bluuebunny/crossref_metadata_2025 huggingface dataset:
The M
3
-Muscarinic Receptor Is Phosphorylated and Regulated by Casein Kinase 2
Authors: Foley John F.
Published: July 2007

After binding to ligands, almost all G protein-coupled receptors (GPCRs) are rapidly phosphorylated--a modification with important functional consequences, such as desensitization and internalization. Many of the kinases involved in the phosphorylation of GPCRs are members of the family of GPCR kinases (GRKs). GPCRs are often phosphorylated at multiple sites and by more than one kinase, so the regulation of GPCRs by phosphorylation is quite complex. Torrecilla
et al
. have extended their previous studies by examining the role of casein kinase 2 (CK2) in the phosphorylation and regulation of the M
3
-muscarinic receptor. Both knockdown of CK2 with siRNA and blocking of its activity with specific inhibitors reduced the amount of phosphorylation of the M
3
-muscarinic receptor in CHO cells, as measured by immunoprecipitation assays. The use of mutant M
3
-muscarinic receptors showed that the sites phosphorylated by CK2 were in the third intracellular loop. This region is also targeted by GRK2 and GRK6, although further analysis showed that the specific sites phosphorylated by CK2 and GRK2 or -6 did not significantly overlap. Inhibitors of CK2 reduced the phosphorylation of the M
3
-muscarinic receptor in mouse cerebellar granule (CG) neurons. GRK-mediated phosphorylation causes internalization of the M
3
-muscarinic receptor, but CK2-mediated phosphorylation had no such effect. Whereas knockdown of CK2 did not change the ability of the M
3
-muscarinic receptor to activate extracellular signal-regulated kinase 1 and 2 (ERK1/2), activation of the c-Jun pathway was increased. Analysis of phosphopeptides from digested M
3
-muscarinic receptors from either CHO cells or CG neurons revealed different patterns of phosphorylation, implying that the phosphorylation of a given receptor is cell type-specific, potentially leading to different functional outcomes.

I. Torrecilla, E. J. Spragg, B. Poulin, P. J. McWilliams, S. C. Mistry, A. Blaukat, A. B. Tobin, Phosphorylation and regulation of a G protein-coupled receptor by protein kinase CK2.
J. Cell Biol
.
177
, 127-137 (2007).
[Abstract]
[Full Text]
Spotlight on the Pre-B Cell Receptor
Authors: Vinson Valda
Published: July 2007

The pre-B cell receptor (pre-BCR), comprising a heavy chain and a heterodimeric surrogate light chain (SLC), is a signaling complex that acts as a checkpoint in B cell development. Bankovich
et al
. report the structure of a pre-BCR Fab-like fragment at 2.7 angstrom resolution. The structure shows how the requirement for pairing with the SLC might constrain the repertoire of heavy chains in the mature antibody population. The crystal structure, together with electron microscopy data and biochemical analysis, supports a model of antigen-independent SLC-mediated dimerization of the pre-BCR to promote pre-B cell activation and expansion.

A. J. Bankovich, S. Raunser, Z. S. Juo, T. Walz, M. M. Davis, K. C. Garcia, Structural insight into pre-B cell receptor function.
Science
316
, 291-294 (2007).
[Abstract]
[Full Text]
IFN-α Finds Its SOCS
Authors: Foley John F.
Published: July 2007

One of the treatments for patients with metastatic malignant melanoma is interferon-α (IFN-α); however, the success rate is only 10 to 15%. Previous studies suggest that IFN-α treatment of T cell lines leads to increased production of members of the suppressor of cytokine signaling (SOCS) family of proteins, which are well characterized in their ability to inhibit cytokine signaling. SOCS1 and SOCS3 inhibit IFN-α-mediated signal transduction. Zimmerer
et al.
investigated the effect of IFN-α treatment on the abundance of SOCS mRNAs and proteins in primary cells from healthy donors and patients with metastatic melanoma. Real-time polymerase chain reaction analysis revealed that peripheral blood mononuclear cells (PBMCs) from healthy donors treated in vitro with IFN-α had increased amounts of mRNAs for SOCS1, SOCS2, SOCS3, and the SOCS family member, cytokine-inducible Src homology 2 (SH2)-containing protein (CIS). Western blot analysis of immunoprecipitates from PBMCs showed that the abundance of these SOCS family members was also increased after IFN-α treatment. Further cell-specific analyses demonstrated that the strongest effect of IFN-α treatment was found within T cells. Analysis of PBMCs from patients with metastatic melanoma 1 hour after intravenous administration of IFN-α showed increased amounts of the mRNAs for some SOCS family members analyzed in healthy donors. Treatment of SOCS1-deficient mice (in a mouse model of malignant melanoma) with IFN-α reduced tumors in 70% of mice, without side effects, whereas all of the IFN-α-treated wild-type mice died. These data suggest that modulating SOCS activity in the context of cytokine treatment may improve clinical outcome.

J. M. Zimmerer, G. B. Lesinski, S. V. Kondadasula, V. I. Karpa, A. Lehman, A. RayChaudhury, B. Becknell, W. E. Carson III, IFN-α-induced signal transduction, gene expression, and antitumor activity of immune effector cells are negatively regulated by suppressor of cytokine signaling proteins.
J. Immunol.
178
, 4832-4845 (2007).
[PubMed]
Does D2 Determine DAT Location?
Authors: Adler Elizabeth M.
Published: July 2007

The presynaptic dopamine transporter (DAT), which facilitates the reuptake of released dopamine, plays a critical role in regulating the synaptic concentration of dopamine and thus the intensity and duration of the dopamine signal. DAT activity, which may be regulated by presynaptic D2 dopamine receptors (D2, a class of G protein-coupled receptors), is therefore pertinent to various neurological and neuropsychiatric disorders associated with altered dopaminergic signaling. Lee
et al
. found that D2 coimmunoprecipitated with DAT from rat striatal tissue. Affinity purification analysis (using fusion proteins containing different regions of D2 or DAT) combined with in vitro binding assays indicated that the two proteins interacted directly through regions in the third intracellular loop of D2 and the DAT N terminus. When D2S (the "short" isoform of D2) was coexpressed with DAT in human embryonic kidney (HEK) 293 cells, dopamine uptake was enhanced compared with that mediated by DAT alone. This facilitated uptake was unaffected by treatment with D2 antagonists and did not involve an increase in DAT abundance but was blocked by overexpression of minigenes encoding DAT or D2 sequences that mediated the interaction between the two proteins. Confocal immunofluorescence analysis indicated that D2S recruited DAT to the plasma membrane; similarly, cell ELISA assays indicated that D2S expression elicited ~20% increase in DAT localization to the plasma membrane. Intriguingly, injection into mice of peptides that blocked the D2-DAT interaction decreased dopamine uptake into striatal synaptosomes and led to increased locomotor activity. Thus, the authors conclude that D2S enhances DAT activity through direct protein interactions that enhance its localization to the plasma membrane.

F. J. S. Lee, L. Pei, A. Moszczynska, B. Vukusic, P. J. Fletcher, F. Liu, Dopamine transporter cell surface localization facilitated by a direct interaction with the dopamine D2 receptor.
EMBO J.
26
, 2127–2136 (2007).
[PubMed]
Giving ITK the SLP
Authors: Foley John F.
Published: July 2007

Tyrosine phosphorylation and activation of phospholipase C-γ (PLC-γ) after stimulation of the T cell antigen receptor (TCR) is dependent on the activities of the tyrosine kinases Lck, ZAP-70 (ζ chain-associated protein kinase of 70 kD), and ITK (IL-2-inducible T cell kinase). Other important components are adaptor molecules, such as SLP-76 (SH2-domain-containing leukocyte protein of 76 kD), which couple Syk family kinases, such as ZAP-70, to PLC-γ activation. Two tyrosine residues important for TCR-mediated phosphorylation and activation of PLC-γ1 are Y
783
and Y
775
. Although previous studies suggest that ZAP-70 and ITK act together to activate PLC-γ1, it is not known which is responsible for phosphorylating the critical tyrosines. Bogin
et al
. performed studies in a SLP-76-deficient T cell line called J14 (derived from the human CD4
+
T cell line, Jurkat) to answer this question. In vitro kinase assays with ITK, but not ZAP-70, immunoprecipitated from TCR-stimulated J14 cells (transfected with a tagged form of SLP-76) resulted in the phosphorylation of both Y
783
and Y
775
in a recombinant form of PLC-γ1, whereas both kinases could phosphorylate SLP-76. The ability of ITK to phosphorylate PLC-γ1 was dependent on SLP-76. Studies with mutants of SLP-76 found that ITK phosphorylation of recombinant PLC-γ1 was dependent on the presence of tyrosines in the N-terminal region of SLP-76. Coimmunoprecipitation studies revealed that stimulation of the TCR resulted in the association of SLP-76 with ITK in J14 cells. Immunoprecipitated ITK in complex with SLP-76 was catalytically more active than uncomplexed ITK. These data suggest that SLP-76 is necessary to initiate and maintain the activity of ITK.

Y. Bogin, C. Ainey, D. Beach, D. Yablonski, SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK.
Proc. Natl. Acad. Sci. U.S.A.
104
, 6638-6643 (2007).
[Abstract]
[Full Text]
The Heart of Stress Responses
Authors: Purnell Beverly
Published: July 2007

Two myosin heavy chain (MHC) genes are expressed in opposing manners in the mouse heart; βMHC is expressed embryonically, whereas αMHC is up-regulated postnatally. Cardiac stress shifts this ratio toward βMHC with negative effects on cardiac function, and previous work has identified microRNAs (miRNAs) as possible regulators of cardiac growth and function. Van Rooij
et al
. now show that miR-208, which is encoded by an intron of the αMHC gene, is a cardiac-specific regulator of βMHC expression in response to stress and hypothyroidism in the heart. Deletion of the coding region of miR-208 resulted in inhibition of βMHC expression and a reduced stress response in the heart. Thus, miR-208 may act through thyroid signaling to regulate βMHC expression, possibly by repressing expression of the thyroid receptor coregulator THRAP1.

E. van Rooij, L. B. Sutherland, X. Qi, J. A. Richardson, J. Hill, E. N. Olson, Control of stress-dependent cardiac growth and gene expression by a microRNA.
Science
316
, 575-579 (2007).
[Abstract]
[Full Text]
Maintaining Polarity in the Face of Energetic Stress
Authors: Gough Nancy R.
Published: July 2007

The serine-threonine LKB1 is a tumor suppressor, and mutations in the encoding gene cause epithelial tissue-derived tumors. Two targets of LKB1 are the polarity-stimulating kinase PAR-1 and a kinase that is activated under conditions of energy depletion, AMPK.
Drosophila
represents a simpler system than mammalian ones to investigate the role of LKB1 and AMPK in epithelial cell function, because there is only a single AMPK complex (not the multiple isoforms found in mammals). Mirouse
et al
. created
Drosophila
mutants of the gene encoding the α subunit of the AMPK complex (
ampkα
) and found that the follicle epithelial cells of the ovary exhibited normal polarity under energy-replete conditions but showed disrupted polarity under conditions of glucose deprivation. In the latter case, apical markers were decreased in abundance and mislocalized, whereas basal markers were more broadly distributed. However, intercellular adhesion did not appear to be affected, because E-cadherin was enriched in adherens junctions. The localization of a tagged form of AMPKα, as well as the LKB1-phosphorylated form of AMPK, showed that AMPK was distributed throughout the cytoplasm and was not enriched at either apical or basal sides of the cells. However, LKB1 appears to be important for the polarity-regulating activity of AMPK under conditions of glucose deprivation. LKB1 functions in energy-replete conditions in the stem cells that produce the follicular epithelium, and when these cells have the
lkb1
mutation, a large cell with disrupted polarity is formed. Mirouse
et al
. showed that small cells that formed after the epithelium was formed that had the
lkb1
mutation did not have polarity defects under energy-replete conditions but did show disruption of polarity in response to glucose deprivation. An activated form of AMPK rescued the polarity defects that occurred in the
lkb1
cells in response to energetic stress. Thus, AMPK appears to serve a role in maintaining polarity under conditions of energy stress, whereas LKB1 appears to participate in both this maintenance pathway and the pathway that establishes epithelial polarity.

V. Mirouse, L. L. Swick, N. Kazgan, D. St. Johnston, J. E. Brenman, LKB1 and AMPK maintain epithelial cell polarity under energetic stress.
J. Cell Biol.
177
, 387-392 (2007).
[Abstract]
[Full Text]
β-catenin Activates ERK: Two Pathways for the Price of One
Authors: Foley John F.
Published: July 2007

Wnt/β-catenin signaling is important for developmental processes, but this pathway is also associated with the development of certain cancers. Loss-of-function mutations in proteins involved in the negative regulation of Wnt/β-catenin signaling, such as Axin, result in the accumulation of β-catenin in the nucleus. This leads to the increased expression of β-catenin-responsive genes and to cellular proliferation. Dysregulation of the extracellular signal-regulated kinase (ERK) pathway, such as in the case of cells with mutations in
ras
, also leads to cancers. To investigate a possible link between these two pathways in carcinogenesis, Jeon
et al
. established an L929 fibroblast cell line in which production of Axin was inducible by the addition of doxycycline (L929-Axin). Western blot analysis revealed that the increased abundance of Axin in L929-Axin cells inhibited the accumulation of β-catenin, as expected, but also inhibited the phosphorylation of components of the ERK pathway, including Raf-1, MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), and ERK. L929-Axin cells showed a smaller proliferative response to epidermal growth factor (EGF) than did control cells, as measured by phosphorylation of Raf-1, MEK, and ERK. Flow-cytometry studies revealed that Axin also inhibited EGF-stimulated progression from the G1 to the S phase of the cell cycle. Negative regulation by Axin of the ERK pathway did not occur in cells containing a nondegradable mutant form of β-catenin. Ras, a small guanosine triphosphatase that responds to growth factor signals and activates the ERK pathway, was less abundant in L929-Axin cells than in control cells. This reduction in Ras (which was mediated by lysosomal degradation) did not occur in cells containing nondegradable β-catenin. These data suggest that β-catenin signaling can also activate the ERK signaling pathway, thereby enhancing the likelihood of carcinogenesis.

S. H. Jeon, J.-Y. Yoon, Y.-N. Park, W.-J. Jeong, S. Kim, E.-H. Jho, Y.-J. Surh, K.-Y. Choi, Axin inhibits extracellular signal-regulated kinase pathway by Ras degradation via β-catenin.
J. Biol. Chem.
282
, 14482-14492 (2007).
[Abstract]
[Full Text]
IP
4
Recruits Signaling Proteins
Authors: Simpson Stephen
Published: July 2007

Inositol phosphates are important intracellular second messengers in eukaryotic cells. In particular, higher-order inositol polyphosphates regulate a range of biological processes, from chromatin remodeling to calcium signaling. Huang
et al
. (see the Perspective by Irvine) now report that inositol (1,3,4,5)-tetrakisphosphate (IP
4
) plays an unexpected role in T cells by modifying a well-established protein recruitment pathway. Soluble IP
4
in the cell was found to lock onto plekstrin homology domains that regulate the recruitment of signaling proteins to the cell membrane for activation during T cell development.

Y. H. Huang, J. A. Grasis, A. T. Miller, R. Xu, S. Soonthornvacharin, A. H. Andreotti, C. D. Tsoukas, M. P. Cooke, K. Sauer, Positive regulation of Itk PH domain function by soluble IP
4
.
Science
316
, 886-889 (2007).
[Abstract]
[Full Text]

R. Irvine, The art of the soluble.
Science
316
, 845-846 (2007).
[Summary]
[Full Text]
Masterful Decisions
Authors: Simpson Stephen
Published: July 2007

In the immune system, B and T lymphocytes develop via distinct pathways from common bone marrow progenitors, and the signaling protein Notch plays a crucial role in deciding T cell fate determination. Maeda
et al
. now find that a proto-oncogene called
LRF
represses this Notch signal and in so doing induces progenitors to undergo a B cell developmental program. Thus,
LRF
may act as a master regulator in the cell fate decision that generates the two main arms of the adaptive immune system.

T. Maeda, T. Merghoub, R. M. Hobbs, L. Dong, M. Maeda, J. Zakrzewski, M. R. M. van den Brink, A. Zelent, H. Shigematsu, K. Akashi, J. Teruya-Feldstein, G. Cattoretti, P. P. Pandolfi, Regulation of B versus T lymphoid lineage fate decision by the proto-oncogene LRF.
Science
316
, 860-866 (2007).
[Abstract]
[Full Text]

I. Maillard, W. S. Pear, Keeping a tight leash on Notch.
Science
316
, 840-842 (2007).
[Summary]
[Full Text]
Micromanagement During the Innate Immune Response
Authors: Dahlberg James E. , Lund Elsebet
Published: July 2007

The innate immune response can be initiated by the binding of various pathogen-associated compounds or cytokines to receptors on the surfaces of dendritic cells. These interactions result in the activation of many genes and gene products. Several different pathways converge to raise the abundance of specific microRNAs (miRNAs). In particular, activation of the transcription factors AP-1 and NF-κB results in an increase in the amount of miR-155. High levels of this miRNA are associated with several types of cancer. However, the mRNAs that may be targeted by miR-155 in the innate immune response remain to be determined.
Decisions of Life or Death for Retinoic Acid
Authors: Ray L. Bryan
Published: July 2007

The hormone and vitamin A metabolite retinoic acid (RA) is best known for its actions promoting cell differentiation, arrest of the cell division cycle, or apoptosis, and it has even been proposed as an anticancer agent. These effects are mediated by the nuclear hormone receptor known as the retinoic acid receptor (RAR). But, in some tissues, RA can promote cell survival and even enhance formation of tumors, for example, in the skin. Shug
et al
. propose that these nearly opposite effects of RA can be explained by its association with and activation of another nuclear receptor called PPARβ (or, alternatively, PPARδ). Their experiments monitoring transcription in cultured cells confirmed that RA could act through both RAR and PPARβ. Furthermore, the choice of which receptor RA activates appeared to be dictated by a set of proteins that work with the receptors, carrying ligands from the cytosol to the nucleus where they pass them off to nuclear receptors. The proteins CRABP-II (cellular retinoic acid-binding protein II) and FABP5 (fatty acid-binding protein 5, also sometimes called K-FABP, eFABP, or mal1) work selectively with RAR or PPARβ, respectively. The authors showed that altering the ratio of these binding proteins in a cell (with RNAi or overexpression in transfected cells) determined which receptor was primarily engaged by RA and, thus, what the ultimate biological effect would be. When amounts of CRABP-II were high, RA acted primarily through RAR in cultured keratinocytes and promoted apoptosis. But if FABP5 was more abundant, RA acted through PPARβ and favored cell survival. Thus, the authors propose that in most cells, high affinity interactions of RA with CRABP-II and RAR [with Kds (dissociation constants) of about 0.1nM] predominate, but in certain cell types, PPARβ and FABP5 (which bind RA with Kds of 10 to 50 nM) are sufficiently abundant to take over and produce the "nontraditional" proliferative effects of the hormone.

T. T. Schug, D. C. Berry, N. S. Shaw, S. N. Travis, N. Noy, Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors.
Cell
129
, 723-733 (2007).
[Online Journal]
Building a Better Interferon
Authors: Gough Nancy R.
Published: July 2007

Type I interferons (IFNs) are encoded by multiple genes (up to 20 in mice) and, although each of these binds to the same receptor, they produce different cellular responses. Each has some antiviral, immunomodulatory, and antiproliferative activity. Because of these important properties, IFNs are used clinically to treat chronic viral infection and cancer. However, high antiviral activity with low antiproliferative activity would be desirable to minimize the side effects of IFN treatment in chronic infection, whereas maximizing antiproliferative activity while minimizing immunoregulatory activity would be desirable in cancer treatment. Brideau-Andersen
et al
. engineered IFNs by gene shuffling that possessed high antiviral activity but minimal antiproliferative activity. They created a series of IFNs that they call the B9X series and assayed these in three different viral infection assays in cultured cells. They also tested the proteins for stimulation of the differentiation of Th1 cells, which is a desirable response to viral infection, and for antiproliferative effects on a human-derived B cell line (Daudi). By these criteria, two specific engineered IFNs, B9X14 and B9X25, were identified that have very high ratios of antiviral:antiproliferative and Th1-inducing:antiproliferative activities. Receptor binding assays with the Daudi cells indicated that these proteins bound the interferon receptor complex with very high affinity (greater than that of several natural IFN-αs). These results have clinical implications for IFN treatment of chronic viral infection, as well as providing insight into the interaction of IFNs with their receptor.

A. D. Brideau-Andersen, X. Huang, S.-C. Chang Sun, T. T. Chen, D. Stark, I. J. Sas, L. Zadik, G. N. Dawes, D. R. Guptill, R. McCord, S. Govindarajan, A. Roy, S. Yang, J. Gao, Y. H. Chen, N. J. Ø. Skartved, A. K. Pedersen, D. Lin, C. P. Locher, I. Rebbapragada, A. D. Jensen, S. H. Bass, T. L. Straight Nissen, S. Viswanathan, G. R. Foster, J. A. Symons, P. A. Patten, Directed evolution of gene-shuffled IFN-α molecules with activity profiles tailored for treatment of chronic viral diseases.
Proc. Natl. Acad. Sci. U.S.A.
104
, 8269-8274 (2007).
[Abstract]
[Full Text]
Synaptic Sumoylation
Authors: Adler Elizabeth M.
Published: July 2007

Sumoylation--the posttranslational modification of proteins with the small ubiquitin-like modifier protein (SUMO)--is most familiar in the context of transcriptional regulation and nuclear transport; increasingly, however, roles for extranuclear sumoylation have begun to surface. Martin
et al
. found that the SUMO-conjugating enzyme Ubc9 and the SUMO ligase PIAS3 interacted with the GluR6 subunit of the kainate type of glutamate receptor (KAR) in a yeast two-hybrid screen of rat brain cDNA and coimmunoprecipitated with GluR6 from cultured hippocampal neuronal extracts. Truncation analysis indicated that interaction depended on a region of GluR6 containing the consensus sumoylation motif. Immunocytochemical analysis indicated that there was limited colocalization of GluR6 and SUMO-1 in cultured hippocampal neurons and that most of the sumoylated GluR6 was intracellular. Kainate, which stimulates KAR internalization and degradation, increased GluR6 sumoylation (as did glutamate), whereas desumoylation with the SUMO-specific isopeptidase SENP-1 inhibited kainate-dependent GluR6 endocytosis. Moreover, unlike fluorescently labeled wild-type GluR6a, a fluorescently labeled non-sumoylatable GluR6a point mutant expressed in COS-7 cells failed to undergo kainate-dependent endocytosis. Infusion of SUMO-1 into CA3 neurons in hippocampal slices decreased the amplitude of KAR-mediated excitatory postsynaptic currents (KAR-EPSCs) without affecting AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor-mediated EPSCs, whereas infusion of SENP-1 increased the amplitude of KAR-EPSCs. Thus, the authors conclude that, under certain conditions, sumoylation may modify the postsynaptic response to glutamate and propose that sumoylation may regulate the function of many other synaptic proteins as well. Coussen and Choquet discuss some of the implications of this research.

S. Martin, A. Nishimune, J. R. Mellor, J. M. Henley, SUMOylation regulates kainate-receptor-mediated synaptic transmission.
Nature
447
, 321-325 (2007).
[PubMed]
TGF-β
1
Uses Semaphorin
Authors: Foley John F.
Published: July 2007

The transforming growth factor β (TGF-β) family of cytokines perform many important functions in processes such as inflammation, cell growth, and tissue remodeling, to name but a few. TGF-β
1
is essential for wound healing and has been implicated in the pathogenesis of fibrotic disorders, such as idiopathic pulmonary fibrosis. The semaphorins are another large family of proteins, members of which are known to be critical for axon guidance and have been implicated in pathologies such as Alzheimer’s disease. Kang
et al
. investigated a possible role for the semaphorin SEMA7A in contributing to TGF-β
1
-induced fibrosis in the lung. They established two transgenic mouse strains with doxycycline-inducible expression of TGF-β
1
, one of which was also a SEMA7A-deficient mouse (SEMA7A KO), whereas the other TGF-β
1
transgenic animal was wild type (WT) with respect to SEMA7A. In this way the authors could investigate the effects of overproduction of TGF-β
1
(through the administration of doxycycline-water to mice) in the presence or absence of SEMA7A. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting analyses showed that TGF-β
1
stimulated an increase in the production of SEMA7A and its receptors, plexin C1 and integrin β1. TGF-β
1
caused fibrosis in the lungs of WT mice, as shown by histological analysis, but this was much reduced in SEMA7A KO mice. The collagen content of the lungs of wild-type mice was much higher than that in the lungs of SEMA7A KO mice, further demonstrating a role for SEMA 7A in mediating the fibrotic effects of TGF-β
1
. Loss of SEMA7A did not adversely affect the ability of TGF-β
1
to activate its target transcription factors Smads 2 and 3, indicating that SEMA7A did not have a role in mediating TGF-β
1
-induced inflammation. The ability of TGF-β
1
to stimulate the production of extracellular matrix-associated proteins such as collagens, elastin, and fibronectin, and fibroregulatory cytokines including IL-18, was completely abolished in the absence of SEMA7A. Deletion of SEMA7A also blocked the ability of TGF-β
1
to activate phosphatidylinositol 3-kinase and protein kinase B. Finally, bleomycin-induced fibrosis (which is dependent on TGF-β
1
) was greatly diminished in SEMA7A KO mice compared with WT mice, as measured by histochemistry and collagen content. This study provides evidence for a previously unappreciated role for SEMA7A in meditating the effects of TGF-β
1
in tissue fibrosis.

H.-R. Kang, C. G. Lee, R. J. Homer, J. A. Elias, Semaphorin 7A plays a critical role in TGF-β
1
-induced pulmonary fibrosis.
J. Exp. Med.
204
, 1083-1093 (2007).
[Abstract]
[Full Text]
Tumor Suppressor Joined to Wnt Network
Authors: Kiberstis Paula A.
Published: July 2007

Elucidation of the cellular signaling pathways that contribute to cancer development often begins with the identification of a gene mutated in human tumors. Complementary biochemical approaches become especially important when the sequence of the newly identified gene provides few clues as to its function. Major
et al
. used analysis of protein interaction networks to define the function of
WTX
, a tumor suppressor gene found very recently to be mutated in an inherited kidney cancer called Wilms tumor. The WTX protein forms a complex with several proteins in the Wnt signaling cascade, including β-catenin, Axin1, β-TRCP2 (β-transducin repeat-containing protein 2), and APC (adenomatous polyposis coli), and antagonizes Wnt signaling by promoting β-catenin degradation.

M. B. Major, N. D. Camp, J. D. Berndt, X. Yi, S. J. Goldenberg, C. Hubbert, T. L. Biechele, A.-C. Gingras, N. Zheng, M. J. MacCoss, S. Angers, R. T. Moon, Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
Science
316
, 1043-1046 (2007).
[Abstract]
[Full Text]

R. Nusse, Converging on β-catenin in Wilms tumor.
Science
316
, 988-989 (2007).
[Summary]
[Full Text]
From Leaf to Flower
Authors: Hines Pamela J.
Published: July 2007

As spring begins, many plants turn to flower under the control of florigen. The molecular nature of florigen has long been unknown, but the signal was known to originate in leaves, and thus had to travel through the plant to the growing buds. Now Corbesier
et al
. and Tamaki
et al
. show that it is a protein rather than its coding RNA that is the likely florigen signal that moves within the plant. The florigen RNA and protein are encoded by the
FLOWERING LOCUS T
gene in
Arabidopsis
and the
Hd3a
gene in rice.

L. Corbesier, C. Vincent, S. Jang, F. Fornara, Q. Fan, I. Searle, A. Giakountis, S. Farrona, L. Gissot, C. Turnbull, G. Coupland, FT protein movement contributes to long-distance signaling in floral induction of
Arabidopsis
.
Science
316
, 1030-1033 (2007).
[Abstract]
[Full Text]

S. Tamaki, S. Matsuo, H. L. Wong, S. Yokoi, K. Shimamoto, Hd3a Protein is a mobile flowering signal in rice.
Science
316
, 1033-1036 (2007).
[Abstract]
[Full Text]
Focus Issue: Single Molecules—Zooming in for a Closer Look
Authors: Foley John F. , Gough Nancy R.
Published: July 2007

This Focus Issue of
Science’s
STKE complements the
Science
Special Issue on Single Molecules by highlighting technical advances that allow signaling events to be analyzed at the level of individual molecules within a single cell. Moving away from studies of populations of cells provides a number of benefits, including the reduction of biological "noise" and the easier detection of low-frequency events. Increasing the resolution with which cellular signaling is monitored will provide structural and mechanistic information of a more subtle and individual nature than was previously feasible, thereby providing better understanding of cellular variation, of molecular dynamics, and of how molecular structure influences function.
The Impact of X-ray Crystallography and NMR on Intracellular Calcium Signal Transduction by EF-Hand Proteins: Crossing the Threshold from Structure to Biology and Medicine
Authors: Chazin Walter J.
Published: July 2007

The use of x-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy has revolutionized our understanding of the transduction of intracellular calcium signals into cellular responses. Indeed, the ability to cross a critical threshold from basic descriptive structural biochemistry to real-world applications in therapeutics and biotechnology now looms on the experimental horizon. The ~500 atomic resolution structures determined by x-ray and NMR approaches and deposited in the Protein Data Bank, many of which are of complexes of EF-hand proteins with peptide fragments of cellular targets, have yielded an extremely thorough description for how EF-hand proteins respond to the binding of calcium. Although this database of structures is a powerful structural tool to describe EF-hand protein function, it is limited in its ability to have a significant impact on biology and medicine because the structural effects on the downstream target are not determined. The opportunity that now lies before us is to extend this EF-hand–centric structural information so that the alterations in the target proteins are defined and the structural basis for functional consequences downstream is understood.
NEMO: Master of Interferon Production
Authors: Gough Nancy R.
Published: July 2007

NEMO, also known as IKKγ, is a regulatory subunit of the complex involved in activation of nuclear factor κB (NF-κB). Zhao
et al
. now show that in addition this function in NF-κB activation, NEMO participates in the activation of transcription factors called interferon regulatory factors (IRFs), specifically IRF3 and IRF7, which, in combination with NF-kB and activator protein 1, stimulate the production of type I interferons (IFN-α and -β) in virally infected cells. IRF3 and IRF7 are especially important for the initial production of IFN-α that occurs in virally infected cells. Experiments with mouse embryo fibroblasts (MEFs) in which the gene encoding NEMO (
Ikgkb
) or IKKβ (a kinase required for NF-κB activation) that were infected with virus showed that efficient production of IFN-α and the subsequent expression of signal transducer and activator of transcription 1 (STAT1) required NEMO, but not IKKβ, suggesting that NEMO was not acting through the NF-κB pathway. Reporter gene assays also demonstrated the dependence of IRF3- and IRF7-mediated gene expression on NEMO. Phosphorylation and dimerization is required for activation of IRF3 and IRF7, and NEMO was required as the knockout cells exhibited a lack of phosphorylation, dimerization, and DNA binding in response to viral infection. In the NEMO knockout cells, overexpression of TBK1 (an IKK-related kinase that is upstream of IRF3 and IRF7 activation) restored reporter gene activation, and NEMO was also required for activation of the kinase activity of TBK1, placing NEMO upstream of TBK1 in the path to IRF3 and IRF7 activation. However, NEMO did not interact directly with TBK1 (or the related kinase IKKε). Instead, immunoprecipitation studies with transfected cells showed that TANK (TRAF family member-associated NF-κB activator) was required for the formation of a trimeric complex of TANK, NEMO, and IKKε or TBK1. Mutational analysis and deletion analysis suggest that different regions of NEMO participate in NF-κB or IRF activation and may explain some of the diseases associated with various point mutations in the
Ikgkb
in humans.

T. Zhao, L. Yang, Q. Sun, M. Arguello, D. W. Ballard, J. Hiscott, R. Lin, The NEMO adaptor bridges the nuclear factor-κB and interferon regulatory factor signaling pathways.
Nat. Immunol.
8
, 592-600 (2007).
[PubMed]
TLR2 Stimulates Th1 Effector Functions
Authors: Foley John F.
Published: July 2007

Toll-like receptors (TLRs) are pattern-recognition receptors that recognize microbial products and are crucial components of the innate immune system. TLR ligands have costimulatory roles with stimulation of the T cell antigen receptor (TCR) of naïve CD4
+
T cells (although TLR ligands alone cannot stimulate naïve cells), but the actions of TLR ligands on established effector (memory) T cells are poorly understood. T helper (Th) cells are distinct lineages of memory CD4
+
T cells. Th1 cells secrete the effector cytokine interferon-γ (IFN-γ) and are important in the immune response to intracellular pathogens, whereas Th2 cells secrete interleukin-4 (IL-4) and protect against infection by extracellular pathogens. Imanishi
et al
. investigated the effects of TLR ligands on mouse Th1 and Th2 cell cultures established after TCR stimulation of naïve CD4
+
T cells under the appropriate conditions. When Th1 cells were restimulated with various TLR ligands, only the TLR2 ligands MALP-2 (macrophage-activating lipopeptide 2) and Pam3 [
N
-palmitoyl-
S
-(2,3-bis(palmitoyloxy)-(2
RS
)-propyl)-Cys-Ser-Lys
4
] stimulated the secretion of IFN-γ, as measured by enzyme-linked immunosorbant assay. TLR2 stimulation also resulted in the activation and proliferation of Th1 cells. In contrast, TLR2 stimulation of Th2 cells did not increase IL-4 secretion or cause cellular activation. TLR2-mediated production of IFN-γ also occurred in Th1 cells that were treated with the TCR inhibitor cyclosporine A, indicating that TCR signaling was not necessary for IFN-γ production. Western blot analyses demonstrated that Pam3 treatment of Th1 cells, but not Th2 cells, resulted in strong and sustained activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Activation of these pathways by TLR2 requires the adaptor molecule MyD88 and IRAK4 (IL-1 receptor-associated kinase 4). Th1 cells from mice deficient in either MyD88 or IRAK4 did not secrete IFN-γ after TLR2 stimulation. The secretion of IFN-γ by Th1 cells after stimulation with Pam3 was enhanced by either IL-2 or IL-12, and these effects were mediated by enhanced MAPK activation. Together, these data establish TLR2 as a direct stimulator of Th1 functions and suggest that TLR2 signaling differs in naïve and memory CD4
+
T cells.

T. Imanishi, H. Hara, S. Suzuki, N. Suzuki, S. Akira, T. Saito, Cutting Edge: TLR2 directly triggers Th1 effector functions.
J. Immunol.
178
, 6715-6719 (2007).
[PubMed]
Shifting into Reverse
Authors: Gough Nancy R.
Published: July 2007

Dictyostelium discoideum
are a widely used model system for the study of cellular motility. Like many mammalian cells, this single-celled organism exhibits both chemoattractive and chemorepulsive responses. It is well understood that chemoattraction involves polarization of membrane lipids with phosphatidylinositol-(3,4,5)-trisphosphate (PIP
3
) accumulating at the front of the cell, which is oriented toward the area with higher concentration of chemoattractant, and the lipid phosphatase PTEN at the back of the cell, where lower concentration of the attractant is present; however, the mechanisms that control chemorepulsion are less well understood. Keizer-Gunnink
et al
. used analogs of cAMP, such as 8CPT-cAMP, to stimulate chemorepulsion of
D. discoideum
and monitored the accumulation of PIP
3
, phosphatidylinositol 3-kinase (PI3K), and PTEN by immunofluorescence techniques. In the presence of the chemorepulsive gradient, PIP3 and PI3K were enriched at the front of the cells (where concentrations of the repellent were lower), whereas PTEN was enriched at the back of the cell (where the repellent concentration was higher). Thus, the polarity of the cells with respect to the chemorepulsive gradient was opposite that of cells in a chemoattractive gradient. Cells in which two of three genes encoding PI3K or the gene encoding phospholipase C (PLC) were knocked out showed a chemoattractant response to cAMP but no response to the chemorepulsive 8CPT-cAMP. Exposure of the cells to cAMP stimulates PLC activity, which metabolizes phosphatidylinositol-(4,5)-diphosphate (PIP
2
) to produce diacylglycerol and inositol trisphosphate, whereas 8CPT-cAMP leads to inhibition of PLC activity. Thus, in a chemoattractive gradient, the combination of PI3K and PLC activity at the front of the cell leads to a loss of PIP
2
to which PTEN binds, thereby causing PTEN to accumulate in the rear of the cell and PIP
3
to accumulate at the front of the cell. The authors propose that in a chemorepulsive gradient, PLC activity is inhibited, which allows PIP
2
to remain high in the side of the cell where the repellent concentration is highest, thereby recruiting PTEN to this side of the cell and allowing the cell to polarize PIP
3
in a direction away from the highest concentrations of repellent and thus move away. In the life of
D. discoideum
, both chemorepulsion and chemoattraction are involved in the response to starvation with a switch from a repulsive to an attractive behavior. After a short period of starvation (~1 hour), the cells migrate away from each other, and during this time they have the G protein that inhibits PLC. After longer periods of starvation (~5 hours), the cells secrete cAMP and produce the G protein that stimulates PLC, thereby allowing the cells to coalesce and form a multicellular structure

I. Keizer-Gunnink, A. Kortholt, P. J. M. Van Haastert, Chemoattractants and chemorepellents act by inducing opposite polarity in phospholipase C and PI3-kinase signaling.
J. Cell Biol.
177
, 579-585 (2007).
[Abstract]
[Full Text]
DNA Damage-Response Teams
Authors: Ray L. Bryan
Published: July 2007

DNA damage is often a key event in triggering malignancy (see the Perspective by Petrini). Much of the cellular response to DNA damage is mediated by two protein kinases, ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related kinase). Matsuoka
et al
. report a proteomic screen that implicates more than 700 proteins in the cellular response to DNA damage caused by ionizing radiation. Antibodies that recognized the phosphorylated forms of peptides containing consensus phosphorylation sites recognized by ATM or ATR were used to search for previously unrecognized substrates. These results provide a resource for identification of previously unrecognized proteins that function in control of DNA damage in mammalian cells. Three reports, Wang
et al
., Sobhian
et al
., and Kim
et al
., describe a complex of proteins that interact with the breast cancer-associated tumor suppressor gene product BRCA1 and implicate covalent modification of proteins by ubiquitination in regulating the functions of BRCA1 and its partners in the cellular response to DNA damage. A complex of BRCA1 with the protein Bard1 is known to have ubiquitin ligase activity. In the present work, BRCA1 formed a complex at sites of DNA damage with RAP80, a protein with a ubiquitin-interacting motif domain, and RAP80 contributed to localization of BRCA1 to sites of DNA damage. A third protein, Abraxas, appears to mediate interaction of BRCA1 with Rap80. BRCA1 complexes also contained BRCC36, a deubiquitinating enzyme. The DNA damage checkpoint that halts division of cells with damaged DNA was defective in cells lacking RAP80. Thus, the BRCA1-Abraxas-RAP80 complex appears to target BRCA1 to sites of DNA damage.

S. Matsuoka, B. B. Ballif, A. Smogorzewska, E. R. McDonald, III, K. E. Hurov, J. Luo, C. E. Bakalarski, Z. Zhao, N. Solimini, Y. Lerenthal, Y. Shiloh, S. P. Gygi, S. J. Elledge, ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.
Science
316
, 1160-1166 (2007).
[Abstract]
[Full Text]

B. Wang, S. Matsuoka, B. A. Ballif, D. Zhang, A. Smogorzewska, S. P. Gygi, S. J. Elledge, Abraxs and RAP80 form a BRCA1 protein complex required for the DNA damage response.
Science
316
, 1194-1198 (2007).
[Abstract]
[Full Text]

B. Sobhian, G. Shao, D. R. Lilli, A. C. Culhane, L. A. Moreau, B. Xia, D. M. Livingston, R. A. Greenberg, RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites.
Science
316
, 1198-1202 (2007).
[Abstract]
[Full Text]

H. Kim, J. Chen, X. Yu, Ubiquitin-binding protein RAP80 mediates BRCA1-dependent DNA damage response.
Science
316
, 1202-1205 (2007).
[Abstract]
[Full Text]

J. H. J. Petrini, A touching response to damage.
Science
316
, 1138-1139 (2007).
[Summary]
[Full Text]
The Biology of Regenerative MedicineReview and commentary on
Regeneration Biology and Medicine
, by David L. Stocum. Academic Press, an imprint of Elsevier, 2006, 448 pp., ISBN 13: 978-0-12-369371-6.
Authors: Muneoka Ken
Published: July 2007

The goal of regenerative medicine is to restore the structure and function of human body parts compromised by injury or disease. Regenerative medicine is a relatively new field that uses multiple approaches and has its roots largely in tissue engineering, tissue transplantation, and stem cell biology. It is becoming increasingly clear that advances in the areas of regeneration biology and developmental biology have the potential to transform regenerative medicine, and David Stocum has produced a textbook that attempts to pull everything together for the next generation of regeneration scientists.
Regeneration Biology and Medicine
is a terrific reference for anyone toying with the idea of moving into this field. The text offers an enormous breath of coverage of different systems currently under investigation, and Stocum provides a central theme that pits regenerative ability against fibrosis as a way to conceptually untangle the limited regenerative capacity of humans. In writing this textbook, Stocum has erected an umbrella large enough for scientists of diverse backgrounds to initiate conceptual cross-talk between those actively involved in the biology of regeneration and those targeting its clinical application.
Silencing Cell Death?
Authors: Adler Elizabeth M.
Published: July 2007

Although most cases of chronic lymphocytic leukemia (CLL, one of the most common forms of adult leukemia) are sporadic, perhaps 10 to 20% are familial (see Debatin). Noting that aberrant DNA methylation--and thereby abnormal gene silencing--was emerging as a factor in CLL, Raval
et al
. performed quantitative high-throughput analysis to investigate DNA methylation in the CpG island of
DAPK1
(
death-associated protein kinase 1
). DNA methylation of
DAPK1
gene, which encodes a serine/threonine kinase implicated in promoting apoptosis in response to Fas, interferon-γ, and TNF-α, was increased in peripheral blood mononuclear cells and CD19+ B cells from people with CLL compared with that in cells from healthy volunteers. Reverse transcription polymerase chain reaction analysis indicated that
DAPK1
expression was decreased in CD19+ CLL cells compared with that in B lymphocytes, and methylation reduced activity of a gene reporter containing a region of the
DAPK1
promoter. Genome-wide linkage analysis of a large family with several members with CLL, combined with high-resolution genotyping, identified a 707-kb haplotype on chromosome 9 common to all individuals affected by CLL that included
DAPK1
. Genomic sequencing identified a single-nucleotide polymorphism (SNP) in the CLL allele that was not found in 383 control tissue samples from Europe and the United States; analysis of 263 CLL tissue samples revealed a single additional CLL case with this SNP. Transcriptional activation of gene reporters suggested that the CLL SNP enhanced binding of a transcriptional suppressor; electrophoretic mobility shift analysis also indicated that the SNP enhanced protein binding. Supershift assays suggested that HOXB7 bound to this region, and transfection of fibroblasts with
HOXB7
siRNA led to an increase in
DAPK1
expression that was more pronounced with the CLL than the wild-type allele. Thus, the authors concluded that a combination of genetic and epigenetic factors leads to a decrease in DAPK1 expression and that the decrease in DAPK1 activity may increase susceptibility to CLL.

A. Raval, S. M. Tanner, J. C. Byrd, E. B. Angerman, J. D. Perko, S.-S. Chen, B. Hackanson, M. R. Grever, D. M. Lucas, J. J. Matkovic, T. S. Lin, T. J. Kipps, F. Murray, D. Weisenburger, W. Sanger, J. Lynch, P. Watson, M. Jansen, Y. Yoshinaga, R. Rosenquist, P. J. de Jong, P. Coggill, S. Beck, H. Lynch, A. de la Chapelle, C. Plass, Downregulation of
death-associated protein kinase 1
(
DAPK1
) in chronic lymphocytic leukemia.
Cell
129
, 879-890 (2007).
[PubMed]

K.-M. Debatin, Chronic lymphocytic leukemia: Keeping cell death at bay.
Cell
129
, 853-855 (2007).
[PubMed]