Grb2 is a negative modulator of the intrinsic Ras-GEF activity of hSos1

hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modulation of hSos1 activity. Our results demonstrate that the first two canonical Grb2 binding sites, inside the carboxyl-terminal region of hSos1, are responsible for this regulation. Following different approaches, such as displacement of Grb2 from the hSos1-Grb2 complex or depletion of Grb2 levels by small interfering RNA, we found that the full-length Grb2 proteins mediate negative regulation of the intrinsic Ras guanine-nucleotide exchange activity of hSos1.     

Stanniocalcin 2 is a negative modulator of store-operated calcium entry

The regulation of cellular Ca(2+) homeostasis is essential for innumerable physiological and pathological processes. Stanniocalcin 1, a secreted glycoprotein hormone originally described in fish, is a well-established endocrine regulator of gill Ca(2+) uptake during hypercalcemia. While there are two mammalian Stanniocalcin homologs (STC1 and STC2), their precise molecular functions remain unknown. Notably, STC2 is a prosurvival component of the unfolded protein response. Here, we demonstrate a cell-intrinsic role for STC2 in the regulation of store-operated Ca(2+) entry (SOCE). Fibroblasts cultured from Stc2 knockout mice accumulate higher levels of cytosolic Ca(2+) following endoplasmic reticulum (ER) Ca(2+) store depletion, specifically due to an increase in extracellular Ca(2+) influx through store-operated Ca(2+) channels (SOC). The knockdown of STC2 expression in a hippocampal cell line also potentiates SOCE, and the overexpression of STC2 attenuates SOCE. Moreover, STC2 interacts with the ER Ca(2+) sensor STIM1, which activates SOCs following ER store depletion. These results define a novel molecular function for STC2 as a negative modulator of SOCE and provide the first direct evidence for the regulation of Ca(2+) homeostasis by mammalian STC2. Furthermore, our findings implicate the modulation of SOCE through STC2 expression as one of the prosurvival measures of the unfolded protein response.     

SH3BP4 is a negative regulator of amino acid-Rag GTPase-mTORC1 signaling

Amino acids stimulate cell growth and suppress autophagy through activation of mTORC1. The activation of mTORC1 by amino acids is mediated by Rag guanosine triphosphatase (GTPase) heterodimers on the lysosome. The molecular mechanism by which amino acids regulate the Rag GTPase heterodimers remains to be elucidated. Here, we identify SH3 domain-binding protein 4 (SH3BP4) as a binding protein and a negative regulator of Rag GTPase complex. SH3BP4 binds to the inactive Rag GTPase complex through its Src homology 3 (SH3) domain under conditions of amino acid starvation and inhibits the formation of active Rag GTPase complex. As a consequence, the binding abrogates the interaction of mTORC1 with Rag GTPase complex and the recruitment of mTORC1 to the lysosome, thus inhibiting amino acid-induced mTORC1 activation and cell growth and promoting autophagy. These results demonstrate that SH3BP4 is a negative regulator of the Rag GTPase complex and amino acid-dependent mTORC1 signaling.     

Platelet derived growth factor receptor alpha is essential for establishing a microenvironment that supports definitive erythropoiesis

The hematopoietic system undergoes a qualitative change during the embryogenesis of most vertebrates. It is designated as the shift of primitive to definitive hematopoiesis and suitable microenvironment must be established to support this shift. While studying the role of platelet derived growth factor receptor alpha (PDGFR alpha) in embryonic hematopoiesis, we found that it was expressed in a stromal cell component of liver, a major site of this shift, but not in the yolk sac, the site of primitive hematopoiesis. Thus, we considered that development of PDGFRalpha positive stromal cells is an essential requirement for this shift. Without PDFGRalpha positive cell component, erythropoiesis was suppressed in the culture of fetal liver. Moreover, injection of an antagonistic anti-PDGFRalpha monoclonal antibody during embryogenesis suppressed the production of definitive erythrocytes. These indicated that PDGF exerts its effect on a subset of stromal components to prepare a microenvironment that can support the definitive erythropoiesis.     

HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading

Background: Class II molecules of the major histocompatibility complex become loaded with antigenic peptides after dissociation of invariant chainderived peptides (CLIP) from the peptide-binding groove. The human leukocyte antigen (HLA)-DM is a prerequisite for this process, which takes place in specialised intracellular compartments. HLA-DM catalyses the peptide-exchange process, simultaneously functioning as a peptide ‘editor’, favouring the presentation of stably binding peptides. Recently, HLA-DO, an unconventional class II molecule, has been found associated with HLA-DM in B cells, yet its function has remained elusive.Results: The function of the HLA-DO complex was investigated by expression of both chains of the HLA-DO heterodimer (either alone or fused to green fluorescent protein) in human Mel JuSo cells. Expression of HLA-DO resulted in greatly enhanced surface expression of CLIP via HLA-DR3, the conversion of class II complexes to the SDS-unstable phenotype and reduced antigen presentation to T-cell clones. Analysis of peptides eluted from HLA-DR3 demonstrated that CLIP was the major peptide bound to class II in the HLA-DO transfectants. Peptide exchange assays in vitro revealed that HLA-DO functions directly at the level of class II peptide loading by inhibiting the catalytic action of HLA-DM.Conclusions: HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.     

RGS9-2 is a negative modulator of mu-opioid receptor function

Regulators of G-protein signaling (RGS) 9-2 is a striatal enriched protein that controls G protein coupled receptor signaling duration by accelerating Galpha subunit guanosine triphosphate hydrolysis. We have previously demonstrated that mice lacking the RGS9 gene show enhanced morphine analgesia and delayed development of tolerance. Here we extend these studies to understand the mechanism via which RGS9-2 modulates opiate actions. Our data suggest that RGS9-2 prevents several events triggered by mu-opioid receptor (MOR) activation. In transiently transfected PC12 cells, RGS9-2 delays agonist induced internalization of epitope HA-tagged mu-opioid receptor. This action of RGS9-2 requires localization of the protein near the cell membrane. Co-immunoprecipitation studies reveal that RGS9-2 interacts with HA-tagged mu-opioid receptor, and that this interaction is enhanced by morphine treatment. In addition, morphine promotes the association of RGS9-2 with another essential component of MOR desensitization, beta-arrestin-2. We also show that over-expression of RGS9-2 prevents opiate-induced extracellular signal-regulated kinase phosphorylation. Our data indicate that RGS9-2 plays an essential role in opiate actions, by negatively modulating MOR downstream signaling as well as the rate of MOR endocytosis.     

GPRC5B a putative glutamate-receptor candidate is negative modulator of insulin secretion

GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). GPRC5B is abundantly expressed in both human and mouse pancreatic islets, and both GPRC5B mRNA and protein are up-regulated 2.5-fold in islets from organ donors with type 2 diabetes. Expression of Gprc5b is 50% lower in islets isolated from newborn (<3 weeks) than in adult (>36 weeks) mice. Lentiviral shRNA-mediated down-regulation of Gprc5b in intact islets from 12 to 16 week-old mice strongly (2.5-fold) increased basal (1 mmol/l) and moderately (40%) potentiated glucose (20 mmol/l) stimulated insulin secretion and also enhanced the potentiating effect of glutamate on insulin secretion. Down-regulation of Gprc5b protected murine insulin-secreting clonal MIN6 cells against cytokine-induced apoptosis. We propose that increased expression of GPRC5B contributes to the reduced insulin secretion and β-cell viability observed in type-2 diabetes. Thus, pharmacological targeting of GPRC5B might provide a novel means therapy for the treatment and prevention of type-2 diabetes.     

U2552 methylation at the ribosomal A-site is a negative modulator of translational accuracy

We have recently identified RrmJ, the first encoded protein of the rrmJ-ftsH heat shock operon, as being the Um(2552) methyltransferase of 23S rRNA, and reported that rrmJ-deficient strains exhibit growth defects, reduced translation rates and reduced stability of 70S ribosomes. U2552 is an ubiquitously methylated residue. It belongs to the A loop of 23S RNA which is an essential component of the ribosome peptidyltransferase centre and interacts directly with aminoacyl(A)-site tRNA. In the present study, we show that a lack of U2552 methylation, obtained in rrmJ-deficient mutants, results in a decrease in programmed +1 and -1 translational frameshifing and a decrease in readthrough of UAA and UGA stop codons. The increased translational accuracy of rrmJ-deficient strains suggests that the interaction between aminoacyl-tRNA and U2552 is important for selection of the correct tRNA at the ribosomal A site, and supports the idea that translational accuracy in vivo is optimal rather than maximal, thus pointing to the participation of recoding events in the normal cell physiology.     

LRP1B is a negative modulator of increased migration activity of intimal smooth muscle cells from rabbit aortic plaques

The migration of cultured cultured smooth muscle cells (SMCs) is regulated by the time-specific expression of members of the LDL receptor family (LRs). LRP1B, a member of LRs, modulates the catabolism of PDGF beta-receptor, affecting the migration of SMCs. An involvement of PDGF beta-receptor in atherosclerosis is focused because of its abundant expression in intimal SMCs. Here, in order to know a functional significance of LRP1B in the increased migration of intimal SMCs, the functions of three groups of cultured SMCs with different origins in atherosclerotic arteries were studied. Each group of SMCs (central, marginal or medial SMCs) was isolated from explanted pieces of central or marginal area of thickened intima, or media prepared from rabbit aortic plaques. The LRP1B expression levels were significantly decreased in intimal SMCs, particularly in marginal SMCs, compared to medial SMCs. The expression levels of LRP1B in SMCs were negatively correlated with those of PDGF beta-receptor. The level of PDGF beta-receptor-mediated phosphorylation of ERK 1/2 in central SMCs was increased to 5.2-fold with the functional inhibition of LRP1B using anti-LRP1B IgY. The antibody increased the PDGF-BB-stimulated migration and invasion activities in SMCs. The increase in the PDGF beta-receptor-mediated outgrowth activity of SMCs from the explants was also inhibited by the functional inhibition of LRP1B. These results indicate that LRP1B regulated the migration activity of SMCs through the modulation of PDGF beta-receptor-mediated pathway. The regulation of LRP1B expression is possibly involved in the activated migration of intimal SMCs in the course of atherosclerosis.     

Mkp3 is a negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizer

The pivotal mechanisms that govern the correct patterning and regionalization of the distinct areas of the mammalian CNS are driven by key molecules that emanate from the so-called secondary organizers at neural plate and tube stages. FGF8 is the candidate morphogenetic molecule to pattern the mesencephalon and rhombencephalon in the isthmic organizer (IsO). Recognizable relevance has been given to the intracellular pathways by which Fgf8 is regulated and modulated. In chick limb bud development, a dual mitogen-activated protein kinase phosphatase-3 (Mkp3) plays a role as a negative feedback modulator of Fgf8 signaling. We have investigated the role of Mkp3 and its functional relationship with the Fgf8 signaling pathway in the mouse IsO using gene transfer microelectroporation assays and protein-soaked bead experiments. Here, we demonstrate that MKP3 has a negative feedback action on the MAPK/ERK-mediated FGF8 pathway in the mouse neuroepithelium.     

53BP1 is a Positive Regulator of the BRCA1 Promoter

Germline mutations in the BRCA1 tumor suppressor gene contribute to familial breast and ovarian tumor formation. Sporadic breast and ovarian cancer, however, which accounts for more than 90% of total cases and virtually lacks BRCA1 mutations, exhibits reduced expression of the BRCA1 gene. The magnitude of this reduction correlates with disease progression. In this report we have identified an imperfect palindrome sequence for binding of the 53BP1-containing complex, -40TTCCGTGG CAACGGAA-25, within the BRCA1 minimal promoter. Overexpression of 53BP1 activates a luciferase reporter driven by the wild type BRCA1 minimal promoter, but not by the BRCA1 minimal promoter with mutated palindrome sequence. Depletion of endogenous 53BP1 by siRNA suppresses activity of the BRCA1 minimal promoter. In vitro and in vivo DNA-protein interaction studies demonstrate that this palindrome sequence binds to the 53BP1-containing complex. These findings establish a positive regulation of the BRCA1 promoter by 53BP1. Disruption of this regulation in cancer cells may provide a molecular mechanistic basis for sporadic breast and ovarian tumor formation.     

Neuraminidase 1 is a negative regulator of lysosomal exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.