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PubMed2000

New steps in the Wnt/beta-catenin signal transduction pathway.

Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. Wnt family genes encode secreted glycoproteins that act as ligands for membrane receptors belonging to the Frizzled family of proteins. Wnt-1 originally was found as a proto-oncogene that was upregulated in tumors caused by the mouse mammary tumor virus. The Drosophila homologue of Wnt-1, wingless, is a segment polarity gene that regulates body patterning of the fly embryo. In Xenopus, the Wnt pathway regulates formation of the ventral-dorsal axis. Although Wnt proteins are expressed widely in mammals, the function of the Wnt signaling pathway in normal adult mammalian tissues is not understood. Downstream components of the Wnt pathway, APC (adenomatous polyposis coli) and beta-catenin, clearly are involved in human cancer. There are also several reports that Wnt ligands are highly expressed in tumors. Wnt stabilizes cytoplasmic beta-catenin and activates beta-catenin/Lef-1 (lymphoid enhancer factor), Tcf (T-cell factor)-dependent gene transcription. This regulation of cytosolic beta-catenin is mediated by glycogen synthase kinase-3 (GSK-3) activity but in neither case is the mechanism known. The mechanism by which Wnt inhibits GSK-3 is unknown. Recent studies have shown that some of the intracellular signaling molecules that mediate the Wnt pathway are in complexes, including Dishevelled (Dsh or Dvl), GSK-3beta, and APC protein. However, little is known about how Wnt or other upstream stimuli regulate these complexes to stabilize beta-catenin. We took a variety of approaches to identify new components of the Wnt pathway. Using an expression-cloning technique, we isolated casein kinase I (CKI)epsilon as a positive regulator of beta-catenin in the Wnt pathway. Overexpression of CKIepsilon mimics Wnt by stabilizing beta-catenin, thereby increasing expression of beta-catenin-dependent genes. Inhibition of endogenous CKIepsilon attenuated gene transcription stimulated by Wnt or by Dsh. CKIepsilon forms a complex with Axin and the other downstream components of the Wnt pathway. CKIepsilon is a positive regulator of the Wnt pathway and a possible functional link between upstream signals and the intracellular Axin signaling complex that regulates beta-catenin. In separate experiments, we have identified a Dishevelled-associated kinase (DAK) that binds to Dsh and regulates its functions. Dsh is required for two different pathways, the Wnt pathway and planar polarity pathway in Drosophila. DAK dramatically enhances the function of Dsh in the Wnt pathway and inhibits its function in the planar polarity pathway. This chapter will discuss these newly identified components of the Wnt pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1997

ArgBP2, a multiple Src homology 3 domain-containing, Arg/Abl-interacting protein, is phosphorylated in v-Abl-transformed cells and localized in stress fibers and cardiocyte Z-disks.

Arg and c-Abl represent the mammalian members of the Abelson family of protein-tyrosine kinases. A novel Arg/Abl-binding protein, ArgBP2, was isolated using a segment of the Arg COOH-terminal domain as bait in the yeast two-hybrid system. ArgBP2 contains three COOH-terminal Src homology 3 domains, a serine/threonine-rich domain, and several potential Abl phosphorylation sites. ArgBP2 associates with and is a substrate of Arg and v-Abl, and is phosphorylated on tyrosine in v-Abl-transformed cells. ArgBP2 is widely expressed in human tissues and extremely abundant in heart. In epithelial cells ArgBP2 is located in stress fibers and the nucleus, similar to the reported localization of c-Abl. In cardiac muscle cells ArgBP2 is located in the Z-disks of sarcomeres. These observations suggest that ArgBP2 functions as an adapter protein to assemble signaling complexes in stress fibers, and that ArgBP2 is a potential link between Abl family kinases and the actin cytoskeleton. In addition, the localization of ArgBP2 to Z-disks suggests that ArgBP2 may influence the contractile or elastic properties of cardiac sarcomeres and that the Z-disk is a target of signal transduction cascades.

3T3 Cells↗
PubMed2000

Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy.

The interaction of activated epidermal growth factor receptor (EGFR) with the Src homology 2 (SH2) domain of the growth-factor-receptor binding protein Grb2 initiates signaling through Ras and mitogen-activated protein kinase (MAP kinase) [1,2]. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyze the spatiotemporal regulation of EGFR-Grb2 interactions in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) and Grb2 fused to yellow fluorescent protein (YFP). Efficient energy transfer between CFP and YFP should only occur if CFP and YFP are less than 50A apart, which requires direct interaction of the EGFR and Grb2 fused to these fluorescent moieties [3]. Stimulation by EGF resulted in the recruitment of Grb2-YFP to cellular compartments that contained EGFR-CFP and a large increase in FRET signal amplitude. In particular, FRET measurements indicated that activated EGFR-CFP interacted with Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signaling via EGFRs can occur in the endosomal compartment. The work also highlights the potential of FRET microscopy in the study of subcellular compartmentalization of protein-protein interactions in living cells.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The docking protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway.

BACKGROUND: Gab1 is a docking protein that recruits phosphatidylinositol-3 kinase (PI-3 kinase) and other effector proteins in response to the activation of many receptor tyrosine kinases (RTKs). As the autophosphorylation sites on EGF-receptor (EGFR) do not include canonical PI-3 kinase binding sites, it is thought that EGF stimulation of PI-3 kinase and its downstream effector Akt is mediated by an indirect mechanism. RESULTS: We used fibroblasts isolated from Gab1-/- mouse embryos to explore the mechanism of EGF stimulation of the PI-3 kinase/Akt anti-apoptotic cell signaling pathway. We demonstrate that Gab1 is essential for EGF stimulation of PI-3 kinase and Akt in these cells and that these responses are mediated by complex formation between p85, the regulatory subunit of PI-3 kinase, and three canonical tyrosine phosphorylation sites on Gab1. Furthermore, complex formation between Gab1 and the protein tyrosine phosphatase Shp2 negatively regulates Gab1 mediated PI-3 kinase and Akt activation following EGF-receptor stimulation. We also demonstrate that tyrosine phosphorylation of ErbB3 may lead to recruitment and activation of PI-3 kinase and Akt in Gab1-/- MEFs. CONCLUSIONS: The primary mechanism of EGF-induced stimulation of the PI-3 kinase/Akt anti-apoptotic pathway occurs via the docking protein Gab1. However, in cells expressing ErbB3, EGF and neuroregulin can stimulate PI-3 kinase and Akt activation in a Gab1-dependent or Gab1-independent manner.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The inflammasome: first line of the immune response to cell stress.

The NALP3-inflammasome is a protein complex that stimulates caspase-1 activation to promote the processing and secretion of proinflammatory cytokines. Recent work indicates that the NALP3-inflammasome can be activated by endogenous "danger signals" as well as compounds associated with pathogens (Kanneganti et al., 2006; Mariathasan et al., 2006, Martinon et al., 2006; Sutterwala et al., 2006). Here, we discuss new insights into the regulation of caspase-1 activity in the inflammatory response.

Adenosine Triphosphate↗
PubMed2000

c-Cbl is a suppressor of the neu oncogene.

A rodent oncogenic mutant of the Neu receptor tyrosine kinase is a useful experimental model because overexpression of the respective receptor, namely HER2/ErbB-2, in human malignancies is associated with relatively aggressive diseases. Here we show that the oncogenic form of Neu is constitutively associated with the product of the c-cbl proto-oncogene and is part of a large complex that includes the phosphoinositide 3-kinase and Shc. Ectopic expression of c-Cbl, a ubiquitin-protein isopeptide ligase specific to activated tyrosine kinases, causes rapid removal of Neu from the cell surface and severely reduces signaling downstream of oncogenic Neu. c-Cbl-induced down-regulation of Neu involves covalent attachment of ubiquitin molecules and requires the carboxyl-terminal domain of Neu. The negative effect of c-Cbl is antagonized by v-Cbl, a virus-encoded oncogenic truncated form of c-Cbl. In an in vivo model, infection of a Neu-transformed neuroblastoma with a c-Cbl-encoding retrovirus caused enhanced down-regulation of Neu and correlated with tumor retardation. Our results implicate c-Cbl in negative regulation of Neu and offer a potential target for treatment of HER2/ErbB-2-positive human malignancies.

3T3 Cells↗
PubMed1999

Structural requirements for Tyr in the consensus sequence Y-E-N of a novel nonphosphorylated inhibitor to the Grb2-SH2 domain.

The phage library derived, nonphosphorylated and thioether-cyclized peptide, termed G1TE, cyclo(CH(2)CO-Glu(1)-Leu-Tyr(3)-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amid e, represents a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, with an IC(50) of 20 microM. The retention of binding affinity is very sensitive with respect to peptide ring-size alterations and Ala mutations. We demonstrated previously that the Glu(1) side chain and its closely related analogs partially compensate for the absence of the phosphate functionality on Tyr(3), and, based on molecular modeling, these acidic side-chains complex with the Arg67 and Arg86 side-chains of the protein in the binding cavity. In this study we judiciously altered and incorporated various natural and unnatural amino acids as Tyr replacements within the -YEN- motif, and we demonstrate the functional importance and structural requirement of Tyr(3) for effective binding of this novel non-phosphorylated ligand to the Grb2-SH2 domain. The phenyl side-chain moiety and a polar functional group with specific orientation in position Y(3) of the peptide are particularly required. Using SPR binding assays, a submicromolar inhibitor (IC(50) = 0.70 microM) was obtained when Glu(1) was replaced with alpha-aminoadipate and Tyr(3) was replaced with 4-carboxymethyl-Phe, providing peptide 14, G1TE(Adi(1), cmPhe(3)). Peptide 14 also inhibited Grb2/p185(erb)(B-2) protein association in cell homogenates of erbB-2-overexpressing MDA-MA-453 cancer cells at near one micromolar concentrations.

Adaptor Proteins, Signal Transducing↗
PubMed2000

A new pathway of translational regulation mediated by eukaryotic initiation factor 3.

We report a new pathway of translation regulation that may operate in interferon-treated or virus-infected mammalian cells. This pathway is activated by P56, a protein whose synthesis is strongly induced by interferons or double-stranded RNA. Using a yeast two-hybrid screen, we identified the P48 subunit of the mammalian translation initiation factor eIF-3 as a protein that interacts with P56. The P56-P48 interaction was confirmed in human cells by co-immunoprecipitation assays and confocal microscopy. Gel filtration assays revealed that P56 binds to the large eIF-3 complex that contains P48. Purified recombinant P56 inhibited in vitro translation of reporter mRNAs in a dose-dependent fashion, and that inhibition was reversed by the addition of purified eIF-3. In vivo, expression of transfected P56 or induction of the endogenous P56 by interferon caused an inhibition of overall cellular protein synthesis and the synthesis of a transfected reporter protein. As expected, a P56 mutant that does not interact with P48 and eIF-3 failed to inhibit protein synthesis in vitro and in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Reduction of dietary obesity in aP2-Ucp transgenic mice: physiology and adipose tissue distribution.

We seek to determine whether increased energy dissipation in adipose tissue can prevent obesity. Transgenic mice with C57BL6/J background and the adipocyte lipid-binding protein (aP2) gene promoter directing expression of the mitochondrial uncoupling protein (UCP) gene in white and brown fat were used. Physiologically, UCP is essential for nonshivering thermogenesis in brown fat. Mice were assigned to a chow or a high-fat (HF) diet at 3 mo of age. Over the next 25 wk, gains of body weight were similar in corresponding subgroups (n = 6-8) of female and male mice: 4-5 g in chow nontransgenic and transgenic, 20 g in HF nontransgenic, and 9-11 g in HF transgenic mice. The lower body weight gain in the HF transgenic vs. nontransgenic mice corresponded to a twofold lower feed efficiency. Gonadal fat was enlarged, but subcutaneous white fat was decreased in the transgenic vs. nontransgenic mice in both dietary conditions. The results suggest that UCP synthesized from the aP2 gene promoter is capable of reducing dietary obesity.

Adaptor Protein Complex 2↗
PubMed2003

Activation of discoidin domain receptor 1 facilitates the maturation of human monocyte-derived dendritic cells through the TNF receptor associated factor 6/TGF-beta-activated protein kinase 1 binding protein 1 beta/p38 alpha mitogen-activated protein kinase signaling cascade.

Maturation of dendritic cells (DCs) is critical for their ability to stimulate resting naive T cells in primary immune responses. Previous studies demonstrated that collagen, such as type I collagen, could facilitate DC maturation; however, the basis of collagen-mediated DC maturation remains unclear. Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor constitutively expressed in a variety of epithelial cells, including tumor cells, and is inducible in leukocytes. In this study, we evaluated the role of DDR1 in DC maturation using human monocyte-derived DCs. Two DDR1 isoforms, DDR1a and DDR1b, were expressed in both immature and mature DCs. Activation of DDR1 on immature DCs resulted in their partial maturation; however, DDR1 activation markedly amplified TNF-alpha- and LPS-induced phenotypic and functional maturation of DCs through activation of p38 mitogen-activated protein kinase (MAPK), suggesting the involvement of DDR1b in this process. Activation of DDR1b on differentiated DDR1b-overexpressing THP-1 cells or DDR1 on mature DCs induced the formation of TNFR associated factor 6 (TRAF6)/TGF-beta-activated kinase 1 binding protein 1beta/p38alpha MAPK complex and p38alpha autophosphorylation. Transfection of differentiated DDR1b-overexpressing THP-1 cells with dominant negative TRAF6 completely abrogated DDR1b-mediated p38 MAPK phosphorylation, indicating a critical role of TRAF6 in DDR1b-mediated p38 MAPK activation. Taken together, our data suggest that DDR1b-collagen interaction augments the maturation of DCs in a tissue microenvironment through a unique TRAF6/TGF-beta-activated kinase 1 binding protein 1beta/p38alpha MAPK signaling cascade and contributes to the development of adaptive immune responses.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Membrane proteinase 3 and its interactions within microdomains of neutrophil membranes.

Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the phagocytic vesicle upon neutrophil stimulation. A fraction of the enzyme is thought to associate with the cell membrane yielding membrane PR3 (mPR3). In autoimmune disorders characterized by the presence of antineutrophil cytoplasmic antibodies (ANCA), the reaction of the latter with their target antigen mPR3 activates the cell inflicting injuries on the surrounding tissues. In a previous communication we provided evidence for the presence of mPR3 in lipid rafts obtained by lysis of neutrophils in Triton X-100 and for the mediation of PR3 binding to the membrane by a glycosylphosphatidylinositol (GPI)-anchored neutrophil protein, possibly FcgammaRIIIb. In the current study we employed the mild detergent Brij 58 to isolate high molecular weight (HMW) protein complexes in the void volume of a Sepharose 4B gel filtration minicolumn. HMW complexes of unstimulated neutrophils comprised PR3, FcgammaRIIIb, the beta2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C (PI-PLC) reduced amounts of PR3 and FcgammaRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcgammaRIIIb acts as a membrane adaptor for PR3. FcgammaRIIIb of HMW fractions co-immunoprecipitated with PR3, indicating their presence in the same protein complex. Since HMW fractions contained also the majority of biotinylated proteins obtained by the reaction of neutrophils with a membrane impermeable biotinylating agent Sulfo-NHS-biotin, it was concluded that HMW proteins were derived from cell membranes. Lipid rafts isolated from Brij 58-lysed neutrophils were similar in their protein composition to the HMW complexes but not identical.

Antigens, CD↗
PubMed2001

Ligand-dependent formation of retinoid receptors, receptor-interacting protein 140 (RIP140), and histone deacetylase complex is mediated by a novel receptor-interacting motif of RIP140.

Receptor-interacting protein 140 (RIP140) interacts with retinoic acid receptor and retinoid X receptor in a ligand-dependent manner and suppresses retinoic acid (RA) induction of its target genes. The receptor-interacting motif is mapped to a C-terminal peptide sequence (LTKTNPILYYMLQK) of RIP140. The functional role of this motif in mediating the suppressive effects of RIP140 on RA induction is demonstrated in mutation studies. RA induces coimmunoprecipitation of histone deacetylase 3 with retinoic acid receptor/retinoid X receptor in the presence of wild type RIP140, but not in the presence of the C-terminal motif-deleted RIP140. A decrease in histone acetylation on the promoter region that carries a RA response element is associated with the expression of wild type RIP140, but not with expression of the mutant RIP140, in a dose-dependent manner. These data provide a molecular explanation for RIP140 acting as a novel ligand-dependent, negative modulator of RA-regulated gene expression.

Acetylation↗
PubMed2002

Oxidative stress inactivates the human DNA mismatch repair system.

In the human DNA mismatch repair (MMR) system, hMSH2 forms the hMutSalpha and hMutSbeta complexes with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the hMutLalpha heterodimer. These complexes, together with other components in the MMR system, correct single-base mismatches and small insertion/deletion loops that occur during DNA replication. Microsatellite instability (MSI) occurs when the loops in DNA microsatellites are not corrected because of a malfunctioning MMR system. Low-frequency MSI (MSI-L) is seen in some chronically inflamed tissues in the absence of genetic inactivation of the MMR system. We hypothesize that oxidative stress associated with chronic inflammation might damage protein components of the MMR system, leading to its functional inactivation. In this study, we demonstrate that noncytotoxic levels of H2O2 inactivate both single-base mismatch and loop repair activities of the MMR system in a dose-dependent fashion. On the basis of in vitro complementation assays using recombinant MMR proteins, we show that this inactivation is most likely due to oxidative damage to hMutSalpha, hMutSbeta, and hMutLalpha protein complexes. We speculate that inactivation of the MMR function in response to oxidative stress may be responsible for the MSI-L seen in nonneoplastic and cancer tissues associated with chronic inflammation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

AAT-1, a novel testis-specific AMY-1-binding protein, forms a quaternary complex with AMY-1, A-kinase anchor protein 84, and a regulatory subunit of cAMP-dependent protein kinase and is phosphorylated by its kinase.

AMY-1 has been identified by us as a c-Myc-binding protein and was found to stimulate c-Myc transcription activity. AMY-1 was also found to be associated with protein kinase A anchor protein 84/149 (S-AKAP84/AKAP149) in the mitochondria in somatic cells and sperm, suggesting that it plays a role in spermatogenesis. To determine the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human testis cDNA library, and a clone encoding a novel protein, AAT-1, was obtained. Three isoforms of AAT-1, AAT-1alpha, -beta, and -gamma, were found to be derived from an alternative splicing of the transcripts of the aat-1 gene, which was mapped at human chromosome 3q13-3q21. AAT-1 was found to be specifically expressed in the testis during the course of spermatogenesis and also to be present in the spermatid and mature sperm, as was AMY-1. AAT-1alpha was found to bind to and be colocalized in mitochondria with AMY-1 in human HeLa and mouse GC-1 cells. Furthermore, AAT-1alpha was found to bind to the N-terminal half of S-AKAP84/AKAP149 in a quaternary complex with AMY-1 and a regulatory subunit (RII) of cAMP-dependent kinase (PKA), in which AAT-1alpha was associated with RII via S-AKAP84/AKAP149, in rat testis and HeLa cells. It was then found that AAT-1alpha weakly stimulated a phosphorylation activity of PKA and also that AAT-1 itself was phosphorylated by PKA in vivo and in vitro. These results suggest that both AAT-1 and AMY-1 play roles in spermatogenesis.

A Kinase Anchor Proteins↗
PubMed2001

Signaling complexes of the FERM domain-containing protein GRSP1 bound to ARF exchange factor GRP1.

GRP1 is a member of a family of proteins that contain a coiled-coil region, a Sec7 homology domain with guanosine nucleotide exchange activity for the ARF GTP-binding proteins, and a pleckstrin homology domain at the C terminus. The pleckstrin homology domain of GRP1 binds phosphatidylinositol (3,4,5) trisphosphate and mediates the translocation of GRP1 to the plasma membrane upon agonist stimulation of PI 3-kinase activity. Using a (32)P-labeled GRP1 probe to screen a mouse brain cDNA expression library, we isolated a cDNA clone encoding a GRP1-binding partner (GRSP1) that exists as two different splice variants in brain and lung. The GRSP1 protein contains a FERM protein interaction domain as well as two coiled coil domains and may therefore function as a scaffolding protein. Mapping experiments revealed that the interaction of GRP1 and GRSP1 occurs through the coiled coil domains in the two proteins. Immunodepletion experiments indicate that virtually all of the endogenous GRSP1 protein exists as a complex with GRP1 in lung. When co-expressed in Chinese hamster ovary cells expressing the human insulin receptor, both proteins display a diffuse, cytoplasmic localization. Acute translocation and co-localization of GRSP1 and GRP1 to ruffles in the plasma membrane was evident after insulin stimulation. These results identify GRSP1 as a novel member of GRP1 signaling complexes that are acutely recruited to plasma membrane ruffles in response to insulin receptor signaling.

ADP-Ribosylation Factors↗
PubMed2000

Tyrosine phosphatase SHP-2 binding to CTLA-4: absence of direct YVKM/YFIP motif recognition.

CTLA-4 is well documented in its negative regulation of T-cell proliferation. However, little is known regarding the signaling mechanisms induced by CTLA-4. CTLA-4 associates with the phosphatidylinositol 3-kinase, the phosphatase SHP-2 and the clathrin adaptor complexes AP-1 and AP-2. SHP-2 SH2 domain binding to CTLA-4 is unusual given the absence of a I/VxYxxI/V/L motif. Here, we demonstrate that the phosphorylation of CTLA-4 tyrosines (YVKM and YFIP) fails to allow for single or tandem SHP-2 SH2 domain binding. This was observed using wild-type and inactive SHP-2 as well as a construct with the isolated two SH2 domains. The phosphorylated YVKM and YFIP motifs therefore do not appear to represent novel binding motifs for SHP-2 SH2 domains. At the same time, we could confirm that SHP-2 can associate with CTLA-4 in murine T-cells indicating that the interaction between the phosphatase and CTLA-4 is an indirect event, possibly mediated by PI 3-kinase/SHP-2 binding.

Abatacept↗
PubMed2004

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes.

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.

Adaptor Proteins, Signal Transducing↗
PubMed2026

Glutathione acts as an exometabolite that promotes growth recovery in fission yeast with defects in amino acid metabolism and cell polarity.

UNLABELLED: Microorganisms in nature form communities through diverse interactions, such as mutualism and competition, to adapt to their ecological environments. These interactions seem to be mediated by extracellular metabolites (exometabolites), yet the chemical and biological diversity underlying these processes remains largely unexplored. In this study, we examined the chemical basis of exometabolite-mediated interactions in the fission yeast Schizosaccharomyces pombe by a genome-wide screen employing 3,420 viable gene deletion mutants. We identified 37 strains that exhibited growth defects in monoculture on a minimal medium but exhibited growth recovery in the vicinity of wild-type colonies (co-culture), suggesting that exometabolites derived from wild-type cells compensated for the gene deletion. Both lipophilic and water-soluble fractions obtained by solvent partitioning of the wild-type culture supernatant promoted growth recovery. Among the 11 mutants rescued by the water-soluble fraction, 6 were cysteine auxotrophs, prompting analyses of thiol-containing metabolites by liquid chromatography-mass spectrometry (LC-MS), revealing the presence of glutathione (GSH) in the culture supernatant. GSH restored growth in most strains as a nutrient source. In contrast, GSH rescued cell morphology defects in the hob3∆ mutant, lacking the Bin/amphiphysin/Rvs (BAR) adaptor protein Hob3, through a mechanism independent of nutrition. This research advances understanding of exometabolite-mediated interactions in S. pombe by identifying GSH as an exometabolite that influences cellular processes and potentially shapes microbial communities. IMPORTANCE: Microorganisms secrete a wide range of metabolites that control microbial community behavior. These extracellular metabolites (exometabolites) include not only well-studied signaling molecules but also diverse primary and secondary metabolites, suggesting complex interactions among microbes. However, the molecular basis of these interactions remains poorly understood, partly due to challenges in detecting them experimentally. In this study, we surveyed exometabolites involved in cell-cell interactions in the model eukaryotic microorganism Schizosaccharomyces pombe. S. pombe releases a wide variety of metabolites outside the cells, including previously reported nitrogen signaling factors (NSFs) and glutathione (GSH) identified in this work. By analyzing gene deletion mutants whose growth is supported by extracellular GSH, we provide new insights into how secreted primary exometabolites compensate for specific genetic defects and influence cell physiology in microbial populations.

exometabolite↗