The juxtamembrane region of TrkA kinase is critical for inhibitor selectivity

Although numerous crystal structures for protein kinases have been reported, many include only the kinase domain but not the juxtamembrane (JM) region, a critical activity-controlling segment of receptor tyrosine kinases (RTKs). In this study, we determined the X-ray crystal structure of the tropomyosin receptor kinase (Trk) A selective inhibitor A1 complexed with the TrkA kinase domain and the JM region. This structure revealed that the unique inhibitor-binding pocket created by a novel JM configuration yields significant potency and high selectivity against TrkB and TrkC. Moreover, we validated the importance of the JM region for the potency of A1 using in vitro assays. The introduction of moieties that interact with the JM region will be one of the most effective strategies for producing highly selective RTK inhibitors.     

Proteasome-dependent proteolysis has a critical role in fine-tuning the feedback inhibition of cytokinin signaling

The ubiquitin/26S proteasome-dependent proteolysis of response regulators is a critical element of many plant hormone signaling pathways. We have recently shown that cytokinin signaling requires the AXR1 component of the related to ubiquitin (RUB) protein modification pathway to promote the proteasome-dependent degradation of the cytokinin response inhibitor ARR5. Here, we show that ARR5 also accumulates in the 26S proteasome mutant rpn12a-1, and leads to a marked resistance to cytokinins. Collectively, these results suggest that proteasome-dependent proteolysis of feedback inhibitors such as ARR5 is essential for the maintenance of optimal responsivity and plasticity in cytokinin signaling.     

The length of the flexible SNAREpin juxtamembrane region is a critical determinant of SNARE-dependent fusion.

The topology of a SNARE complex bridging two docked vesicles could act as a mechanical couple to do work on the lipid bilayer resulting in fusion. To test this, we prepared a series of modified SNARE proteins and determined their effects on SNARE-dependent membrane fusion. When two helix-breaking proline residues are introduced into the juxtamembrane region of VAMP, there is little or no effect on fusion, and the same change in syntaxin 1A only reduced the extent and rate of fusion by half. The insertion of a flexible linker between the transmembrane domain and the conserved coiled-coil domain only moderately affected fusion; however, fusion efficiency systematically decreased with increasing length of the linker. Together, these results rule out a requirement for helical continuity and suggest that distance is a critical factor for membrane fusion.     

PTPepsilon has a critical role in signaling transduction pathways and phosphoprotein network topology in red cells

Protein tyrosine phosphatases (PTPs) are crucial components of cellular signal transduction pathways. Here, we report that red blood cells (RBCs) from mice lacking PTPepsilon (Ptpre(-/-)) exhibit (i) abnormal morphology; (ii) increased Ca(2+)-activated-K(+) channel activity, which was partially blocked by the Src family kinases (SFKs) inhibitor PP1; and (iii) market perturbation of the RBC membrane tyrosine (Tyr-) phosphoproteome, indicating an alteration of RBC signal transduction pathways. Using the signaling network computational analysis of the Tyr-phosphoproteomic data, we identified seven topological clusters. We studied cluster 1 containing Fyn, SFK, and Syk another tyrosine kinase. In Ptpre(-/-)mouse RBCs, the activity of Fyn was increased while Syk kinase activity was decreased compared to wild-type RBCs, validating the network computational analysis, and indicating a novel signaling pathway, which involves Fyn and Syk in regulation of red cell morphology.     

The GTPase Rho has a critical regulatory role in thymus development.

The present study employs a genetic approach to explore the role of Rho GTPases in murine thymic development. Inactivation of Rho function in the thymus was achieved by thymic targeting of a transgene encoding C3 transferase from Clostridium botulinum which selectively ADP-ribosylates Rho within its effector domain and thereby abolishes its biological function. Thymi lacking functional Rho isolated from C3 transgenic mice were strikingly smaller and showed a marked (90%) decrease in cellularity compared with their normal litter mates. We also observed a similar decrease in levels of peripheral T cells in C3 transgenic mice. Analysis of the maturation status of thymocytes indicated that differentiation of progenitor cells to mature T cells can occur in the absence of Rho function, and both positive and negative selection of T cells appear to be intact. However, transgenic mice that lack Rho function in the thymus show maturational, proliferative and cell survival defects during T-cell development that severely impair the generation of normal numbers of thymocytes and mature peripheral T cells. The present study thus identifies a role for Rho-dependent signalling pathways in thymocyte development. The data show that the function of Rho GTPases is critical for the proliferative expansion of thymocytes. This defines a selective role for the GTPase Rho in early thymic development as a critical integrator of proliferation and cell survival signals.     

The regulatory role of the juxtamembrane region in the activity of the epidermal growth factor receptor

Although the EGFR (epidermal growth factor receptor) was discovered over 30?years ago, its mechanism of activation is still the subject of intense study. There are many published studies on the mechanism of EGFR activation and regulation, including biochemical and biophysical analyses and crystallographic structures of EGFR in different activation states and conformations, mutated at various amino acids or bound to different pharmacological inhibitors. The cumulative biochemical, biophysical and structural data have led to a nearly complete account of the mechanism of activation of EGFR. The role of the JXM (juxtamembrane) domain in EGFR structure and activity has only recently begun to be elucidated through biochemical, biophysical and structural studies. In the present article, I review the studies that have highlighted the role of the JXM domain in EGFR activation.     

The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections

Although it has been shown that mast cell-deficient mice have diminished innate immune responses against bacteria, the most important immunoprotective factors secreted from activated mast cells have not been identified. Mouse mast cell protease 6 is a tetramer-forming tryptase. This serine protease is abundant in the secretory granules and is exocytosed upon bacterial challenge. Here we have described the generation of a mast cell protease-6-null mouse. Our discovery that mice lacking this neutral protease cannot efficiently clear Klebsiella pneumoniae from their peritoneal cavities reveals an essential role for this serine protease, and presumably its human ortholog, in innate immunity.     

Y1 signalling has a critical role in allergic airway inflammation

Asthma affects 300 million people worldwide, yet the mechanism behind this pathology has only been partially elucidated. The documented connection between psychological stress and airway inflammation strongly suggests the involvement of the nervous system and its secreted mediators, including neuropeptides, on allergic respiratory disease. In this study, we show that neuropeptide Y (NPY), a prominent neurotransmitter, which release is strongly upregulated during stress, exacerbates allergic airway inflammation (AAI) in mice, via its Y1 receptor. Our data indicate that the development of AAI was associated with elevated NPY expression in the lung and that lack of NPY-mediated signalling in NPYKO mice or its Y1 receptor in Y1KO mice significantly improved AAI. In vivo, eosinophilia in the bronchoalveolar fluid as well as circulating immunoglobulin E in response to AAI, were significantly reduced in NPY- and Y1-deficient compared with wild-type mice. These changes correlated with a blunting of the Th2 immune profile that is characteristic for AAI, as shown by the decreased release of interleukin-5 during ex vivo re-stimulation of T cells isolated from the thoracic draining lymph nodes of NPY- or Y1-deficient mice subjected to AAI. Taken together this study demonstrates that signalling through Y1-receptors emerges as a critical pathway for the development of airway inflammation and as such potentially opens novel avenues for therapeutic intervention in asthma.     

Cytoplasmic juxtamembrane region of the insulin receptor: a critical role in ATP binding, endogenous substrate phosphorylation, and insulin-stimulated bioeffects in CHO cells.

We have expressed in CHO cells a mutant receptor (IR delta 960) from which 12 amino acids in the juxtamembrane region (A954-D965), including Tyr960, have been deleted. The mutant receptor bound insulin normally but exhibited an increased Km for ATP during autophosphorylation. Upon prolonged incubation in vitro, or at high ATP concentrations such as those observed in vivo, autophosphorylation of IR delta 960 was similar to wild type, and the in vitro phosphotransferase activity of the autophosphorylated IR delta 960 was normal. These results suggest that the deletion did not cause a nonspecific structural disruption of the catalytic domain of IR delta 960. In vivo autophosphorylation of the IR delta 960 receptor was reduced by 30% after 2 min of insulin stimulation and was similar to the wild-type receptor after 30 min of insulin stimulation. However, the mutant receptor was defective in insulin-stimulated tyrosyl phosphorylation of the endogenous substrate pp185. In addition, IR delta 960 was deficient in mediating insulin stimulation of glycogen and DNA synthesis. Thus, autophosphorylation of the insulin receptor is necessary but not sufficient for signal transmission. These data extend the hypothesis that the cytoplasmic juxtamembrane region of the insulin receptor is important for its interactions with ATP, intracellular substrates, and other proteins and is broadly necessary for biological signal transmission.     

WASH has a critical role in NK cell cytotoxicity through Lck-mediated phosphorylation

Natural killer (NK) cells are important effector cells of the innate immune system to kill certain virus-infected and transformed cells. Wiskott–Aldrich Syndrome protein (WASP) and SCAR homolog (WASH) has been identified as a member of WASP family proteins implicated in regulating the cytoskeletal reorganization, yet little is known about its function in lymphocytes. Here we demonstrate that WASH is crucial for NK cell cytotoxicity. WASH was found to colocalize with lytic granules upon NK cell activation. Knockdown of WASH expression substantially inhibited polarization and release of lytic granules to the immune synapse, resulting in the impairment of NK cell cytotoxicity. More importantly, our data also define a previously unappreciated mechanism for WASH function, in which Src family kinase Lck can interact with WASH and induce WASH phosphorylation. Mutation of tyrosine residue Y141, identified here as the major site of WASH phosphorylation, partially blocked WASH tyrosine phosphorylation and NK cell cytotoxicity. Taken together, these observations suggest that WASH has a pivotal role for regulation of NK cell cytotoxicity through Lck-mediated Y141 tyrosine phosphorylation.Natural killer (NK) cells are the first defense line against viral infections and tumors.¹ NK cell-mediated lysis of target cells requires the formation of immunological synapse between NK cells and target cells and subsequent delivery of lytic granules containing perforin and granzymes.^{2, 3} The importance of the actin cytoskeleton in this process has been well documented.⁴ However, the precise mechanism of actin reorganization in NK cells remains to be elucidated.Wiskott–Aldrich syndrome protein (WASP) is the first identified member of an actin regulator family.⁵ WASP family proteins contain a C-terminal domain that binds to and activates the Arp2/3 complex for cytoskeleton remodeling.⁶ In the absence of WASP, cytotoxic activity of NK cells is defective owing to impaired immune synapse formation and perforin localization.⁷ It has also been shown that WASP may be important for integration of NK cell signaling, particularly for nuclear translocation of NFAT2 and NF-κB during the activating receptor NKp46-dependent activation.⁸WASP and SCAR homolog (WASH) has been discovered as a new WASP family member.⁹ Subsequent studies show that WASH interacts with multiple proteins, including FAM21, to form a large core complex and regulate actin dynamics.¹⁰ WASH localizes to sorting and recycling endosomes, where WASH complex activates Arp2/3-mediated actin polymerization and controls the production of transport intermediates from endosome.¹¹ Unlike other WASP family members, WASH has distinct N-terminal domains, termed WASH homology domain 1 (WHD1) and tubulin-binding region (TBR).¹² Moreover, WASH has been shown to regulate recycling of many surface receptors via endosomal trafficking in activated T cells.¹³In our previous works, we found embryonic lethality and extensive autophagy in WASH-deficient mice. WASH recruits RNF2 to ubiquitinate AMBRA1 and inhibits the ubiquitination of Beclin1, a well-known moderator in autophage.^{14, 15} Of interest, WASH is located in cell nucleus and participates in hematopoietic stem cell differentiation through recruiting NURF complex to c-Myc promoter.¹⁶ However, the role and mechanism of WASH in NK cell function remain poorly understood.In this study, we show that inhibition of WASH expression with RNA interference or an inducible gene targeting system severely impair NK cell cytotoxicity through blockade of lytic granule polarization. In addition, Src family kinase Lck can interact with and induce tyrosine phosphorylation of WASH protein in human NK cells. These analyses provide the cellular and molecular mechanisms involved in the regulation of WASH function during NK cell activation.     

The canonical intrinsic mitochondrial death pathway has a non-apoptotic role in signaling lens cell differentiation

The mitochondrial cell death pathway is known for its role in signaling apoptosis. Here, we describe a novel function for the mitochondrial cell death pathway in signaling initiation of differentiation in the developing lens. Most remarkably, we induced lens cell differentiation by short-term exposure of lens epithelial cells to the apoptogen staurosporine. Activation of apoptosis-related pathways induced lens epithelial cells to express differentiation-specific markers and to undergo morphogenetic changes that led to formation of the lens-like structures known as lentoids. The fact that multiple stages of differentiation are expressed at a single stage of development in the embryonic lens made it possible to precisely determine the timing of expression of proteins associated with the apoptotic pathway. We discovered that there was high expression in the lens equatorial epithelium (the region of the lens in which differentiation is initiated) of pro-apoptotic molecules such as Bax and Bcl-x(S) and release of cytochrome c from mitochondria. Furthermore, we found significant caspase-3-like activity in the equatorial epithelium, yet this activity was far lower than that associated with lens cell apoptosis. These apoptotic pathways are likely regulated by the concurrent expression of prosurvival molecules, including Bcl-2 and Bcl-x(L); phosphorylation of Bad; and high expression of inhibitor of apoptosis proteins chicken IAP1, IAP3, and survivin. This finding suggests that prosurvival pathways allow pro-apoptotic molecules to function as molecular switches in the differentiation process without tipping the balance toward apoptosis. We call this process apoptosis-related Bcl-2- and caspase-dependent (ABC) differentiation.     

Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling

Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.     

A critical role for endocytosis in Wnt signaling

Background  

The Wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. Wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. While numerous Wnts, their cognate receptors of the Frizzled and Arrow/LRP5/6 families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation.     

The active site region plays a critical role in Na+ binding to thrombin

The catalytic activity of thrombin and other enzymes of the blood coagulation and complement cascades is enhanced significantly by binding of Na⁺ to a site >15 Å away from the catalytic residue S195, buried within the 180 and 220 loops that also contribute to the primary specificity of the enzyme. Rapid kinetics support a binding mechanism of conformational selection where the Na⁺-binding site is in equilibrium between open (N) and closed (N^∗) forms and the cation binds selectively to the N form. Allosteric transduction of this binding step produces enhanced catalytic activity. Molecular details on how Na⁺ gains access to this site and communicates allosterically with the active site remain poorly defined. In this study, we show that the rate of the N∗→N transition is strongly correlated with the analogous E∗→E transition that governs the interaction of synthetic and physiologic substrates with the active site. This correlation supports the active site as the likely point of entry for Na⁺ to its binding site. Mutagenesis and structural data rule out an alternative path through the pore defined by the 180 and 220 loops. We suggest that the active site communicates allosterically with the Na⁺ site through a network of H-bonded water molecules that embeds the primary specificity pocket. Perturbation of the mobility of S195 and its H-bonding capabilities alters interaction with this network and influences the kinetics of Na⁺ binding and allosteric transduction. These findings have general mechanistic relevance for Na⁺-activated proteases and allosteric enzymes.     

Mll has a critical role in fetal and adult hematopoietic stem cell self-renewal

The Mixed Lineage Leukemia (Mll) gene is a homolog of Drosophila Trithorax commonly rearranged in infant leukemia. Comprehensive analysis of the role of Mll in hematopoiesis in fetal and adult knockout mice has been prevented by the lethality of Mll(-/-) mice. We have established a conditional deletion model that allows us to study adult hematopoiesis in the absence of Mll. In this study, Mll(-/-) embryos survive to E16.5 and have reduced numbers of HSCs. The quiescent fraction of these HSCs is greatly reduced, and they are unable to compete with wild-type cells in transplantation assays. Mice with Mll expression conditionally deleted in the hematopoietic system have grossly normal hematopoiesis in bone marrow, thymus, and spleen. However, transplanted Mll-deficient bone marrow cells are highly compromised in their ability to competitively reconstitute irradiated recipients. These results suggest a critical role for Mll in regulating stem cell self-renewal.     

A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration

Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.