Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase.

Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.

Eph receptors and ephrins: therapeutic opportunities.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptor tyrosine kinase family plays important roles in developmental processes, adult tissue homeostasis, and various diseases. Interaction with Eph receptor-interacting protein (ephrin) ligands on the surface of neighboring cells triggers Eph receptor kinase-dependent signaling. The ephrins can also transmit signals, leading to bidirectional cell contact-dependent communication. Moreover, Eph receptors and ephrins can function independently of each other through interplay with other signaling systems. Given their involvement in many pathological conditions ranging from neurological disorders to cancer and viral infections, Eph receptors and ephrins are increasingly recognized as attractive therapeutic targets, and various strategies are being explored to modulate their expression and function. Eph receptor/ephrin upregulation in cancer cells, the angiogenic vasculature, and injured or diseased tissues also offer opportunities for Eph/ephrin-based targeted drug delivery and imaging. Thus, despite the challenges presented by the complex biology of the Eph receptor/ephrin system, exciting possibilities exist for therapies exploiting these molecules.

Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei.

African trypanosomes are protozoan parasites transmitted by a tsetse fly vector to a mammalian host. The life cycle includes highly proliferative forms and quiescent forms, the latter being adapted to host transmission. The signaling pathways controlling the developmental switch between the two forms remain unknown. Trypanosoma brucei contains two target of rapamycin (TOR) kinases, TbTOR1 and TbTOR2, and two TOR complexes, TbTORC1 and TbTORC2. Surprisingly, two additional TOR kinases are encoded in the T. brucei genome. We report that TbTOR4 associates with an Armadillo domain-containing protein (TbArmtor), a major vault protein, and LST8 to form a unique TOR complex, TbTORC4. Depletion of TbTOR4 caused irreversible differentiation of the parasite into the quiescent form. AMP and hydrolysable analogs of cAMP inhibited TbTOR4 expression and induced the stumpy quiescent form. Our results reveal unexpected complexity in TOR signaling and show that TbTORC4 negatively regulates differentiation of the proliferative form into the quiescent form.

Target of rapamycin (TOR) kinase in Trypanosoma brucei: an extended family.

The complex life cycle of Trypanosoma brucei provides an excellent model system to understand signalling pathways that regulate development. We described previously the classical functions of TOR (target of rapamycin) 1 and TOR2 in T. brucei. In a more recent study, we described a novel TOR kinase, named TOR4, which regulates differentiation from the proliferative infective form to the quiescent form. In contrast with TOR1 loss-of-function, down-regulation of TOR4 triggers an irreversible differentiation process through the development of the insect pre-adapted quiescent form. TOR4 governs a signalling pathway distinct from those controlled by the conventional TOR complexes TORC1 and TORC2. Depletion of TOR4 induces all well-known characteristics of the quiescent developmental stage in trypanosomes, including expression of the PAD (proteins associated with differentiation) surface proteins and transcriptional down-regulation of the VSG (variant surface glycoprotein) gene. TOR4 kinase forms a structurally and functionally distinct complex named TORC4. TOR4 associates with LST8 (lethal with sec-13 protein 8) and other factors including an armadillo-domain-containing protein and the major vault protein, which probably serves as a scaffold for this kinase. Research in T. brucei, a protozoan parasite that diverged from the eukaryotic tree early in evolution, may help to uncover new functions of TOR kinases.

Target of rapamycin (TOR)-like 1 kinase is involved in the control of polyphosphate levels and acidocalcisome maintenance in Trypanosoma brucei.

Target of rapamycin (TOR) kinases are highly conserved protein kinases that integrate signals from nutrients and growth factors to coordinate cell growth and cell cycle progression. It has been previously described that two TOR kinases control cell growth in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis. Here we studied an unusual TOR-like protein named TbTOR-like 1 containing a PDZ domain and found exclusively in kinetoplastids. TbTOR-like 1 localizes to unique cytosolic granules. After hyperosmotic stress, the localization of the protein shifts to the cell periphery, different from other organelle markers. Ablation of TbTOR-like 1 causes a progressive inhibition of cell proliferation, producing parasites accumulating in the S/G(2) phase of the cell cycle. TbTOR-like 1 knocked down cells have an increased area occupied by acidic vacuoles, known as acidocalcisomes, and are enriched in polyphosphate and pyrophosphate. These results suggest that TbTOR-like 1 might be involved in the control of acidocalcisome and polyphosphate metabolism in T. brucei.

Rapamycin inhibits trypanosome cell growth by preventing TOR complex 2 formation.

Target of rapamycin (TOR) kinases control cell growth through two functionally distinct multiprotein complexes. TOR complex 1 (TORC1) controls temporal cell growth and is sensitive to rapamycin, whereas TOR complex 2 (TORC2) is rapamycin resistant and regulates spatial cell growth. Here, we identified two TOR orthologues, TbTOR1 and TbTOR2, in the protozoan parasite Trypanosoma brucei, as well as orthologues of the well-known TORC1 and TORC2 partners, KOG1/raptor and AVO3/rictor. TbTOR proteins differ in their functions, subcellular localization, and rapamycin sensitivity. TbTOR1 controls cell growth by regulating cell cycle, nucleolus structure, and protein synthesis, whereas TbTOR2 coordinates cell polarization and cytokinesis. Rapamycin treatment of bloodstream trypanosomes resulted in a pronounced reduction of cell proliferation, with an EC(50) of 152 nM. Unique for a eukaryote, we observed that rapamycin acted exclusively by preventing TORC2 formation, with no effect on TORC1. Our findings on TOR signaling in this protozoan, which is located in a distal position in the eukaryotic cell lineage, highlight the clinical possibilities of rapamycin derivates and provide valuable insights into understanding rapamycin-mediated inhibition of TORC2.

Protein kinase A can block EphA2 receptor-mediated cell repulsion by increasing EphA2 S897 phosphorylation.

The EphA2 receptor tyrosine kinase plays key roles in tissue homeostasis and disease processes such as cancer, pathological angiogenesis, and inflammation through two distinct signaling mechanisms. EphA2 "canonical" signaling involves ephrin-A ligand binding, tyrosine autophosphorylation, and kinase activity; EphA2 "noncanonical" signaling involves phosphorylation of serine 897 (S897) by AKT and RSK kinases. To identify small molecules counteracting EphA2 canonical signaling, we developed a high-content screening platform measuring inhibition of ephrin-A1-induced PC3 prostate cancer cell retraction. Surprisingly, most hits from a screened collection of pharmacologically active compounds are agents that elevate intracellular cAMP by activating G protein-coupled receptors such as the ß2-adrenoceptor. We found that cAMP promotes phosphorylation of S897 by protein kinase A (PKA) as well as increases the phosphorylation of several nearby serine/threonine residues, which constitute a phosphorylation hotspot. Whereas EphA2 canonical and noncanonical signaling have been viewed as mutually exclusive, we show that S897 phosphorylation by PKA can coexist with EphA2 tyrosine phosphorylation and block cell retraction induced by EphA2 kinase activity. Our findings reveal a novel paradigm in EphA2 function involving the interplay of canonical and noncanonical signaling and highlight the ability of the ß2-adrenoceptor/cAMP/PKA axis to rewire EphA2 signaling in a subset of cancer cells.

Trypanosome TOR complex 2 functions in cytokinesis.

TOR (target of rapamycin) is a kinase of the phosphatidylinositol kinase-related kinase (PIKK) family that controls cell growth in eukaryotes in response to nutrients, energy conditions and growth factors. We have recently identified two trypanosome TOR orthologs, named TbTOR1 and TbTOR2, and two other proteins with significant homology to yeast or mammalian TORs, named TbTOR-like 1 and TbTOR-like 2. TbTOR1 depletion results in arrest of bloodstream trypanosomes in G(1), concomitant to protein synthesis inhibition; however, TbTOR2 depletion leads to dramatic morphological defects in cell polarization, endocytosis and cytokinesis. Rapamycin inhibits T. brucei cell growth by prevention of TORC2 complex formation, without any effect on TORC1 contrary to what generally occurs in other eukaryotes. Based on the unique features of T. brucei and its distal position in the eukaryotic cell lineage, we describe our views on the function of the TOR protein as a major regulator of cell growth and cytokinesis and discuss a possible role in the developmental differentiation processes.

Trypanosome TOR as a major regulator of cell growth and autophagy.

Trypanosomatid protozoa parasites are responsible for tropical diseases, and undergo complex life cycles involving developmental forms adapted to insect vectors and vertebrate hosts. During their life cycle these parasites proceed through different forms in response to dramatic environmental changes and/or developmentally regulated programs. Successful progression of the parasite through its life cycle is highly dependent on the capacity of adaptation to distinct stresses involving processes such as autophagy. In eukaryotes, target of rapamycin (TOR) protein kinases act as a sensor, which integrates the nutritional and energetic status, adjusting cell metabolism and growth. Compromising cell viability in yeast and mammals leads to a reduction of TOR function, triggering processes aimed to overcome unfavorable conditions. This is partly achieved by TOR-mediated regulation of protein synthesis and recycling of cellular components by autophagy. In the last few years, autophagy has been described during developmental differentiation processes in Trypanosomatidae. However, no link between TOR signalling, autophagy, and differentiation has been described so far. This addendum is a commentary to the work published by our group,(1) in which we discuss the possible role of TOR kinases, as a controller of cell growth and autophagy, in the regulation of differentiation processes during Trypanosomatids life cycles.