bluuebunny/crossref_metadata_2025 huggingface dataset:
A New E1 Activation System for Ubiquitin
Authors: Foley John F.
Published: September 2007

Ubiquitination is an important process in the regulation of protein function and degradation and in the control of cell signaling. A sequence of events results in the modification of a protein with ubiquitin or ubiquitin-like proteins (UBLs) such as Nedd8 or the small ubiquitin-like modifier protein SUMO. The E1 ubiquitin-activating enzyme initiates ubiquitination by modifying ubiquitin (or a UBL) (through adenylation and thioesterification reactions) and then transferring it to the E2-conjugating enzyme (a process known as charging). E2 functions together with the ubiquitin ligase E3 to transfer ubiquitin to the target protein. The current paradigm states that within each species, single E1 enzymes are responsible for charging E2s with UBLs. In humans, this E1 is known as Ube1. Jin
et al
. performed a screen of the human genome for genes encoding proteins that contain a characteristic motif found in E1s and identified a previously uncharacterized protein, Uba6. Uba6 has 40% identity with Ube1, and orthologs of Uba6 are found in vertebrates and in the sea urchin but not in other organisms such as fungi and plants. Western blot analysis showed that Uba6 was as widely expressed as Ube1 in most human tissues and cell types. In vitro experiments with recombinant Uba6 and glutathione fusion proteins of UBLs demonstrated that Uba6 had similar ubiquitin activation properties as Ube1 but did not activate other UBLs. Lysates of 293T cells transfected with epitope-tagged Uba6 were analyzed by Western blotting and mass spectrometry, and Uba6 bound to ubiquitin but not to other UBLs. The authors then screened a panel of recombinant E2-conjugating enzymes for their ability to be charged in vitro by either Ube1 or Uba6 and found one previously uncharacterized E2 (Use1) that was charged by Uba6 but not by Ube1. Western blot analysis of lysates of transfected HeLa cells (which were treated with Ube1- or Uba6-specific siRNAs) confirmed that Use1 was charged by Uba6 but not Ube1. Taken together, these data demonstrate the existence of a previously unknown Ube1-independent mechanism for ubiquitin activation and charging of E2-conjugating enzymes such as Use1. Given that Ube1 and Uba6 are coexpressed in many tissues (although Ube1 is more abundant that Uba6), these two enzymes may act in concert or in sequence to affect various signaling pathways.

J. Jin, X. Li, S. P. Gygi, J. W. Harper, Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.
Nature
447
, 1135-1138 (2007).
[PubMed]
Focus Issue: STNG—Signal Transduction, the Next Generation
Authors: Adler Elizabeth M. , Gough Nancy R.
Published: September 2007

In this Focus Issue of
Science
’s STKE, which complements the
Science
Special Issue on undergraduate education, we present three new Teaching Resources and inaugurate a new section, the STKE Journal Club.
Ceramide: From Embryos to Tumors
Authors: Savtchouk Iaroslav A. , Mattie Floyd J. , Ollis Anne A.
Published: September 2007

Ceramides are ubiquitous lipids that have important functions integral to apoptotic signaling. Several therapeutic agents currently exist that induce ceramide-dependent apoptosis in cancerous cells, and a number of enzymes involved in ceramide metabolism are beginning to be recognized as potential targets for cancer therapy. Recent research shows that evasion of ceramide-dependent apoptosis is essential at the earliest stages of embryonic development and is an important mechanism of multidrug resistance. Although ceramide-based strategies for treating cancer are promising, current data about ceramide-resistant tumors require further understanding of the role of ceramide in apoptosis.
Instructing Graduate Students to Tackle the "Real World": A Course Description
Authors: Patterson Randen L.
Published: September 2007

This Teaching Resource describes a course designed to expose graduate students to various hurdles they will encounter during their development as scientists. This course aims to train students in such elements of scientific life as working within a research group, presenting research, writing proposals, and publishing peer-reviewed manuscripts and to provide them with the tools necessary to accomplish these tasks. A critical element of this course is that it is designed to provide students with valuable "real world" experiences. A course description and a syllabus are provided.
Signaling Endosomes Point the Way
Authors: Gough Nancy R.
Published: September 2007

Brain-derived neurotrophic factor (BDNF) stimulates the migration of cerebellar granule cell precursors (GCPs) from the external granule layer to the internal granule layer. The movement of the GCPs is impaired in BDNF knockout mice. Zhou
et al
. isolated GCPs from postnatal day 6 (P6) mice and monitored their migration in culture in response to BDNF, applied uniformly or as a gradient. BDNF promoted the directional chemotaxis of the GCPs only when presented as a gradient. Although exogenously applied BDNF stimulated movement of GCPs from
bdnf
−/−
mice, these cells lacked directional migration even in response to a gradient of BDNF, which suggests that an autocrine function for BDNF is also important for proper chemotaxis. To understand how autocrine BDNF may contribute to directional movement, cultured GCPs were stimulated with a related neurotrophin NT4, which also stimulates the TrkB receptor, and BDNF was found to be secreted, which could amplify the BDNF gradient and thus contribute to directional migration. GCPs have two receptors for BDNF: TrkB and p75
NTR
. BDNF-induced chemotaxis (in cultured cells and organotypic slices) was blocked if the TrkB receptor activity was inhibited but was unaffected in cells defective for p75
NTR
signaling. In the cultured GCPs, TrkB receptors colocalized with early endosomal markers at the leading edge toward the BDNF gradient, suggesting internalization of the receptor. In the P6 mouse cerebellum, TrkB was found in punctate structures that often colocalized with an endosomal marker; however, only in the wild-type mouse brains was TrkB consistently oriented toward the internal granule layer. Pharmacological inhibition of endocytosis inhibited cultured GCP migration toward a BDNF gradient, and impairment of dynamin function, which is required for clathrin-mediated endocytosis, blocked movement of GCPs from the external granule layer in organotypic slices. BDNF-induced GCP migration involved phosphoinositide 3-kinase (PI3K) signaling and activation of the Rac-specific guanine-nucleotide exchange factor Tiam1 (based on pharmacological inhibition and siRNA knockdown experiments). Thus, BDNF-induced migration appears to involve both the formation of oriented signaling endosomes and endogenous production of BDNF, which may amplify the BDNF gradient to promote motility (see Kaplan and Miller for commentary).

P. Zhou, M. Porcionatto, M. Pilapil, Y. Chen, Y. Choi, K. F. Tolias, J. B. Bikoff, E. J. Hong, M. E. Greenberg, R. A. Segal, Polarized signaling endosomes coordinate BDNF-induced chemotaxis of cerebellar precursors.
Neuron
55
, 53-68 (2007).
[PubMed]

D. R. Kaplan, F. D. Miller, Developing with BDNF: A moving experience.
Neuron
55
, 1-2 (2007).
[PubMed]
Hedgehog Likes (to) Dally
Authors: Foley John F.
Published: September 2007

Hedgehog (Hh) is a morphogen, a secreted signaling protein important in pattern formation during embryonic development in
Drosophila
. The receptor for Hh is Patched (Ptc), a transmembrane protein that, in the absence of Hh binding, inhibits another membrane-bound protein known as Smoothened (Smo). This interaction allows an intracellular complex to trigger the cleavage of the protein Cubitus interruptus (Ci), thereby generating a transcriptional repressor. If Hh binds to Ptc, however, Smo is no longer inhibited and, instead of becoming cleaved, Ci translocates to the nucleus where it activates gene transcription. The
Drosophila
lipoprotein particle Lipophorin binds to Hh and is important for its long-range movement and signaling. Heparan sulfate proteoglycans (HSPGs) are also important for the trafficking of Hh, and the glypicans (lipid-linked HSPGs) Dally and Dally-like (Dlp) are necessary for normal Hh signaling. Eugster
et al
. investigated a possible relationship between Lipophorin and the glypicans in the Hh pathway. The authors expressed green fluorescent protein (GFP) fusions of Dally and Dlp in
Drosophila
imaginal disc cells and used differential centrifugation to show that both GFP:Dally and GFP:Dlp existed in both membrane-bound and released forms. Lipid fractionation followed by Western blotting analyses demonstrated that both GFP:Dally and GFP:Dlp associated with Lipophorin particles, and analysis of mutant forms of GFP:Dally showed that it interacted with Lipophorin through its HS domain. A mutant GFP:Dally that did not contain a glycosylphosphatidylinositol (GPI) anchor (GFP:DallyΔgpi) coimmunoprecipitated with Lipophorin. Immunohistochemical analysis showed that Lipophorin bound to GFP:Dally and GFP:Dlp in the dorsal compartment of the wing disc--an interaction that was dependent on HS. Quantitative immunostaining of Hh targets revealed that Hh signaling in
dally
mutant discs was reduced and that this was due to lower Ci activity in
dally
mutant discs than in wild-type discs. Expression of GFP:DallyΔgpi in
dally
mutant discs extended the range of Hh target production, essentially reversing the
dally
phenotype. Antibodies against GFP coimmunoprecipitated both Lipophorin and Hh when extracts from cells expressing either GFP:Dally or GFP:DallyΔgpi were analyzed. To confirm this association in vivo, the authors performed immunohistochemical analysis of wing imaginal discs of flies expressing GFP:DallyΔgpi and found that it colocalized with Lipohorin, Hh, and Ptc in the endosomes of targeted tissues. The authors suggest that Lipophorin particles pick up secreted Hh and released Dally from the apical lumen and are then internalized at the apical surfaces of receiving cells. They hypothesize that the presence of Dally in these particles enhances Hh signaling, possibly by the formation of a signaling complex with Ptc.

C. Eugster, D. Panáková, A. Mahmoud, S. Eaton, Lipoprotein-heparan sulfate interactions in the Hh pathway.
Dev. Cell
13
, 57-71 (2007).
[PubMed]
Treating Parkinson's the CDNF Way
Authors: Gough Nancy R.
Published: September 2007

Lindholm
et al
. report the cloning, molecular characterization, and tissue expression of a member of a new family of neurotrophins. Through sequence analysis, they identified a gene similar to that encoding mesencephalic-astrocyte-derived neurotrophic factor (MANF, also known as ARMET) and named the new protein conserved dopamine neurotrophic factor (CDNF, which in genomic databases was called ARMETL1 for ARMET-like 1). CDNF was secreted when heterologously expressed in human cultured cell lines or the insect cell line Sf9. Electrospray ionization-mass spectrometry analysis of the secreted protein indicated that the protein formed four disulfide bridges with the eight conserved cysteines. Transcript analysis in mice revealed that
CDNF
was expressed in several tissues during embryogenesis and postnatally. In the brain, in situ protein analysis showed that the protein was present in the somas of neurons. Although MANF was previously reported to promote survival of dopaminergic neurons in vitro, Lindholm
et al
. show that in a rat model of Parkinson's disease, injection of CDNF before or after exposure to the neurotoxin prevented or ameliorated symptoms and reduced the loss of dopaminergic neurons in the substantia nigra.

P. Lindholm, M. H. Voutilainen, J. Laurén, J. Peränen, V.-M. Leppänen, J.-O. Andressoo, M. Lindahl, S. Janhunen, N. Kalkkinen, T. Timmusk, R. K. Tuominen, M. Saarma, Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons
in vivo
.
Nature
448
, 73-77 (2007).
[PubMed]
Inflammation and Tumor Progression
Authors: Ray L. Bryan
Published: September 2007

Hepatocellular carcinoma, a common and deadly cancer of the liver, is 3 to 5 times more likely to occur in men than in women (see the Perspective by Lawrence
et al
.). Working in a mouse model in which liver cancer is induced by exposure to a chemical carcinogen, Naugler
et al
. propose a molecular basis for this phenomenon, explained by the action of the female hormone estrogen and its ability to inhibit inflammatory responses in the liver. Estrogen acts to inhibit secretion of interleukin-6 (IL-6) by liver macrophages known as Kupffer cells. Production of IL-6 was dependent on the signaling adaptor protein MyD88, which in turn may be activated by products of dying cells in the injured liver. Rakoff-Nahoum and Medzhitov implicate MyD88 in promoting another cancer, that of the intestine. Inflammation is known to be a risk factor for colorectal tumors. In a mouse model of intestinal tumorigenesis, mice lacking MyD88 showed inhibited growth and progression of tumors.

W. E. Naugler, T. Sakurai, S. Kim, S. Maeda, K. Kim, A. M. Elsharkawy, M. Karin, Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production.
Science
317
, 121-124 (2007).
[Abstract]
[Full Text]

T. Lawrence, T. Hagemann, F. Balkwill, Sex, cytokines, and cancer.
Science
317
, 51-52 (2007).
[Summary]
[Full Text]

S. Rakoff-Nahoum, R. Medzhitov, Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88.
Science
317
, 124-127 (2007).
[Abstract]
[Full Text]
Does Erythropoietin Have a Dark Side? Epo Signaling and Cancer Cells
Authors: Sytkowski Arthur J.
Published: September 2007

Erythropoietin (Epo) stimulates red blood cell production by docking with its cognate receptor on the erythroid progenitor cell and triggering an array of signaling pathways that inhibit apoptosis and promote cell proliferation and differentiation. In its pharmaceutical forms, epoetin and darbepoetin, Epo is widely used to treat various anemias, including those associated with cancer. The Epo receptor is also expressed by nonhematopoietic cells, including cancer cells, and Epo exhibits a "tissue-protective" effect on nonhematopoietic tissues, possibly mediated through a novel heteroreceptor, blocking apoptosis induced by a variety of insults. The unexpected results of several clinical studies in which Epo was used to treat cancer patients have now raised the question of a potential direct growth-promoting action of Epo on cancer cells.
From Protein Interactions to Complex Physiology
Authors: Ray L. Bryan
Published: September 2007

Docking or adaptor proteins may provide the building blocks from which complex cellular biological regulatory systems could be built during the course of evolution. Hardy
et al
. examined the docking protein ShcA, which has a phosphotyrosine binding (PTB) domain and a Src homology 2 (SH2) domain, each of which can bind to phosphorylated receptors and other proteins. Shc proteins also contain tyrosine residues that become phosphorylated and serve as binding sites for the adaptor protein Grb2 (growth factor receptor-bound protein 2). In mice that expressed mutant proteins with one or more domains altered, the PTB domain was essential for development of the heart, but the SH2 domain and Grb2-binding domains were not. However, successful development of skeletal muscle required that the PTB, SH2, and Grb2-binding domains be present within the same Shc molecule. Thus, acquisition of new protein-protein interaction motifs within proteins and their distinct use in various cell types or organisms may support complex physiological functions.

W. R. Hardy, L. Li, Z. Wang, J. Sedy, J. Fawcett, E. Frank, J. Kucera, T. Pawson, Combinatorial ShcA docking interactions support diversity in tissue morphogenesis.
Science
317
, 251-256 (2007).
[Abstract]
[Full Text]
Balancing Act
Authors: Simpson Stephen
Published: September 2007

In the immune system, T cells are divided into various subsets according to their functions, both as mediators of protective immunity and as regulators of other immune cells. Mucida
et al
. now show that the vitamin A metabolite retinoic acid influences the balance between a major inflammatory and a regulatory phenotype of T cells. Under conditions that would normally favor the development of a recently described inflammatory T helper 17 cell, retinoic acid steered differentiation toward the regulatory phenotype. This ability to tip the balance between two opposing types of T cell response adds to the immune effects being attributed to retinoic acid.

D. Mucida, Y. Park, G. Kim, O. Turovskaya, I. Scott, M. Kronenberg, H. Cheroutre, Reciprocal T
H
17 and regulatory T cell differentiation mediated by retinoic acid.
Science
317
, 256-260 (2007).
[Abstract]
[Full Text]
A Shocking Use for CD6?
Authors: Adler Elizabeth M.
Published: September 2007

CD6, which exists mainly as a transmembrane lymphocyte receptor but has also been detected in a soluble form in serum, is a member of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Noting that some SRCR-SF proteins interact with conserved pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), Sarrias
et al
. incubated a biotin-labeled recombinant protein comprising the extracellular domain of human CD6 (rsCD6) with bacterial suspensions and assessed binding with Western analysis. rsCD6 bound to both
Escherichia coli
and
Staphyloccus aureus
in a calcium-dependent manner; binding to
E. coli
was inhibited by LPS from
E. coli
, whereas rsCD6 binding to
S. aureus
was inhibited by LTA from
S. aureus
. Natural soluble CD6 (nsCD6) purified from human serum was indistinguishable from rsCD6 in terms of its molecular mass (smaller than membrane-associated CD6) and recognition by polyclonal antibodies. Both nsCD6 and rsCD6 bound LPS with an affinity similar to that of CD14 (known for its role in detecting LPS). Less LPS bound to a CD6-deficient line of Jurkat-derived cells (2G5 cells) than bound a daughter line (2G5-CD6.wt) stably transfected with membrane-associated CD6. Phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in response to LPS was enhanced in 2G5-CD6.wt cells compared with 2G5 cells. Furthermore, LPS elicited ERK1/2 phosphorylation in COS-7 cells transfected with CD6 but not a form with a truncated cytoplasmic tail. Intriguingly, administration of rsCD6 to mice before LPS exposure protected against septic shock, an effect associated with decreased concentrations of proinflammatory cytokines in serum. Thus, the authors conclude that CD6 binds PAMPs and that the soluble form may prove useful in the therapy of septic shock.

M.-R. Sarrias, M. Farnós, R. Mota, F. Sánchez-Barbero, A. Ibáñez, I. Gimferrer, J. Vera, R. Fenutría, C. Casals, J. Yélamos, F. Lozano, CD6 binds to pathogen-associated molecular patterns and protects from LPS-induced septic shock.
Proc. Natl. Acad. Sci. U.S.A.
104
, 11724-11729 (2007).
[Abstract]
[Full Text]
Internal P2X Receptors
Authors: Gough Nancy R.
Published: September 2007

Fountain
et al
. report the identification and characterization of an ATP receptor in
Dictyostelium discoideum
that is homologous to vertebrate P2X receptors. When heterologously expressed in the human embryonic kidney (HEK) cell line, the protein was localized to the plasma membrane, was activated by ATP or ATP analogs, was a cation channel permeable to calcium and smaller cations, and appeared to form a trimer. Furthermore, mutagenesis studies showed that certain residues critical for ATP binding and channel activity in the vertebrate P2X receptors were conserved in the DdP2X. One key difference between the known vertebrate receptors and the DdP2X receptors was that in vivo, the DdP2X receptor was localized to the membrane of the contractile vacuole instead of the plasma membrane. Thus far, all vertebrate P2X receptors have been reported to act at the plasma membrane. Pharmacological blockade or genetic knockout of DdP2X function produced
Dictyostelium
that could not osmoregulate properly under hypotonic conditions. They swelled and, although filling of the contractile vacuole was normal, contraction and emptying of the vacuole, which is critical for osmoregulation, was impaired.

S. J. Fountain, K. Parkinson, M. T. Young, L. Cao, C. R. L. Thompson, R. A. North, An intracellular P2X receptor required for osmoregulation in
Dictyostelium discoideum
.
Nature
448
, 200-203 (2007).
[PubMed]
Crystallized Kinase Regulation
Authors: Vinson Valda
Published: September 2007

Many human cancers involve gain-of-function mutations in the phosphoinositide 3-kinase PI3Kα. The kinase is a heterodimer of a catalytic subunit (p110α) and a regulatory subunit (p85α), with both subunits comprising multiple domains. Miled
et al
. (see the Perspective by Lee
et al
.) have determined the crystal structure of the adaptor binding domain of p110α bound to the inter-SH2 domain of p85α at 2.4 angstrom resolution and have performed functional studies to investigate the effect of oncogenic mutations in the helical domain of p110α on its interaction with the N-terminal SH2 domain of p85α. The studies suggest how these two classes of mutations cause the up-regulation of PI3Kα that can lead to cancer.

N. Miled, Y. Yan, W.-C. Hon, O. Perisic, M. Zvelebil, Y. Inbar, D. Schneidman-Duhovny, H. J. Wolfson, J. M. Backer, R. L. Williams, Mechanism of two classes of cancer mutations in the phosphoinositide 3-kinase catalytic subunit.
Science
317
, 239-242 (2007).
[Abstract]
[Full Text]

J. Y. Lee, J. A. Engelman, L. C. Cantley, PI3K charges ahead.
Science
317
, 206-207 (2007).
[Summary]
[Full Text]
Chemotaxis: Navigating by Multiple Signaling Pathways
Authors: Van Haastert Peter J. M. , Veltman Douwe M.
Published: September 2007

During chemotaxis, phosphatidylinositol 3,4,5-trisphosphate (PIP
3
) accumulates at the leading edge of a eukaryotic cell, where it induces the formation of pseudopodia. PIP
3
has been suggested to be the compass of cells navigating in gradients of signaling molecules. Recent observations suggest that chemotaxis is more complex than previously anticipated. Complete inhibition of all PIP
3
signaling has little effect, and alternative pathways have been identified. In addition, selective pseudopod growth and retraction are more important in directing cell movement than is the place where new pseudopodia are formed.
Dispersing Clusters with Calpain
Authors: Gough Nancy R.
Published: September 2007

A reciprocal relationship exists between agrin and acetylcholine (ACh) in controlling acetylcholine receptor (AChR) clustering at the neuromuscular junction: Agrin promotes clustering, and ACh disperses clusters. Chen
et al
. provide insight into how these opposing signals may allow stabilization of AChR at the neuromuscular junction by differential activity of the calcium-activated proteases calpains. Exposure of C2C12 myotubes in culture to the cholinergic agonist carbachol triggered a calcium transient, activation of calpain, and stimulation of cyclin-dependent kinase 5 (CDK5) through production of the activator p25 by cleavage of the CDK5 regulatory partner p35. (CDK5 has been previously implicated in AChR dispersal, and p35 is a known substrate for calpain.) Activation of CDK5 by carbachol was blocked by pharmacological inhibition of calpain with calpeptin, and the dispersal of AChR clusters induced by agrin was also prevented if calpain activity was blocked (pharmacologically or by RNAi). In vivo, pharmacologic inhibition of calpain (by injection of pregnant mice) in mice deficient for agrin rescued the formation of AChR clusters, although not all clusters were apposed to the nerve terminals. In transgenic mice overexpressing a calpain-inhibitory peptide calpastatin in skeletal muscle, p25 abundance was lower in the skeletal muscles from the transgenic mice, and AChR clustering was increased compared with control mice. A yeast two-hybrid screen revealed that rapsyn interacted with the large subunit of m-calpain, and mutagenesis analysis identified that the two proteins interacted through the tetratricopeptide repeat (TPR) domain of rapsyn and the regulatory domain (domain III) of calpain. Rapsyn is a protein that interacts with the AChR and is necessary for cluster stabilization. Calpain activity in vitro or in transfected cells was inhibited by rapsyn, and RNAi experiments to knock down rapsyn in C2C12 myotubes showed that p25 was more abundant in control and carbachol-treated cells. Rapsyn, calpain, and the AChR coimmunoprecipitated from C2C12 cells, and agrin stimulated this interaction. Analysis of the synaptic and nonsynaptic regions of diaphragm suggested that m-calpain was relatively evenly distributed between these two regions, but the activity of calpain was substantially less in the synaptic region (based on the abundance of p25). Thus, the authors propose that agrin promotes the interaction of calpain with the inhibitor rapsyn at the synapse, allowing AChR to cluster, and in nonsynaptic regions, calpain is activated in response to the calcium signals induced by ACh and disperses nonsynaptic ACh clusters.

F. Chen, L. Qian, Z.-H. Yang, Y. Huang, S. T. Ngo, N.-J. Ruan, J. Wang, C. Schneider, P. G. Noakes, Y.-Q. Ding, L. Mei, Z.-G. Luo, Rapsyn interaction with calpain stabilizes AChR clusters at the neuromuscular junction.
Neuron
55
, 247-260 (2007).
[PubMed]
Hearing New Things About Calcium
Authors: Adler Elizabeth M.
Published: September 2007

Our ability to sense gravity--and our sense of balance--depends on otoconia, particles in the inner ear that consist of a proteinaceous core coated with CaCo
3
crystals. Otoconial development depends on otopetrin 1 (Otop1), a member of a protein family defined by a highly conserved multitransmembrane domain of unknown function. Its predicted structure, together with its extracellular location, suggested to Hughes
et al
. that Otop1 was likely associated with extruded membrane vesicles (globular substance vesicles) that were previously shown to respond to adenosine triphosphate (ATP) with an increase in intravesicular Ca
2+
, leading them to investigate its effects on the Ca
2+
response to purinergic stimulation. When overexpressed in COS7 cells, Otop1 abolished an early peak in intracellular Ca
2+
concentration elicited in wild-type cells by extracellular ATP or uridine triphosphate (UTP). ATP and UTP both activate P2Y purinergic receptors, which signal through the Gα
q
family of heterotrimeric guanine nucleotide-binding proteins. Signaling through other Gα
q­
-coupled receptors was maintained in the presence of Otop1, suggesting that an Otop1-mediated decrease in endoplasmic reticulum Ca
2+
stores (determined in experiments with thapsigargin) was not sufficient to abolish all rapid Gα
q
-dependent Ca
2+
signals and that P2Y inhibition occurred upstream of Gα
q
. ATP also elicited a slower sustained phase of increased intracellular Ca
2+
(Ca
2+
plateau). Cells overexpressing Otop1 exhibited a Ca
2+
plateau even after treatment with thapsigargin; this plateau was abolished by chelating extracellular Ca
2+
(unlike the P2Y-mediated rapid Ca
2+
peak in wild-type cells). Moreover, overexpression of Otop1 in rat osteosarcoma cells that do not independently respond to purinergic stimuli enabled an ATP-dependent increase in intracellular Ca
2+
. The effects of Otop1 on the Ca
2+
response to ATP were reversibly inhibited by suramin (previously shown to inhibit the ATP-dependent Ca
2+
response of globular substance vesicles). The authors conclude that Otop1 has marked effects on the Ca
2+
response to ATP that could be critical to its role in otoconia formation.

I. Hughes, M. Saito, P. H. Schlesinger, D. M. Ornitz, Otopetrin 1 activation by purinergic nucleotides regulates intracellular calcium.
Proc. Natl. Acad. Sci. U.S.A.
104
, 12023-12028 (2007).
[Abstract]
[Full Text]
Slowing Senescence with Wnt
Authors: Ray L. Bryan
Published: September 2007

The Wnt signaling pathway has key roles in development and generally promotes proliferation of stem cells and inhibits apoptosis. These effects are essentially opposite to the changes that occur in senescent stem cells. Thus, Ye
et al
. examined whether reduced Wnt signaling might have a role inhibitory in senescence. They monitored the formation of specialized domains of heterochromatin known as senescence-associated heterochromatin foci or SAHF, which are thought to repress transcription of genes that promote proliferation. In human WI38 fibroblasts, expression of
Wnt2
mRNA was decreased as cells approached senescence. Formation of SAHF was inhibited when pharmacological inhibitors were used to decrease activity of glycogen synthase kinase 3β (a kinase activated downstream of Wnt). Furthermore, small hybrid RNAs were used to decrease expression of Wnt2 in young fibroblasts, and this promoted formation of SAHF, the authors’ marker of senescence. Accordingly, exposure of cells to a Wnt ligand delayed formation of SAHF and the onset of senescence. The authors note that their findings implicate Wnt as the second extracellular ligand proposed to regulate senescence, along with plasminogen activator inhibitor-1, which appears to promote senescence.

X. Ye, B. Zerlanko, A. Kennedy, G. Banumathy, R. Zhang, P. D. Adams, Downregulation of Wnt signaling is a trigger for formation of facultative heterochromatin and onset of cell senescence in primary human cells.
Mol. Cell
27
, 183-196 (2007).
[PubMed]
Longevity on the Brain
Authors: Purnell Beverly A.
Published: September 2007

Several studies show that loss-of-function mutations in the insulin-like signaling cascade extend the life span of worms and flies; however, equivalent mutations are associated with metabolic disease and fatal diabetes in mice. In contrast, calorie restriction or genetic strategies in mice that enhance insulin sensitivity lower the risk of age-related disease and extend life span. Taguchi
et al
. resolve these conflicting results by pointing to the brain as the site where reduced insulin-like signaling can extend mouse life span.

A. Taguchi, L. M. Wartschow, M. F. White, Brain IRS2 signaling coordinates life span and nutrient homeostasis.
Science
317
, 369-372 (2007).
[Abstract]
[Full Text]
To Cluster or Not to Cluster
Authors: Vinson Valda J.
Published: September 2007

The PDZ domain is a frequently occurring interaction domain in eukaryotic proteins that binds to the C termini of target proteins. PDZ domains have been thought to cluster into functional classes, each with distinct sequence binding preferences. Using a combination of experimental data and modeling, Stiffler
et al
. predict PDZ domain-peptide interactions across the mouse proteome. Instead of observing clustering, they find that PDZ domains are evenly distributed throughout selectivity space likely to minimize cross-reactivity. The study highlights focusing on families of interaction domains as a productive approach to gain insight into protein function.

M. A. Stiffler, J. R. Chen, V. P. Grantcharova, Y. Lei, D. Fuchs, J. E. Allen, L. A. Zaslavskaia, G. MacBeath, PDZ domain binding selectivity is optimized across the mouse proteome.
Science
317
, 364-369 (2007).
[Abstract]
[Full Text]
What’s the Buzz
Authors: Zahn Laura M.
Published: September 2007

The residents of bee hives are well known to be closely related, but hives can often exhibit more genetic diversity than might be anticipated from theories on the benefits of cooperation among closely related individuals. Mattila and Seeley show that one reason for this is that more genetically diverse hives (those originating from a female mating with multiple males) perform better in the rate of comb building, foraging rates, and honey production than those originating from a single female and male. To advertise her presence in the colony and to exert influence over its members, a honey bee queen produces a complex blend of substances known as queen mandibular pheromone. Vergoz
et al
. (see the Perspective by Galizia) found that exposure to queen pheromone leads to a reduction in aversive learning but not to a reduction in appetitive learning in young honey bees. The queen substance modulates the dopaminergic system of bees, which reduces the capacity of young workers to form aversive memories.

H. R. Mattila, T. D. Seeley, Genetic diversity in honey bee colonies enhances productivity and fitness.
Science
317
, 362-364 (2007).
[Abstract]
[Full Text]

V. Vergoz, H. A. Schreurs, A. R. Mercer, Queen pheromone blocks aversive learning in young worker bees.
Science
317
, 384-386 (2007).
[Abstract]
[Full Text]

C. G. Galizia, Brainwashing, honeybee style.
Science
317
, 326-327 (2007)
[Summary]
[Full Text]
Sensing Cytosolic DNA
Authors: Gough Nancy R.
Published: September 2007

How do cells sense cytosolic viral DNA when infected and trigger a response that helps the body clear these cells? Takaoka
et al
. provide insight into this question by characterizing a protein previously named DLM-1 or ZPB1, which they rename DAI for their discovery that it appears to function as a DNA-dependent activator of interferon (IFN) regulatory factors. Expression of the gene encoding DAI is stimulated by IFN, which is a common positive feedback mechanism for participants of the IFN pathway. Thus, Takaoka
et al
. showed that in cells overexpressing DAI, there were enhanced responses (increased expression of
IFN
genes) to synthetic DNA [poly(dA-dT)•poly(dT-dA)], bacterial DNA, viral DNA, or mammalian DNA, but not to synthetic RNA. Analysis of the response in mouse embryo fibroblasts deficient in various interferon regulatory factors (IRFs) suggested that IRF3 and IRF7 were important for
IFN
gene induction in response to poly(dA-dT)•poly(dT-dA). When DAI was knocked down by RNAi methods in mouse fibroblasts, increased expression of IFN genes, formation of an IRF3 dimer, and activation of the transcription factor NF-κB in response to DNA was reduced. In addition, the response to infection with a DNA virus was impaired, but not that to an RNA virus. DAI directly interacted with poly(dA-dT)•poly(dT-dA) in the cytosol based on fluorescence resonance energy transfer (FRET) experiments in cells containing labeled DAI and labeled synthetic DNA and based on an assay in which DAI pulled down poly(dA-dT)•poly(dT-dA). Coimmunoprecipitation experiments with transfected cells suggested that DNA stimulated the formation of a complex containing DAI, IRF3, and the kinase TBK1. Thus, DAI may be another weapon in the cell's arsenal against viral infection (see commentary by Chi and Flavell).

A. Takaoka, Z. Wang, M. K. Choi, H. Yanai, H. Negishi, T. Ban, Y. Lu, M. Miyagishi, T. Kodama, K. Honda, Y. Ohba, T. Taniguchi, DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response.
Nature
448
, 501-505 (2007).
[PubMed]

H. Chi, R. A. Flavell, Sensing the enemy within.
Nature
448
, 423-424 (2007).
[PubMed]
Focus Issue: Uncovering Immunological Secrets
Authors: Foley John F. , Adler Elizabeth M.
Published: September 2007

This Focus Issue of
Science
’s STKE complements a Special Issue of
Science
, which features a collection of Perspectives that describe obstacles to attaining a more complete understanding of the immune system. Taking stock of where the field now stands, the authors identify areas that warrant further investigation and speculate on the likely clinical benefits of achieving a deeper understanding of immune function. Articles in
Science
’s STKE highlight molecular challenges that immunologists have overcome, new questions that have arisen from their studies, and cutting-edge techniques that will enable immunologists to tackle these problems.
Diacylglycerol Kinases Put the Brakes on Immune Function
Authors: Wattenberg Binks W. , Raben Daniel M.
Published: September 2007

Diacylglycerol kinases (DGKs) are emerging as key negative regulators of immune function, particularly in T cells. DGKs consume diacylglycerol to produce phosphatidic acid. Because both diacylglycerol and phosphatidic acid are important activators of signaling molecules, DGKs have the potential to modulate a number of signaling pathways, and this certainly seems to be the case in T cell function. Studies of T cell signaling demonstrate that DGKs inhibit T cell receptor signaling and thus may serve an important role in limiting the immune response. Other studies have examined the molecular basis of anergy, a state of T cell unresponsiveness that is an important postdevelopmental control over the immune response to self antigens. Two groups have suggested that DGK activity lies at the heart of the anergic phenotype. In addition, DGK activity may limit the response of macrophages and dendritic cells to intracellular pathogens. An overall picture is emerging in which the capacity of DGKs to modulate membrane signaling lipids is used to keep a tight rein on immune responses.
Toxic Mother's Milk
Authors: Gough Nancy R.
Published: September 2007

Wan
et al
. created tissue-specific knockout mice deficient in peroxisome proliferator-activated receptor γ (PPARγ), which is a transcription factor regulated by fatty acids, some prostaglandins, and components of oxidized low-density lipoprotein particles and which regulates lipid biosynthesis. The mice were deficient in PPARγ in the hematopoietic and endothelial tissues. Pups nursed from mothers deficient in PPARγ exhibited growth retardation and hair loss that was completely reversed after the pups were weaned. Furthermore, pups deficient in PPARγ fostered by a wild-type mother did not exhibit these abnormalities, and wild-type pups fostered with the PPARγ-deficient mothers exhibited hair loss and low weight until weaning, confirming that the problem was maternal. Investigation of the skin of the pups nursed by the PPARγ-deficient mothers showed the formation of follicular cysts, which appeared to result from an inflammatory response that was characterized by leukocyte infiltration, elevated expression, and abundance of inflammatory cytokines and cyclooxygenase (COX-1 and COX-2). Hair loss was partially prevented if the pups were treated with aspirin, a COX inhibitor. Inflammation was also noted in the livers of the pups nursed by the PPARγ-deficient mothers. Histological analysis of the mammary tissue of the PPARγ-deficient mice showed increased lipid accumulation. Transcription analysis revealed that the PPARγ-deficient mammary glands exhibited decreased expression of homeobox genes and matrix metalloproteinases involved in mammary gland development. The expression of genes encoding two lipid oxidation enzymes that are involved in the production of inflammatory lipids was elevated in the mutant mice. When a mouse macrophage cell line was exposed to milk from the PPARγ-deficient mice in the presence of lipoprotein lipase, which is involved in lipid digestion, the cells increased production of inflammatory mediators. Furthermore, the skin from the mouse pups fed by the PPARγ-deficient mice exhibited an altered lipid profile compared with pups fed by wild-type mice, and mass analysis suggested that one group of lipids present in the skin of pups fed by the PPARγ-deficient mice was oxidized free fatty acids. Investigation of the gene expression of the macrophages from the mutant mice, which do not express PPARγ in the macrophages but do in the epithelial cells and adipocytes in the mammary gland, revealed that the macrophages had elevated expression of genes encoding enzymes that produce PPAΡγ ligands. The authors propose that these ligands activated PPARγ in the epithelial and adipocyte tissue of the mammary gland, leading to excessive production of inflammatory lipids.

Y. Wan, A. Saghatelian, L.-W. Chong, C.-L. Zhang, B. F. Cravatt, R. M. Evans, Maternal PPARγ protects nursing neonates by suppressing the production of inflammatory milk.
Genes Dev.
21
, 1895-1908 (2007).
[Abstract]
[Full Text]
Switching on the Ras Signal
Authors: Adler Elizabeth M.
Published: September 2007

Ras activation leads to the formation of specialized microdomains--called nanoclusters--tethered to the inner leaflet of the plasma membrane; indeed, the formation of these transient nanoclusters appears to be critical to activation of the Ras-Raf-MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase)-ERK (extracellular signal-regulated kinase) signaling pathway [the mitogen-activated protein kinase (MAPK) pathway]. Tian
et al
. combined immunogold electron microscopic analysis of the plasma membrane distribution of labeled forms of Raf1 and constitutively active K-RasG12V with fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) analysis to show that Raf was specifically recruited to Ras nanoclusters, rather than to freely diffusing Ras monomers. Moreover, experiments with a constitutively active Raf mutant that could not undergo Ras-dependent recruitment to the plasma membrane indicated that Raf-dependent activation of MEK failed to occur in the cytosol and thus occurred at Ras nanoclusters as well. The authors incorporated this information into a spatiotemporal model of epidermal growth factor (EGF) activation of MAPK signaling. In silico analysis combined with experimental results led them to the conclusion that, whereas Ras nanocluster formation was a linear function of EGF concentration, each nanocluster functioned as an on-off "nanoswitch" in activating MAPK signaling. In turn, the existence of these nanoswitches enabled cellular ERK activation to faithfully track EGF input. Thus, the authors conclude that Ras signaling depends on the existence of nanoclusters and that organization of signaling molecules into microdomains may be required for high-fidelity signal transduction. Kenworthy discusses the implications.

T. Tian, A. Harding, K. Inder, S. Plowman, R. G. Parton, J. F. Hancock, Plasma membrane nanoswitches generate high-fidelity Ras signal transduction.
Nat. Cell Biol.
9
, 905-914 (2007).
[PubMed]

A. K. Kenworthy, Nanoclusters digitize Ras signalling.
Nat. Cell Biol.
9
, 875-877 (2007).
[PubMed]
Matchmaking in Plants
Authors: Hines Pamela J.
Published: September 2007

In plants, as in animals, fertilization requires male and female gametes selectively and exclusively to meet and fuse. In plants, unlike in animals, it is not the actual gametes that participate in this dance, but rather the gametophytes, which carry immotile gametes. Escobar-Restrepo
et al
. (see the Perspective by McCormick) have now identified one of the molecules by which the female gametophyte (the embryo sac) recognizes the male gametophyte (the pollen). A receptor kinase, FER, is situated at cell surfaces within the female gametophyte. As the pollen tube approaches, a signaling cascade is initiated in the embryo sac that closes the door to late-arriving pollen tubes and halts further growth of the successful pollen tube, which then releases the male gamete. A good match between FER and its presumed ligand from the pollen tube appears to form the basis of reproductive compatibility.

J.-M. Escobar-Restrepo, N. Huck, S. Kessler, V. Gagliardini, J. Gheyselinck, W.-C. Yang, U. Grossniklaus, The FERONIA receptor-like kinase mediates male-female interactions during pollen tube reception.
Science
317
, 656-660 (2007).
[Abstract]
[Full Text]

S. McCormick, Reproductive dialog.
Science
317
, 606-607 (2007).
[Summary]
[Full Text]
Scratch and See
Authors: Foley John F.
Published: September 2007

Scratching monolayers of tissue culture cells and observing the changes that occur in those cells closest to the scratch have provided important insights into how cells polarize and coordinate their movement to repair the damage. The area of the cell closest to the scratch (essentially the new front of the cell) becomes the site of actin-rich protrusions, whereas rearrangements of the microtubule cytoskeleton push the centrosome and Golgi toward the front of the cell and push the nucleus toward the back. This polarization of the cell is dependent on the activity of the Rho family guanosine triphosphatase, Cdc42, which also leads to the assembly of a complex between the scaffold protein Par6 and atypical protein kinase C (aPKC). This complex is necessary for the accumulation of adenomatous polyposis coli (APC), a tumor suppressor protein involved in Wnt signaling. Inhibition of the activity of glycogen synthase kinase (GSK)-3β by PKC phosphorylation is presumed to allow the accumulation of APC at the leading edge. However, experiments by Schlessinger
et al
. suggest that the interaction between GSK-3β and aPKC is not the relevant one. They generated double knockin mice (GSK-3
SA
) in which the relevant Ser residues of both GSK-3α and GSK-3β were mutated to Ala residues, thus preventing their inhibition by phosphorylation. Unexpectedly, movement of the Golgi and centrosome in response to monolayer scratching of fibroblasts from GSK-3
SA
mice was normal. Dishevelled (Dvl) is another component in the Wnt pathway, and Wnt stimulates the binding of Dvl to the scaffold protein axin, which results in the release of GSK-3β from a complex containing axin, APC, and β-catenin. Knockdown of Dvl or axin with siRNA in rat embryo fibroblasts inhibited reorientation of both the centrosome and Golgi, as did treatment of scratched monolayers with a Wnt-binding protein, demonstrating the importance of Wnt signaling in cell polarization. Inhibition of Wnt5a, but not Wnt1 or Wnt3a, by siRNA blocked reorientation of the fibroblasts in scratched monolayers and also inhibited the accumulation of APC to microtubules at the leading edge, as demonstrated by confocal microscopy. Western blotting demonstrated that scratching of the monolayer resulted in the phosphorylation of Dvl proteins that was dependent on Wnt signaling. Finally, coimmunoprecipitation studies showed that aPKC and Dvl2 formed a complex after scratching of monolayers, which was dependent on both Wnt and Cdc42, as demonstrated by the use of Wnt-specific siRNA and an inhibitor of Cdc42. Thus, Cdc42-dependent Wnt signaling appears to play a role in cell polarization.

K. Schlessinger, E. J. McManus, A. Hall, Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity.
J. Cell Biol.
178
, 355-361 (2007).
[Abstract]
[Full Text]
TGF-β Signaling in Development
Authors: Kitisin Krit , Saha Tapas , Blake Tiffany , Golestaneh Nady , Deng Merlyn , Kim Christine , Tang Yi , Shetty Kirti , Mishra Bibhuti , Mishra Lopa
Published: September 2007

The transforming growth factor–β (TGF-β) superfamily comprises nearly 30 growth and differentiation factors that include TGF-βs, activins, inhibins, and bone morphogenetic proteins (BMPs). Multiple members of the TGF-β superfamily serve key roles in stem cell fate commitment. The various members of the family can exhibit disparate roles in regulating the biology of embryonic stem (ES) cells and tumor suppression. For example, TGF-β inhibits proliferation of multipotent hematopoietic progenitors, promotes lineage commitment of neural precursors, and suppresses epithelial tumors. BMPs block neural differentiation of mouse and human ES cells, contribute to self-renewal of mouse ES cells, and also suppress tumorigenesis. ES cells and tumors may be exposed to multiple TGF-β members, and it is likely that the combination of growth factors and cross-talk among the intracellular signaling pathways is what precisely defines stem cell fate commitment. This Connections Map Pathway in the Database of Cell Signaling integrates signaling not only from TGF-β and BMP but also from the ligands nodal and activin, and describes the role of the signaling pathways activated by these ligands in mammalian development. Much of the evidence for the connections shown comes from studies on mouse and human ES cells or mouse knockouts. This pathway is important for understanding not only stem cell biology, but also the molecular effectors of TGF-β and BMP signaling that may contribute to cancer suppression or progression and thus are potential targets for therapeutic intervention.