Y-470 Phosphorylation-Dependent Gastric Epithelial Cell Scattering via Abl, Vav2 and Rac1 Activation.

Simple SummaryVarious microbial pathogens target the actin-binding protein cortactin to promote their own uptake, proliferation and spread, and exhibit proposed roles in human cancerogenesis. We aimed to study the molecular mechanisms of how the gastric pathogen Helicobacter pylori hijacks cortactin phosphorylation via tyrosine kinase Abl to trigger cancer-related signal transduction events. We discovered that cortactin phosphorylated at Y-470 recruits the signaling factor Vav2 to activate the small Rho GTPase Rac1, and finally, a cancer cell motility phenotype. We also demonstrate that phosphorylation of cortactin at Y-470 can be completely inhibited by the well-known Abl inhibitor imatinib. Imatinib is an established oral chemotherapy medication, employed for efficient systemic treatment of various cancers. These results reveal a comprehensive novel pathway for how precisely H. pylori manipulates host signaling in gastric disease development, and may pave the way for new opportunities of treatment of the outcome of infections with this pathogen, i.e., through using imatinib.The pathogen Helicobacter pylori is the first reported bacterial type-1 carcinogen playing a role in the development of human malignancies, including gastric adenocarcinoma. Cancer cell motility is an important process in this scenario, however, the molecular mechanisms are still not fully understood. Here, we demonstrate that H. pylori subverts the actin-binding protein cortactin through its type-IV secretion system and injected oncoprotein CagA, e.g., by inducing tyrosine phosphorylation of cortactin at Y-470, which triggers gastric epithelial cell scattering and motility. During infection of AGS cells, cortactin was discovered to undergo tyrosine dephosphorylation at residues Y-421 and Y-486, which is mediated through inactivation of Src kinase. However, H. pylori also profoundly activates tyrosine kinase Abl, which simultaneously phosphorylates cortactin at Y-470. Phosphorylated cortactin interacts with the SH2-domain of Vav2, a guanine nucleotide exchange factor for the Rho-family of GTPases. The cortactin/Vav2 complex then stimulates a previously unrecognized activation cascade including the small GTPase Rac1, to effect actin rearrangements and cell scattering. We hypothesize that injected CagA targets cortactin to locally open the gastric epithelium in order to get access to certain nutrients. This may disturb the cellular barrier functions, likely contributing to the induction of cell motility, which is important in gastric cancer development.

Novel Tyrosine Phosphorylation Signals in the Nucleus and on Mitotic Spindle Fibers and Lysosomes Revealed by Strong Inhibition of Tyrosine Dephosphorylation

Protein-tyrosine phosphorylation is one of the posttranslational modifications and plays critical roles in regulating a wide variety of cellular processes, such as cell proliferation, differentiation, adhesion, migration, survival, and apoptosis. Protein-tyrosine phosphorylation is reversibly regulated by protein-tyrosine kinases and protein-tyrosine phosphatases. Strong inhibition of protein-tyrosine phosphatase activities is required to undoubtedly detect tyrosine phosphorylation. Our extremely careful usage of Na3VO4, a potent protein-tyrosine phosphatase inhibitor, has revealed not only the different intracellular trafficking pathways of Src-family tyrosine kinase members but also novel tyrosine phosphorylation signals in the nucleus and on mitotic spindle fibers and lysosomes. Furthermore, despite that the first identified oncogene product v-Src is generally believed to induce transformation through continuous stimulation of proliferation signaling by its strong tyrosine kinase activity, v-Src-driven transformation was found to be caused not by continuous proliferation signaling but by v-Src tyrosine kinase activity-dependent stochastic genome alterations. Here, I summarize our findings regarding novel tyrosine phosphorylation signaling in a spatiotemporal sense and highlight the significance of the roles of tyrosine phosphorylation in transcriptional regulation inside the nucleus and chromosome dynamics.

Inhibition of the Shear-Induced Platelet Microparticle Formation by a mAb Against GPIb alpha Is Associated with Protein Tyrosine Dephosphorylation and the Blockade of Filamin Cleavage.

Platelet microparticle (MP) formation during shear-induced platelet aggregation (SIPA) contributes to a relevant part of thrombin generation. A monoclonal antibody (mAb) Ib-23 against platelet GPIb alpha (provided by B.S.) inhibits von Willebrand factor binding, ristocetin-induced platelet aggregation (RIPA), shear induced MP formation and thrombin generation. Platelet calpain affects several functions including MP-formation. One of several target substrates of calpain, filamin, binds to the cytoplasmic tail of GPIbalpha and mediates the assembly of the contractile elements during platelet activation. We therefore hypothesized that the inhibitory mechanism of our mAb Ib-23 on MP formation is a “calpeptin-like effect”, i.e. an inhibition of calpain affecting the protein tyrosine phosphorylation.PPACK-anticoagulated platelet-rich plasma or washed platelets (250′000/μl) were exposed to a shear rate of 5000 sec−1 for 5 min in a cone-plate viscometer. The mAb Ib-23 was added at the RIPA-inhibiting IC80–90. The samples were fluorescence-labelled with an anti-GPIIb-PE mAb to monitor SIPA and MP formation and analyzed by flow cytometry. Resting and sheared platelets were lysed in SDS-PAGE loading buffer for the analysis of filamin degradation and protein tyrosine phosphorylation.Results: Epitope mapping showed that mAb Ib-23 binds between residues 234 and 456 on GP Ib alpha. Exposure of platelets to high shear rates induced the activation of calpain as demonstrated by the degradation of filamin, a phenomenon blocked by the mAb Ib-23. Ib-23 did not inhibit the SIPA induced tyrosine phosphorylation of high molecular weight proteins like FAK, Syk and alpha-actinin, it even weakly enhanced the tyrosine phosphorylation of these proteins. Moreover, Ib-23 prevented the dephosphorylation of low molecular weight proteins (appr. 40 and 33kDa). Annexin V-binding was unaffected by Ib-23. In contrast, abciximab, a GP IIb/IIIa antagonist, partially inhibited SIPA and annexin binding, but not MP generation. Abciximab weakly inhibited the degradation of filamin, inhibited SIPA-induced tyrosine phosphorylation of FAK and Syk, but did not prevent the tyrosine dephosphorylation of the low molecular weight proteins. Control-experiments with calpeptin demonstrated a similar inhibition of filamin degradation as with mAb Ib-23. An isotype control antibody was without effects. The intact antibody Ib-23 was more effective than the Fab2 fragment. A mAb against the FcgammaRII receptor (IV.3, to exclude a bridging of the FcgammaRII and GpIbalpha via the antibody's Fc part) did not affect MP-formation.We conclude that binding of mAb Ib-23 on the outside of GP Ib alpha inhibits SIPA-induced MP formation and thrombin generation possibly via a “calpeptin-like effect” which inhibits the activation of calpain and of protein tyrosine phosphatases.

Sirtuin 6 is a negative regulator of FcεRI signaling and anaphylactic responses

Binding IgE to a cognate allergen causes aggregation of Fcε receptor I (FcεRI) in mast cells, resulting in activation of receptor-associated Src family tyrosine kinases, including Lyn and Syk. Protein tyrosine phosphatase, receptor type C (PTPRC), also known as CD45, has emerged as a positive regulator of FcεRI signaling by dephosphorylation of the inhibitory tyrosine of Lyn. Sirtuin 6 (Sirt6), a NAD+-dependent deacetylase, exhibits an anti-inflammatory property. It remains to be determined, however, whether Sirt6 attenuates mast cell-associated diseases, including anaphylaxis. FcεRI signaling and mast cell degranulation were measured after IgE cross-linking in murine bone marrow-derived mast cells (BMMCs) and human cord blood-derived mast cells. To investigate the function of Sirt6 in mast cell activation invivo, we used mast cell-dependent animal models of passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA). Sirt6-deficient BMMCs augmented IgE-FcεRI-mediated signaling and degranulation compared to wild-type BMMCs. Reconstitution of mast cell-deficient KitW-sh/W-sh mice with BMMCs received from Sirt6 knockout mice developed more severe PSA and PCA compared to mice engrafted with wild-type BMMCs. Similarly, genetic overexpression or pharmacologic activation of Sirt6 suppressed mast cell degranulation and blunted responses to PCA. Mechanistically, Sirt6 deficiency increased PTPRC transcription via acetylating histone H3, leading to enhanced aggregation of FcεRI in BMMCs. Finally, we recapitulated the Sirt6 regulation of PTPRC and FcεRI signaling in human mast cells. Sirt6 acts as a negative regulator of FcεRI signaling cascade in mast cells by suppressing PTPRC transcription. Activation of Sirt6 may therefore represent apromising and novel therapeutic strategy for anaphylaxis.

A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases Can Be A Potential Therapeutic Target for Anti-cancer Therapy.

While protein tyrosine kinases (PTKs) play an initiative role in growth factor-mediated cellular processes, protein tyrosine phosphatases (PTPs) negatively regulates these processes, acting as tumor suppressors. Besides selective tyrosine dephosphorylation of PTKs via PTPs may affect oncogenic pathways during carcinogenesis. The PTP family contains a group of dual-specificity phosphatases (DUSPs) that regulate the activity of Mitogen-activated protein kinases (MAPKs), which are key effectors in the control of cell growth, proliferation and survival. Abnormal MAPK signaling is critical for initiation and progression stages of carcinogenesis. Since depletion of DUSP-MAPK phosphatases (MKPs) can reduce tumorigenicity, altering MAPK signaling by DUSP-MKP inhibitors could be a novel strategy in anti-cancer therapy. Moreover, Cdc25A is, a DUSP and a key regulator of the cell cycle, promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases (CDK). Cdc25A-CDK pathway is a novel mechanism in carcinogenesis. Besides the mammalian target of rapamycin (mTOR) kinase inhibitors or mammalian target of rapamycin complex 1 (mTORC1) inhibition in combination with the dual phosphatidylinositol 3 kinase (PI3K)/mTOR or AKT kinase inhibitors are more effective in inhibiting the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and cap-dependent translation. Dual targeting of the Akt and mTOR signaling pathways regulates cellular growth, proliferation and survival. Like the Cdc2-like kinases (CLK), dual-specific tyrosine phosphorylation-regulated kinases (DYRKs) are essential for the regulation of cell fate. The crosstalk between dual-specific phosphatases and dual- specific protein kinases is a novel drug target for anti-cancer therapy. Therefore, the focus of this chapter involves protein kinase modules, critical biochemical checkpoints of cancer therapy and the synergistic effects of protein kinases and anti-cancer molecules.

Lipid-derived electrophiles mediate the effects of chemotherapeutic topoisomerase I poisons

Topoisomerase 1 (Top1) reversibly nicks chromosomal DNA to relax strain accumulated during transcription, replication, chromatin assembly, and chromosome condensation. The Top1 poison camptothecin targets cancer cells by trapping the enzyme in the covalent complex Top1cc, tethered to cleaved DNA by a tyrosine-3'-phosphate bond. Invitro mechanistic studies point to interfacial inhibition, where camptothecin binding to the Top1-DNA interface stabilizes Top1cc. Here we present a complementary covalent mechanism that is critical invivo. We observed that camptothecins induce oxidative stress, leading to lipid peroxidation, lipid-derived electrophile accumulation, and Top1 poisoning via covalent modification. The electrophile 4-hydroxy-2-nonenal can induce Top1cc on its own and forms a Michael adduct to a cysteine thiol in the Top1 active site, potentially blocking tyrosine dephosphorylation and 3' DNA phosphate release. Thereby, camptothecins may leverage a physiological cysteine-based redox switch in Top1 to mediate their selective toxicity to rapidly proliferating cancer cells.

Identification of PTPRσ-interacting proteins by proximity-labelling assay

Receptor protein tyrosine phosphatases (RPTPs) are type-I transmembrane proteins and involved in various biological and pathological processes. Their functions are supposed to be exerted through tyrosine dephosphorylation of their specific substrates. However, our comprehensive understanding of specific substrates or interacting proteins for RPTPs is poor. PTPRσ belongs to class 2a RPTP family, dephosphorylates cortactin, and leads to autophagy flux disruption and axonal regeneration inhibition in response to its ligand chondroitin sulphate. Here, we applied proximity-dependent biotin identification (BioID) assay, a proximity-labelling assay, to PTPRσ and reproducibly identified the 99 candidates as interactors for PTPRσ including already-known interactors such as Liprin-α and Trio. Of note, cortactin was also listed up in our assay. Our results suggest that the BioID assay is a powerful and reliable tool to identify RPTP-interacting proteins including its specific substrate.

Protein tyrosine phosphatases in multiple myeloma

Many cell signaling pathways are activated or deactivated by protein tyrosine phosphorylation and dephosphorylation, catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively. Even though PTPs are as important as PTKs in this process, their role has been neglected for a long time. Multiple myeloma (MM) is a cancer of plasma cells, which is characterized by production of monoclonal immunoglobulin, anemia and destruction of bone. MM is still incurable with high relapse frequency after treatment. In this review, we highlight the PTPs that were previously described in MM or have a role that can be relevant in a myeloma context. Our purpose is to show that despite the importance of PTPs in MM pathogenesis, many unanswered questions in this field need to be addressed. This might help to detect novel treatment strategies for MM patients.

Neuronal surface P antigen (NSPA) modulates postsynaptic NMDAR stability through ubiquitination of tyrosine phosphatase PTPMEG

BackgroundCognitive dysfunction (CD) is common among patients with the autoimmune disease systemic lupus erythematosus (SLE). Anti-ribosomal P autoantibodies associate with this dysfunction and have neuropathogenic effects that are mediated by cross-reacting with neuronal surface P antigen (NSPA) protein. Elucidating the function of NSPA can then reveal CD pathogenic mechanisms and treatment opportunities. In the brain, NSPA somehow contributes to glutamatergic NMDA receptor (NMDAR) activity in synaptic plasticity and memory. Here we analyze the consequences of NSPA absence in KO mice considering its structural features shared with E3 ubiquitin ligases and the crucial role of ubiquitination in synaptic plasticity.ResultsElectrophysiological studies revealed a decreased long-term potentiation in CA3-CA1 and medial perforant pathway-dentate gyrus (MPP-DG) hippocampal circuits, reflecting glutamatergic synaptic plasticity impairment in NSPA-KO mice. The hippocampal dentate gyrus of these mice showed a lower number of Arc-positive cells indicative of decreased synaptic activity and also showed proliferation defects of neural progenitors underlying less adult neurogenesis. All this translates into poor spatial and recognition memory when NSPA is absent. A cell-based assay demonstrated ubiquitination of NSPA as a property of RBR-type E3 ligases, while biochemical analysis of synaptic regions disclosed the tyrosine phosphatase PTPMEG as a potential substrate. Mice lacking NSPA have increased levels of PTPMEG due to its reduced ubiquitination and proteasomal degradation, which correlated with lower levels of GluN2A and GluN2B NMDAR subunits only at postsynaptic densities (PSDs), indicating selective trafficking of these proteins out of PSDs. As both GluN2A and GluN2B interact with PTPMEG, tyrosine (Tyr) dephosphorylation likely drives their endocytic removal from the PSD. Actually, immunoblot analysis showed reduced phosphorylation of the GluN2B endocytic signal Tyr1472 in NSPA-KO mice.ConclusionsNSPA contributes to hippocampal plasticity and memory processes ensuring appropriate levels of adult neurogenesis and PSD-located NMDAR. PTPMEG qualifies as NSPA ubiquitination substrate that regulates Tyr phosphorylation-dependent NMDAR stability at PSDs. The NSPA/PTPMEG pathway emerges as a new regulator of glutamatergic transmission and plasticity and may provide mechanistic clues and therapeutic opportunities for anti-P-mediated pathogenicity in SLE, a still unmet need.

Roles of the PTP61F Gene in Regulating Energy Metabolism of Tribolium castaneum (Coleoptera: Tenebrionidae)

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator in the insulin signaling pathway. It belongs to a class of non-receptor phosphatases of protein tyrosine phosphatase and can catalyze the dephosphorylation of tyrosine to regulate cell differentiation, growth, and metabolism. However, few studies have focused on the role of PTP1B in regulating energy metabolism of insects. In this study, we investigated the expression profiles and the functions of a PTP1B gene (designated TcPTP61F) in the red flour beetle Tribolium castaneum. Quantitative real-time PCR analyzed showed that TcPTP61F was highly expressed in the pupal and adult stages. In adult tissues, TcPTP61F was prominently expressed in the tarsus and head. RNA interference-mediated silencing of TcPTP61F reduced the expression of eight genes in trehalose metabolic and glycolytic pathways. TcPTP61F depletion also caused a significant change in the distribution of trehalose, glucose, and glycogen. Additionally, knockdown of TcPTP61F inhibited the pyruvate kinase (PK) activity and significantly decreased the adenosine triphosphate (ATP) level. The results suggest that TcPTP61F is indispensible for trehalose and energy metabolism of T. castaneum.

Annexin A2-S100A10 Represents the Regulatory Component of Maxi-Cl Channel Dependent on Protein Tyrosine Dephosphorylation and Intracellular Ca²⁺.

Maxi-anion channel (Maxi-Cl) is ubiquitously expressed and involved in a number of important cell functions especially by serving as an ATP release pathway. We recently identified SLCO2A1 as its essential core component. However, the regulatory component required for the channel activation/inactivation remains unidentified. In the present study, to identify the regulatory component, we made genome-wide analysis combined with siRNA screening and performed patch-clamp studies and ATP release assay after gene silencing and overexpression. Comparative microarray analysis between Maxi-Cl-rich C127 and -deficient C1300 cells revealed highly differential expression not only of SLCO2A1 but also of four annexin family members. Gene silencing study showed that Anxa2 is involved in Maxi-Cl activity. The Maxi-Cl events appeared in C1300 cells by overexpression of Slco2a1 and more efficiently by that of Slco2a1 plus Anxa2. Immunoprecipitation assay supported the interaction between ANXA2 and SLCO2A1. Suppressive effects of overexpression of a phospho-mimicking mutant of Anxa2, Anxa2-Y23E, indicated that protein tyrosine dephosphorylation dependence of Maxi-Cl is conferred by ANXA2. Maxi-Cl activity was suppressed by gene silencing of S100A10, a binding partner of ANXA2, and by applying a synthetic ANXA2 peptide, Ac-(1-14), which interferes with the ANXA2-S100A10 complex formation. Intracellular Ca2+ dependence of Maxi-Cl activity was abolished by S100a10 knockdown. The ANXA2-S100A10 complex represents the regulatory component of Maxi-Cl conferring protein tyrosine dephosphorylation dependence and intracellular Ca2+ sensitivity on this channel.

Sensitivity of colorectal or pancreatic cancers with high DDX5 and mutations in Kras and p53 genes to FL118 treatment.

e16102 Background: FL118 is a small molecule anticancer drug with novel mechanisms of action, although its chemical structure is similar to camptothecins. Currently, FL118 is completing its IND process to enter clinical trials for patients with advanced colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) cancers. Precision medicine is becoming a trend for modern cancer treatment. Thus, identification of drug efficacy-relevant biomarker(s) for patient selection is critical for enhancing patients’ response rates and avoiding unnecessary treatment in the clinic. Methods: Animal models of CRC and PDAC patient-derived xenograft (PDX) tumors were used to test FL118 efficacy; next generation sequencing (NGS) was used to determine PDX tumor molecular profiles; Western blots (WB) and real time RT-PCR were used to determine gene expression and/or apoptosis; Immunoprecipitation (IP) plus WB was used to determine protein phophorylation. Proteasome inhibitor was used to determine protein expression pathway. FL118 affinity purification was used to identify FL118 binding protein(s). Results: In association with WuXi Apptec, we have completed FL118 CMC and produced a clinical compatible product formulation. In this new product format, FL118 exhibits excess toxicity at a dose of ≥ 7.5 mg/kg via weekly x 4 schedules in mice. However, dosages of ≤ 6 mg/kg via the same schedule are tolerable. FL118 can eliminate the Kras/p53/APC triple-mutated SW620-established CRC tumors in a high percentage of mice at doses of 6 (maximum tolerated dose, MTD), 5, 4, 3, and 2 mg/kg. Tumor elimination under MTD is a typical characteristic of targeted drugs, which do not need MTD but only need the target to be saturated with drug for maximal efficacy. High expression of DDX5 (also called p68), a multifunctional oncogenic transcriptional co-activator and a DEAD-box RNA helicase, is associated with high FL118 efficacy in both CRC and PDAC PDX tumors. Mechanistically, FL118 binds to DDX5 to induce DDX5 tyrosine dephosphorylation and protein degradation without decreasing its mRNA. Tumors with Kras mutation (mKras) and/or TP53/p53 mutation (mp53) possess intrinsic resistance to multiple classic chemotherapeutic agents. However, PDAC PDX tumors with mp53 exhibit higher mKras expression and greater sensitivity to FL118 than tumors with wild type p53 and mKras. Conclusions: DDX5, mKras and mp53 appears to be favorable biomarkers for predicting FL118 high efficacy. However, further studies with a large cohort will be needed for confirmation.

Splice‐dependent trans‐synaptic PTPδ–IL1RAPL1 interaction regulates synapse formation and non‐REM sleep

Alternative splicing regulates trans‐synaptic adhesions and synapse development, but supporting in vivo evidence is limited. PTPδ, a receptor tyrosine phosphatase adhering to multiple synaptic adhesion molecules, is associated with various neuropsychiatric disorders; however, its in vivo functions remain unclear. Here, we show that PTPδ is mainly present at excitatory presynaptic sites by endogenous PTPδ tagging. Global PTPδ deletion in mice leads to input‐specific decreases in excitatory synapse development and strength. This involves tyrosine dephosphorylation and synaptic loss of IL1RAPL1, a postsynaptic partner of PTPδ requiring the PTPδ‐meA splice insert for binding. Importantly, PTPδ‐mutant mice lacking the PTPδ‐meA insert, and thus lacking the PTPδ interaction with IL1RAPL1 but not other postsynaptic partners, recapitulate biochemical and synaptic phenotypes of global PTPδ‐mutant mice. Behaviorally, both global and meA‐specific PTPδ‐mutant mice display abnormal sleep behavior and non‐REM rhythms. Therefore, alternative splicing in PTPδ regulates excitatory synapse development and sleep by modulating a specific trans‐synaptic adhesion.

SHP2-Independent Tyrosine Dephosphorylation of Cortactin and Vinculin during Infection with Helicobacter pylori.

The gastric pathogen Helicobacter pylori colonizes approximately half of the human world population. The bacterium injects the effector protein cytotoxin associated gene A (CagA) via a type-IV secretion system into host epithelial cells, where the protein becomes phosphorylated at specific EPIYA-motifs by cellular kinases. Inside the host cell, CagA can interact with over 25 different proteins in both phosphorylation-dependent and phosphorylation-independent manners, resulting in manipulation of host-cell signaling pathways. During the course of an H. pylori infection, certain host-cell proteins undergo tyrosine dephosphorylation in a CagA-dependent manner, including the actin-binding proteins cortactin and vinculin. A predominant response of intracellular CagA is the binding and activation of tyrosine phosphatase, the human Src-homology-region-2-domain-containing-phosphatase-2 (SHP2). Here, we considered the possibility that activated SHP2 might be responsible for the dephosphorylation of cortactin and vinculin. To investigate this, phosphatase inhibitor studies were performed. Additionally, a complete knockout mutant of SHP2 in AGS cells was created by CRISPR/Cas9 technology, and these cells were infected with H. pylori. However, neither the presence of an inhibitor nor the inactivation of SHP2 prevented the dephosphorylation of cortactin and vinculin upon CagA delivery. Tyrosine dephosphorylation of these proteins is therefore independent of SHP2 and instead must be caused by another, as yet unidentified, protein tyrosine phosphatase.

Structural basis of liprin-\u03b1-promoted LAR-RPTP clustering for modulation of phosphatase activity

Leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cell adhesion molecules involved in mediating neuronal development. The binding of LAR-RPTPs to extracellular ligands induces local clustering of LAR-RPTPs to regulate axon growth and synaptogenesis. LAR-RPTPs interact with synaptic liprin-α proteins via the two cytoplasmic phosphatase domains, D1 and D2. Here we solve the crystal structure of LAR_D1D2 in complex with the SAM repeats of liprin-α3, uncovering a conserved two-site binding mode. Cellular analysis shows that liprin-αs robustly promote clustering of LAR in cells by both the liprin-α/LAR interaction and the oligomerization of liprin-α. Structural analysis reveals a unique homophilic interaction of LAR via the catalytically active D1 domains. Disruption of the D1/D1 interaction diminishes the liprin-α-promoted LAR clustering and increases tyrosine dephosphorylation, demonstrating that the phosphatase activity of LAR is negatively regulated by forming clusters. Additionally, we find that the binding of LAR to liprin-α allosterically regulates the liprin-α/liprin-β interaction.

The protein tyrosine phosphatase PTP-PEST mediates hypoxia-induced endothelial autophagy and angiogenesis via AMPK activation.

Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500 µM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200 nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.

Inhibition of Trypanosoma evansi Protein-Tyrosine Phosphatase by Myristic Acid Analogues Isolated from Khaya senegalensis and Tamarindus indica.

BackgroundTrypanosome infections still pose severe health and economic consequences, especially in the endemic regions of Sub-Saharan Africa. Trypanosome differentiation to the procyclic forms which lack the immune evasion mechanisms for survival in the bloodstream is prevented by tyrosine dephosphorylation which is catalyzed by protein-tyrosine phosphatase; thereby promoting survival of the parasites in the host. Inhibition of Protein-tyrosine phosphatase is a strategic therapeutic target that could attenuate trypanosomiasis. This study investigated the in vitro inhibitory effect of stem bark extracts of Khaya senegalensis and Tamarindus indica on the enzymatic activity of protein-tyrosine phosphatase.MethodsAll determinations were carried out following standard procedures for analytical experiments. The analogues of myristic acid that inhibited the enzymatic activity of protein-tyrosine phosphatase were isolated by bioassay-guided fractionation of stem bark extracts of Khaya senegalensis and Tamarindus indica.ResultsAnalogues of myristic acid proved to be potent inhibitors of protein-tyrosine phosphatase. Double reciprocal (Lineweaver–Burk) plots of the initial velocity data indicated non-competitive inhibition with Ki of 0.67 mg/mL for Khaya senegalensis and 2.17 mg/mL for Tamarindus indica. The kinetic parameters for the cleavage of para-nitrophenylphosphate by the enzyme showed a KM of 3.44 mM and Vmax of 0.19 µmol/min. Sodium orthovanadate, the enzymes’ specific inhibitor, inhibited the enzyme competitively with Ki of 0.20 mg/mL. Gas chromatography-mass spectrometry analysis of the stem bark bioactive fractions of Khaya senegalensis and Tamarindus indica revealed the presence of myristic acid analogues.ConclusionAnalogues of myristic acid are potent inhibitors of protein-tyrosine phosphatase that could be developed as trypanocide to inhibit the enzymatic activity of protein-tyrosine phosphatase in order to prevent transmission of trypanosomes.

DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis.

Dual-specificity phosphatases (DUSPs) regulate the activity of various downstream kinases through serine or threonine or tyrosine dephosphorylation. Loss of function and aberrant expression of DUSPs has been implicated in cancer progression and poor survival, yet the function of DUSP22 in prostate cancer (PCa) cells is not clear. Gene Expression Omnibus and cBioPortal microarray database analyses showed that DUSP22 expression was lower in PCa tissues than normal prostate tissues, and altered DUSP22 expression was associated with shorter progression-free and disease-free survival of patients with PCa. Exogenous DUSP22 expression in LNCaP, PC3, and C4-2B PCa cells inhibited cellular proliferation and colony formation, supporting a growth inhibitory role for DUSP22 in PCa cells. DUSP22 expression significantly attenuated epidermal growth factor (EGF) receptor (EGFR) and its downstream ERK1/2 signaling by dephosphorylation. However, DUSP22 failed to suppress the growth of CWR22Rv1 and DU145 cells with elevated phosphorylated (p-)ERK1/2 levels. A serine-to-alanine mutation at position 58, a potential ERK1/2-targeted phosphorylation site in DUSP22, was sufficient to suppress growth of CWR22Rv1 cells with elevated p-ERK1/2 levels, suggesting a mutually antagonistic relationship between DUSP22 and ERK1/2 dependent on phosphorylation status. We showed that DUSP22 can suppress prostate-specific antigen gene expression through phosphatase-dependent pathways, suggesting that DUSP22 is an important regulator of the androgen receptor (AR) in PCa cells. Mechanistically, DUSP22 can interact with AR as a regulatory partner and interfere with EGF-induced AR phosphorylation at Tyr534, suggesting that DUSP22 serves as a crucial suppressor of both EGFR and AR-dependent signaling in PCa cells via dephosphorylation. Our findings indicate that loss of function of DUSP22 in PCa cells leads to aberrant activation of both EGFR-ERKs and AR signaling and ultimately progression of PCa, supporting the potential for novel therapeutic design of harnessing DUSP22 in the treatment of PCa.-Lin, H.-P., Ho, H.-M., Chang, C.-W., Yeh, S.-D., Su, Y.-W., Tan, T.-H., Lin, W.-J. DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis.