Advancing therapeutics using antibody-induced dimerization of receptor tyrosine phosphatases.

Receptor protein tyrosine phosphatases (RPTPs) are involved in a broad list of cellular, developmental, and physiological functions. Altering their expression leads to significant changes in protein phosphorylation linked to a growing list of human diseases, including cancers and neurological disorders. In this issue of Genes & Development, Qian and colleagues (pp. 743-759) present the identification of a monoclonal antibody targeting PTPRD extracellular domain-inducing dimerization and inhibition of the phosphatase activities, causing the proteolysis of dimeric PTPRD by a mechanism involving intracellular degradation pathways. Their study supports the potential of modulating PTPRD via its extracellular domains. This opens a new framework in the clinical manipulation of PTPRD and its closely related family members.

PRL-1/2 phosphatases control TRPM7 magnesium-dependent function to regulate cellular bioenergetics.

Phosphatases of regenerating liver (PRL-1, PRL-2, PRL-3; also known as PTP4A1, PTP4A2, PTP4A3, respectively) control intracellular magnesium levels by interacting with the CNNM magnesium transport regulators. Still, the exact mechanism governing magnesium transport by this protein complex is not well understood. Herein, we have developed a genetically encoded intracellular magnesium-specific reporter and demonstrate that the CNNM family inhibits the function of the TRPM7 magnesium channel. We show that the small GTPase ARL15 increases CNNM3/TRPM7 protein complex formation to reduce TRPM7 activity. Conversely, PRL-2 overexpression counteracts ARL15 binding to CNNM3 and enhances the function of TRPM7 by preventing the interaction between CNNM3 and TRPM7. Moreover, while TRPM7-induced cell signaling is promoted by PRL-1/2, it is reduced when CNNM3 is overexpressed. Lowering cellular magnesium levels reduces the interaction of CNNM3 with TRPM7 in a PRL-dependent manner, whereby knockdown of PRL-1/2 restores the protein complex formation. Cotargeting of TRPM7 and PRL-1/2 alters mitochondrial function and sensitizes cells to metabolic stress induced by magnesium depletion. These findings reveal the dynamic regulation of TRPM7 function in response to PRL-1/2 levels, to coordinate magnesium transport and reprogram cellular metabolism.

Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS(-) pDCs produced IFN-α. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation.

The PTP family photo album.

Protein tyrosine phosphatases (PTPs) are central players in many biological processes. In this issue, Barr et al. (2009) analyze 22 different PTP structures to define their common and unique features. This effort provides key insights into the regulation of PTP activity that could lead to the development of new therapeutics.

Pharmacological potentiation of monocyte-derived dendritic cell cancer immunotherapy.

Dendritic cells have been at the forefront of cancer-immunotherapy research for over 2 decades. They elicited that attention by having an unprecedented capacity to mount T cells responses against tumors. However, the clinical use of DC-based vaccination against established malignancies has resulted in limited clinical benefits. Several factors are responsible for limiting the efficacy of DC-based immunotherapy, such as the harmful influence of the tumor microenvironment on DCs activity. New insights into the inner process of DC-mediated T cell activation have supported the development of new strategies that potentiate DCs-based therapies. Herein, we identify signaling cascades that have recently been targeted by small molecules and biologicals to promote the activation of monocyte-derived DCs and decrease their susceptibility to becoming tolerogenic. While Statins can markedly enhance antigen presentation, protein kinase inhibitors can be used to increase the expression of co-receptors and adhesion molecules. STAT3 and IDO can be modulated to limit the production of regulatory factors that work against differentiation and activation. The targeting of multiple pathways simultaneously has also been found to produce synergism and drastically enhance DCs activity. Some of these strategies have recently yielded positive results in clinical settings against established malignancies such as non-small cell lung cancer. The emergence of these approaches opens the door for a new generation of potent dendritic cell-based therapeutics to fight cancer.

TAOK3 limits age-associated inflammation by negatively modulating macrophage differentiation and their production of TNFα.

BACKGROUND: Human aging is characterized by a state of chronic inflammation, termed inflammaging, for which the causes are incompletely understood. It is known, however, that macrophages play a driving role in establishing inflammaging by promoting pro-inflammatory rather than anti-inflammatory responses. Numerous genetic and environmental risk factors have been implicated with inflammaging, most of which are directly linked to pro-inflammatory mediators IL-6, IL1Ra, and TNFα. Genes involved in the signaling and production of those molecules have also been highlighted as essential contributors. TAOK3 is a serine/threonine kinase of the STE-20 kinase family that has been associated with an increased risk of developing auto-immune conditions in several genome-wide association studies (GWAS). Yet, the functional role of TAOK3 in inflammation has remained unexplored. RESULTS: We found that mice deficient in the serine/Threonine kinase Taok3 developed severe inflammatory disorders with age, which was more pronounced in female animals. Further analyses revealed a drastic shift from lymphoid to myeloid cells in the spleens of those aged mice. This shift was accompanied by hematopoietic progenitor cells skewing in Taok3-/- mice that favored myeloid lineage commitment. Finally, we identified that the kinase activity of the enzyme plays a vital role in limiting the establishment of proinflammatory responses in macrophages. CONCLUSIONS: Essentially, Taok3 deficiency promotes the accumulation of monocytes in the periphery and their adoption of a pro-inflammatory phenotype. These findings illustrate the role of Taok3 in age-related inflammation and highlight the importance of genetic risk factors in this condition.

Autoimmune susceptibility gene PTPN2 is required for clearance of adherent-invasive Escherichia coli by integrating bacterial uptake and lysosomal defence.

OBJECTIVES: Alterations in the intestinal microbiota are linked with a wide range of autoimmune and inflammatory conditions, including inflammatory bowel diseases (IBD), where pathobionts penetrate the intestinal barrier and promote inflammatory reactions. In patients with IBD, the ability of intestinal macrophages to efficiently clear invading pathogens is compromised resulting in increased bacterial translocation and excessive immune reactions. Here, we investigated how an IBD-associated loss-of-function variant in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene, or loss of PTPN2 expression affected the ability of macrophages to respond to invading bacteria. DESIGN: IBD patient-derived macrophages with wild-type (WT) PTPN2 or carrying the IBD-associated PTPN2 SNP, peritoneal macrophages from WT and constitutive PTPN2-knockout mice, as well as mice specifically lacking PTPN2 in macrophages were infected with non-invasive K12 Escherichia coli, the human adherent-invasive E. coli (AIEC) LF82, or a novel mouse AIEC (mAIEC) strain. RESULTS: Loss of PTPN2 severely compromises the ability of macrophages to clear invading bacteria. Specifically, loss of functional PTPN2 promoted pathobiont invasion/uptake into macrophages and intracellular survival/proliferation by three distinct mechanisms: Increased bacterial uptake was mediated by enhanced expression of carcinoembryonic antigen cellular adhesion molecule (CEACAM)1 and CEACAM6 in PTPN2-deficient cells, while reduced bacterial clearance resulted from defects in autophagy coupled with compromised lysosomal acidification. In vivo, mice lacking PTPN2 in macrophages were more susceptible to mAIEC infection and mAIEC-induced disease. CONCLUSIONS: Our findings reveal a tripartite regulatory mechanism by which PTPN2 preserves macrophage antibacterial function, thus crucially contributing to host defence against invading bacteria.

Protein tyrosine phosphatome metabolic screen identifies TC-PTP as a positive regulator of cancer cell bioenergetics and mitochondrial dynamics.

Metabolic reprogramming occurs in cancer cells and is regulated partly by the opposing actions of tyrosine kinases and tyrosine phosphatases. Several members of the protein tyrosine phosphatase (PTP) superfamily have been linked to cancer as either pro-oncogenic or tumor-suppressive enzymes. In order to investigate which PTPs can modulate the metabolic state of cancer cells, we performed an shRNA screen of PTPs in HCT116 human colorectal cancer cells. Among the 72 PTPs efficiently targeted, 24 were found to regulate mitochondrial respiration, 8 as negative and 16 as positive regulators. Of the latter, we selected TC-PTP (PTPN2) for further characterization since inhibition of this PTP resulted in major functional defects in oxidative metabolism without affecting glycolytic flux. Transmission electron microscopy revealed an increase in the number of damaged mitochondria in TC-PTP-null cells, demonstrating the potential role of this PTP in regulating mitochondrial homeostasis. Downregulation of STAT3 by siRNA-mediated silencing partially rescued the mitochondrial respiration defect observed in TC-PTP-deficient cells, supporting the role of this signaling axis in regulating mitochondrial activity. In addition, mitochondrial stress prevented an increased expression of electron transport chain-related genes in cells with TC-PTP silencing, correlating with decreased ATP production, cellular proliferation, and migration. Our shRNA-based metabolic screen revealed that PTPs can serve as either positive or negative regulators of cancer cell metabolism. Taken together, our findings uncover a new role for TC-PTP as an activator of mitochondrial metabolism, validating this PTP as a key target for cancer therapeutics.

4E-BP-Dependent Translational Control of Irf8 Mediates Adipose Tissue Macrophage Inflammatory Response.

Deregulation of mRNA translation engenders many human disorders, including obesity, neurodegenerative diseases, and cancer, and is associated with pathogen infections. The role of eIF4E-dependent translational control in macrophage inflammatory responses in vivo is largely unexplored. In this study, we investigated the involvement of the translation inhibitors eIF4E-binding proteins (4E-BPs) in the regulation of macrophage inflammatory responses in vitro and in vivo. We show that the lack of 4E-BPs exacerbates inflammatory polarization of bone marrow-derived macrophages and that 4E-BP-null adipose tissue macrophages display enhanced inflammatory gene expression following exposure to a high-fat diet (HFD). The exaggerated inflammatory response in HFD-fed 4E-BP-null mice coincides with significantly higher weight gain, higher Irf8 mRNA translation, and increased expression of IRF8 in adipose tissue compared with wild-type mice. Thus, 4E-BP-dependent translational control limits, in part, the proinflammatory response during HFD. These data underscore the activity of the 4E-BP-IRF8 axis as a paramount regulatory mechanism of proinflammatory responses in adipose tissue macrophages.

ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs.

Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-ß-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.

Magnesium-sensitive upstream ORF controls PRL phosphatase expression to mediate energy metabolism.

Phosphatases of regenerating liver (PRL-1, PRL-2, and PRL-3, also known as PTP4A1, PTP4A2, and PTP4A3) control magnesium homeostasis through an association with the CNNM magnesium transport regulators. Although high PRL levels have been linked to cancer progression, regulation of their expression is poorly understood. Here we show that modulating intracellular magnesium levels correlates with a rapid change of PRL expression by a mechanism involving its 5'UTR mRNA region. Mutations or CRISPR-Cas9 targeting of the conserved upstream ORF present in the mRNA leader derepress PRL protein synthesis and attenuate the translational response to magnesium levels. Mechanistically, magnesium depletion reduces intracellular ATP but up-regulates PRL protein expression via activation of the AMPK/mTORC2 pathway, which controls cellular energy status. Hence, altered PRL-2 expression leads to metabolic reprogramming of the cells. These findings uncover a magnesium-sensitive mechanism controlling PRL expression, which plays a role in cellular bioenergetics.

Mechanisms of invasion in glioblastoma.

PURPOSE OF REVIEW: This review provides an overview of recent updates in understanding the mechanisms by which glioblastoma cells interact with their cellular and molecular partners within the microenvironment. RECENT FINDINGS: We have now a better knowledge of the cell populations involved in Glioblastoma (GBM) invasion. Recent works discovered the role of new molecular players in GBM invasion, and, most importantly, better models are emerging which better recapitulate GBM invasion. SUMMARY: Invasive properties of glioblastoma make complete surgical resection impossible and highly invasive cells are responsible for tumor recurrence. In this review, we focus on recent updates describing how invasive cells progress in the surrounding tissue along brain structures. We also provide an overview of the current knowledge on key cells and molecular players within the microenvironment that contribute to the invasive process. VIDEO ABSTRACT.

Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4.

The four member family of "Cyclin and Cystathionine ß-synthase (CBS) domain divalent metal cation transport mediators", CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2.

Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine ß-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling.

Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.

Mining the Complex Family of Protein Tyrosine Phosphatases for Checkpoint Regulators in Immunity.

The family of protein tyrosine phosphatases (PTPs) includes 107 genes in humans that are diverse in their structures and expression profiles. The majority are present in immune cells and play various roles in either inhibiting or promoting the duration and amplitude of signaling cascades. Several PTPs, including TC-PTP (PTPN2) and SHP-1 (PTPN6), have been recognized as being crucial for maintaining proper immune response and self-tolerance, and have gained recognition as true immune system checkpoint modulators. This chapter details the most recent literature on PTPs and immunity by examining their known functions in regulating signaling from either established checkpoint inhibitors or by their intrinsic properties, as modulators of the immune response. Notably, we review PTP regulatory properties in macrophages, antigen-presenting dendritic cells, and T cells. Overall, we present the PTP gene family as a remarkable source of novel checkpoint inhibitors wherein lies a great number of new targets for immunotherapies.

Inhibition of PRL-2·CNNM3 Protein Complex Formation Decreases Breast Cancer Proliferation and Tumor Growth.

The oncogenic phosphatase of regenerating liver 2 (PRL-2) has been shown to regulate intracellular magnesium levels by forming a complex through an extended amino acid loop present in the Bateman module of the CNNM3 magnesium transporter. Here we identified highly conserved residues located on this amino acid loop critical for the binding with PRL-2. A single point mutation (D426A) of one of those critical amino acids was found to completely disrupt PRL-2·human Cyclin M 3 (CNNM3) complex formation. Whole-cell voltage clamping revealed that expression of CNNM3 influenced the surface current, whereas overexpression of the binding mutant had no effect, indicating that the binding of PRL-2 to CNNM3 is important for the activity of the complex. Interestingly, overexpression of the CNNM3 D426A-binding mutant in cancer cells decreased their ability to proliferate under magnesium-deprived situations and under anchorage-independent growth conditions, demonstrating a PRL-2·CNNM3 complex-dependent oncogenic advantage in a more stringent environment. We further confirmed the importance of this complex in vivo using an orthotopic xenograft breast cancer model. Finally, because molecular modeling showed that the Asp-426 side chain in CNNM3 buries into the catalytic cavity of PRL-2, we showed that a PRL inhibitor could abrogate complex formation, resulting in a decrease in proliferation of human breast cancer cells. In summary, we provide evidence that this fundamental regulatory aspect of PRL-2 in cancer cells could potentially lead to broadly applicable and innovative therapeutic avenues.

Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-ß production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity.

Protein tyrosine phosphatase σ regulates autoimmune encephalomyelitis development.

Protein tyrosine phosphatases (PTPs) play essential roles in regulating signaling events in multiple cells by tyrosine dephosphorylation. One of them, PTPσ, appears important in regulating function of plasmacytoid dendritic cells (pDC). Here we report that PTPσ deletion in knockout mice and inhibition with a selective antagonist peptide exacerbated symptoms of experimental autoimmune encephalomyelitis (EAE) by enhancing axon and myelin damage in the spinal cord. PTPσ-/- mice displayed pro-inflammatory profiles in the spinal cord and lymphoid organs following MOG peptide immunization. PTPσ deletion promoted a pro-inflammatory phenotype in conventional DCs and directly regulated differentiation of CD4+ T cells. It also facilitated infiltration of T lymphocytes, activation of macrophages in the CNS and development of EAE. Therefore, PTPσ is a key negative regulator in EAE initiation and progression, which acts by regulating functions of DCs, T cells, and other immune cells. PTPσ may become an important molecular target for treating autoimmune disorders.

Redox regulation of interleukin-4 signaling.

The physiologic control of cytokine receptor activation is primarily mediated by reciprocal activation of receptor-associated protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Here, we show that immediately after ligand-dependent activation, interleukin (IL)-4 receptor generated reactive oxygen species (ROS) via phosphatidylinositol 3-kinase-dependent activation of NAD(P)H oxidase (NOX)1 and NOX5L. ROS, in turn, promoted IL-4 receptor activation by oxidatively inactivating PTP1B that physically associated with and deactivated IL-4 receptor. However, ROS were not required for the initiation of IL-4 receptor activation. ROS generated by other cytokine receptors, including those for erythropoietin, tumor necrosis factor-alpha, or IL-3, also promoted IL-4 signaling. These data indicate that inactivation of receptor-associated PTP activity by cytokine-generated ROS is a physiologic mechanism for the amplification of cytokine receptor activation in both cis and trans, revealing a role for ROS in cytokine crosstalk.