Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 129
Filter
Add more filters










Publication year range
1.
Genes Immun ; 11(5): 384-96, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20508603

ABSTRACT

Ligand bound chemoattractant receptors activate the heterotrimeric G-protein G(i) to stimulate downstream signaling pathways to properly position lymphocytes in lymphoid organs. Here, we show how variations in the expression of a chemokine receptor and in two components in the signaling pathway, Galpha(i2) and RGS1, affect the output fidelity of the signaling pathway. Examination of B cells from mice with varying numbers of intact alleles of Ccr7, Rgs1, Gnai2, and Gnai3 provided the basis for these results. Loss of a single allele of either Gnai2 or Rgs1 affected CCL19 triggered chemotaxis, whereas the loss of a single allele of Ccr7, which encodes the cognate CCL19 receptor, had little effect. Emphasizing the importance of Gnai2, B cells lacking Gnai3 expression responded to chemokines better than did wild-type B cells. At an organismal level, variations in Rgs1 and Gnai2 expression affected marginal zone B-cell development, splenic architecture, lymphoid follicle size, and germinal center morphology. Gnai2 expression was also needed for the proper alignment of MOMA-1(+) macrophages and MAdCAM-1(+) endothelial cells along marginal zone sinuses in the spleen. These data indicate that chemoattractant receptors, heterotrimeric G-proteins, and RGS protein expression levels have a complex interrelationship that affects the responses to chemoattractant exposure.


Subject(s)
B-Lymphocytes/immunology , GTP-Binding Protein alpha Subunit, Gi2/metabolism , Lymphoid Tissue/anatomy & histology , RGS Proteins/metabolism , Receptors, Chemokine/metabolism , Signal Transduction/immunology , Animals , Cell Adhesion Molecules/metabolism , Chemokine CCL19/immunology , Chemokine CXCL12/immunology , Chemokine CXCL13/immunology , Chemotaxis/immunology , GTP-Binding Protein alpha Subunit, Gi2/genetics , Immunohistochemistry , Lymphoid Tissue/cytology , Mice , Mice, Knockout , Mucoproteins , RGS Proteins/genetics , Receptors, CCR7/genetics , Receptors, CCR7/metabolism
2.
Article in English | MEDLINE | ID: mdl-15180451

ABSTRACT

Heterotrimeric G-protein-coupled receptors (GPCRs) mediate a wide variety of organismal functions ranging from vision, olfaction, and gustation to the development and physiology of the cardiovascular, neuronal, and immune system. Naturally they are targets of a large number of therapeutic drugs. The regulators of G protein signaling (RGS) are a family of diverse proteins that regulate the GPCR-mediated signaling pathways principally by acting as GTPase activating proteins (GAPs) for the alpha subunit of the heterotrimeric G-proteins. Certain members of the RGS family contain multiple domains and motifs that mediate interactions with other signaling molecules, thus linking GPCR-dependent and GPCR-independent signaling pathways. Because of their ability to fine-tune vital GPCR-mediated processes and recent findings linking them to brain disorders, retinitis pigmentosa, and cancer RGS proteins have become excellent candidates for new drug discovery. The focus of this review is to discuss the roles of the RGS proteins in the development and normal physiology of cardiovascular and immune system, and to explore their potential as drug targets useful for the treatment of pathological conditions of the cardiovascular and immune systems.


Subject(s)
Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/metabolism , Drug Delivery Systems/methods , GTP-Binding Protein Regulators/metabolism , Immunity/drug effects , Animals , Cardiovascular Diseases/genetics , Cardiovascular Diseases/immunology , GTP-Binding Protein Regulators/genetics , GTP-Binding Protein Regulators/immunology , GTP-Binding Protein Regulators/physiology , Humans , Immunity/genetics , Signal Transduction/drug effects
3.
BMC Cell Biol ; 2: 21, 2001.
Article in English | MEDLINE | ID: mdl-11716781

ABSTRACT

BACKGROUND: Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs). These proteins act by antagonizing or abbreviating interaction of Galpha proteins with effectors such as phospholipase Cbeta. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells. RESULTS: A truncated version of RGS3 (RGS3T = RGS3 314-519) inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqalpha than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqalpha protein. CONCLUSIONS: RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate) as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqalpha protein function. A version of RGS3 that is amino-terminally truncated is even more potent than intact RGS3 at inhibiting gonadotropin releasing hormone-stimulated inositol trisphosphate production.


Subject(s)
GTP-Binding Proteins , GTPase-Activating Proteins , Gonadotropin-Releasing Hormone/antagonists & inhibitors , Luteinizing Hormone/metabolism , Pituitary Gland, Anterior/metabolism , RGS Proteins/physiology , Repressor Proteins , Animals , COS Cells , Calcium Signaling , Cells, Cultured , Female , Heterotrimeric GTP-Binding Proteins/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , RGS Proteins/genetics , Rats , Receptors, LHRH/metabolism , Sequence Deletion
4.
J Biol Chem ; 276(34): 31845-50, 2001 Aug 24.
Article in English | MEDLINE | ID: mdl-11435419

ABSTRACT

Signaling via a variety of G-protein-coupled receptors (GPCRs) leads to activation of nuclear factor (NF)-kappa B. Evidence exists for a signaling pathway initiated by the B2 type bradykinin receptor via G(q) activation, which leads to the sequential stimulation of phosphoinositide 3-kinase (PI3K), the serine/threonine kinase Akt, I kappa B kinases, and finally nuclear factor NF-kappa B-dependent transcription. GPCR-mediated G(q)alpha or G(13)alpha activation also potently stimulates the tyrosine kinase PYK2. In this study we tested whether G(q)alpha- and/or G(13)alpha-induced PYK2 activation contributes to GPCR-mediated NF-kappa B activation. Among the GTPase-deficient forms of G alpha tested, G(13)alpha and G(q)alpha most potently stimulated an NF-kappa B-dependent reporter gene. PYK2 activated the same reporter gene and synergized with either G(q)alpha Q209L (QL) or G(13)alpha Q226L (QL). Placing PYK2 upstream of both PI3K and Akt activation, PYK2 activated Akt through a PI3K-dependent pathway, and either a dominant negative form of Akt or the PI3K inhibitor LY294002 blocked PYK2-stimulated NF-kappa B-dependent transcription. Placing PYK2 downstream of G-protein activation, a kinase-dead form of PYK2, PYK2 (KD), blocked NF-kappa B-dependent transcription triggered by signaling through the muscarinic receptor type 1 and either G(q)alpha QL or G(13)alpha QL. PYK2 (KD) also blocked Akt activation by the same stimuli. These results indicate that PYK2 can link G-protein activation through PI3K, Akt, and I kappa B kinase to NF-kappa B activation.


Subject(s)
GTP-Binding Proteins/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Focal Adhesion Kinase 2 , HeLa Cells , Humans , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt
5.
Naunyn Schmiedebergs Arch Pharmacol ; 363(4): 456-63, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11330340

ABSTRACT

RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase-activating proteins (GAP) for G protein alpha-subunits. The existence of mRNAs of different size for some of the RGS proteins, e.g. RGS3, suggests that these proteins may exist in isoforms due to alternative splicing. We therefore investigated RGS3 mRNA and protein expression in different human tissues. Ribonuclease protection assays and Northern blot analysis showed two specific mRNAs for RGS3 (RGS3L, RGS3S) in human myocardium, suggesting an additional, N-terminally truncated form of approximately 168 aa. When expressed as a recombinant protein RGS3S was recognized at approximately 23 kDa by an antipeptide antiserum originally raised against an RGS2 sequence. In membranes of human tissues this antiserum detected specific signals for RGS3L (approximately 70 kDa), RGS2 (approximately 30 kDa) and a 25-kDa protein, most likely RGS3S. Both RGS3S mRNA and the 25 kDa protein were abundant in human heart, whereas expression in liver, brain and myometrium was much weaker. To characterize RGS3S functionally, single turnover GTPase, adenylyl cyclase (AC) and phospholipase C (PLC) activities were determined. Both recombinant RGS3S and RGS16 increased Pi release from Galphai1 by about 150% and increased GTP- and GTP plus isoprenaline-stimulated AC activity by 20-30% in human left ventricular myocardial membranes. Additionally, both RGS proteins reduced basal and endothelin-stimulated PLC activity in these membranes by about 40%. We conclude that an additional truncated form of RGS3 is expressed in the human heart. As described for the full-length protein, RGS3S negatively regulates the activity of Gi/o- and Gq-, but not Gs-subfamily members.


Subject(s)
Brain/metabolism , GTP-Binding Proteins/metabolism , GTPase-Activating Proteins , Liver/metabolism , Myocardium/metabolism , RGS Proteins/physiology , Humans , Protein Isoforms , RGS Proteins/genetics
6.
J Biol Chem ; 276(26): 24293-300, 2001 Jun 29.
Article in English | MEDLINE | ID: mdl-11294858

ABSTRACT

Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which Galpha(i) and Galpha(q) hydrolyze GTP to GDP, thereby limiting the duration in which GTP-Galpha(i) and GTP-Galpha(q) can activate effectors. Since GDP-Galpha subunits rapidly combine with free Gbetagamma subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of Gbetagamma subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind Gbeta(5) in the absence of a Ggamma subunit, RGS proteins are not known to directly influence Gbetagamma signaling. Here we show that RGS3 binds Gbeta(1)gamma(2) subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with Gbeta(1)gamma(2) inhibits Gbeta(1)gamma(2)-induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of Gbeta(1)gamma(2) signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect Gbeta(1)gamma(2) signaling in these assays. Consistent with the in vivo results, RGS3 inhibits Gbetagamma-mediated activation of phospholipase Cbeta in vitro. Thus, RGS3 may limit Gbetagamma signaling not only by virtue of its GTPase-activating protein activity for Galpha subunits, but also by directly interfering with the activation of effectors.


Subject(s)
GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/antagonists & inhibitors , GTPase-Activating Proteins , Heterotrimeric GTP-Binding Proteins/antagonists & inhibitors , Inositol Phosphates/biosynthesis , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/antagonists & inhibitors , RGS Proteins/physiology , Animals , Binding Sites , COS Cells , Cell Line , Enzyme Activation , Humans , Isoenzymes/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3 , Phospholipase C beta , Precipitin Tests , Proto-Oncogene Proteins c-akt , RGS Proteins/chemistry , RGS Proteins/genetics , Transfection , Type C Phospholipases/antagonists & inhibitors
7.
Nature ; 409(6823): 1051-5, 2001 Feb 22.
Article in English | MEDLINE | ID: mdl-11234015

ABSTRACT

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.


Subject(s)
Adenylyl Cyclases/metabolism , Isoenzymes/metabolism , Olfactory Receptor Neurons/metabolism , RGS Proteins/physiology , Signal Transduction , Adenylyl Cyclase Inhibitors , Adenylyl Cyclases/genetics , Animals , Cell Line , Cell Membrane/enzymology , Cell Membrane/metabolism , Cyclic AMP/metabolism , Enzyme Activation , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Patch-Clamp Techniques , Rats , Recombinant Proteins , Transfection
8.
Curr Protoc Immunol ; Chapter 11: Unit 11.9A, 2001 Nov.
Article in English | MEDLINE | ID: mdl-18432708

ABSTRACT

This unit, along with Unit 11.9B, provides a summary of our current knowledge about various signaling pathways critical to the function of immune cells. Here, our understanding of T cell receptor (TCR)- and B cell receptor (BCR)-mediated signaling is summarized. A schematic representation of immunologically relevant cytokine receptors and the Janus Family Kinases (JAKs) that is activated through these receptors is provided, along with details about molecules involved in interleukin 2 mediated signal transduction.


Subject(s)
Cytokines/metabolism , Lymphocyte Activation , Protein Kinases/metabolism , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, T-Cell/immunology , Animals , B-Lymphocytes/immunology , Cytokines/immunology , Humans , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, T-Cell/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , T-Lymphocytes/immunology
9.
Mol Biol Cell ; 11(9): 3155-68, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10982407

ABSTRACT

COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit beta'-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant beta'-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of beta'-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on G(ialpha). In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi-plasma membrane or intra-Golgi transport.


Subject(s)
Coat Protein Complex I/metabolism , Coatomer Protein/metabolism , RGS Proteins/metabolism , Alkaline Phosphatase/metabolism , Amino Acid Sequence , Animals , Cell Line , Coat Protein Complex I/antagonists & inhibitors , Coat Protein Complex I/chemistry , Consensus Sequence , Humans , Mice , Molecular Sequence Data , Protein Subunits , RGS Proteins/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Sequence Alignment , Stem Cells/metabolism , Transfection
10.
Mol Pharmacol ; 58(4): 719-28, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10999941

ABSTRACT

Many Regulators of G protein Signaling (RGS) proteins accelerate the intrinsic GTPase activity of G(ialpha) and G(qalpha)-subunits [i.e., behave as GTPase-activating proteins (GAPs)] and several act as G(qalpha)-effector antagonists. RGS3, a structurally distinct RGS member with a unique N-terminal domain and a C-terminal RGS domain, and an N-terminally truncated version of RGS3 (RGS3CT) both stimulated the GTPase activity of G(ialpha) (except G(zalpha)) and G(qalpha) but not that of G(salpha) or G(12alpha). RGS3 and RGS3CT had G(qalpha) GAP activity similar to that of RGS4. RGS3 impaired signaling through G(q)-linked receptors, although RGS3CT invariably inhibited better than did full-length RGS3. RGS3 potently inhibited G(qalpha)Q209L- and G(11alpha)Q209L-mediated activation of a cAMP-response element-binding protein reporter gene and G(qalpha)Q209L induced inositol phosphate production, suggesting that RGS3 efficiently blocks G(qalpha) from activating its downstream effector phospholipase C-beta. Whereas RGS2 and to a lesser extent RGS10 also inhibited signaling by these GTPase-deficient G proteins, other RGS proteins including RGS4 did not. Mutation of residues in RGS3 similar to those required for RGS4 G(ialpha) GAP activity, as well as several residues N terminal to its RGS domain impaired RGS3 function. A greater percentage of RGS3CT localized at the cell membrane than the full-length version, potentially explaining why RGS3CT blocked signaling better than did full-length RGS3. Thus, RGS3 can impair Gi- (but not Gz-) and Gq-mediated signaling in hematopoietic and other cell types by acting as a GAP for G(ialpha) and G(qalpha) subfamily members and as a potent G(qalpha) subfamily effector antagonist.


Subject(s)
GTP Phosphohydrolases/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Proteins , GTPase-Activating Proteins , Heterotrimeric GTP-Binding Proteins/metabolism , RGS Proteins/physiology , Cells, Cultured , Enzyme Activation , GTP Phosphohydrolases/deficiency , GTP-Binding Protein alpha Subunits, Gq-G11 , HeLa Cells , Humans , Jurkat Cells , K562 Cells , RGS Proteins/metabolism , Receptor, Muscarinic M1 , Receptors, Adrenergic, beta/metabolism , Receptors, Muscarinic/metabolism , Signal Transduction
11.
Mol Pharmacol ; 58(3): 569-76, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10953050

ABSTRACT

Regulator of G protein signaling (RGS) proteins are a family of approximately 20 proteins that negatively regulate signaling through heterotrimeric G protein-coupled receptors. The RGS proteins act as GTPase-activating proteins (GAPs) for certain Galpha subunits and as effector antagonists for Gqalpha. Mouse RGS14 encodes a 547-amino-acid protein with an N-terminal RGS domain, which is highly expressed in lymphoid tissues. In this study, we demonstrate that RGS14 is a GAP for Gialpha subfamily members and it attenuates interleukin-8 receptor-mediated mitogen-activated protein kinase activation. However, RGS14 does not exhibit GAP activity toward Gsalpha or Gqalpha nor does it regulate Gsalpha- or Gqalpha-mediated signaling pathways. Although RGS14 does not act as a GAP for G12/13alpha, it impairs c-fos serum response element activation induced by either a constitutively active mutant of G13alpha (G13alphaQ226L) or by carbachol stimulation of muscarinic type 1 receptors. An RGS14 mutant (EN92/93AA), which does not block Gialpha-linked signaling, also inhibits serum response element activation. RGS14 localizes predominantly in the cytosol, but it can be recruited to membranes by expression of G13alphaQ226L. Although RGS14 is constitutively expressed in lymphoid cells, agents that activate B or T lymphocytes further enhance its levels. Taken together, our results suggest that signals generated after lymphocyte activation may via RGS14 directly impinge on Gialpha- or G13alpha-mediated cellular processes in lymphocytes, such as adhesion and migration.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTPase-Activating Proteins/metabolism , RGS Proteins/metabolism , Animals , B-Lymphocytes/metabolism , COS Cells , Cell Membrane/metabolism , Cells, Cultured , Cytoplasm/metabolism , Gene Expression , Humans , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , RGS Proteins/genetics , Signal Transduction , Subcellular Fractions , T-Lymphocytes/metabolism
12.
J Biol Chem ; 275(32): 24470-6, 2000 Aug 11.
Article in English | MEDLINE | ID: mdl-10821841

ABSTRACT

G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Amino Acid Substitution , Animals , COS Cells , Focal Adhesion Kinase 2 , GTP Phosphohydrolases/metabolism , GTP-Binding Protein alpha Subunits, G12-G13 , Genes, Reporter , HeLa Cells , Heterotrimeric GTP-Binding Proteins/chemistry , Humans , Luciferases/genetics , Mutagenesis, Site-Directed , Phosphorylation , Recombinant Proteins/metabolism , Sequence Deletion , Transcription, Genetic , Transfection , rhoA GTP-Binding Protein/metabolism
13.
Oncogene ; 19(7): 933-42, 2000 Feb 17.
Article in English | MEDLINE | ID: mdl-10702802

ABSTRACT

Radiation resistance is a hallmark of human melanoma, and yet mechanisms underlying this resistance are not well understood. We recently established the role of ATF2 in this process, suggesting that stress kinases, which contribute to regulation of ATF2 stability and activity, play an important role in the acquisition of such resistance. Here we demonstrate that changes in the expression and respective activities of TRAF2/GCK occur during melanoma development and regulate its sensitivity to UV-induced apoptosis. Comparing early- and late-stage melanoma cells revealed low expression of TRAF2 and GCK in early-stage melanoma, which coincided with poor resistance to UV-induced, TNF-mediated apoptosis; forced expression of GCK alone or in combination with TRAF2 efficiently increased JNK and NF-kappaB activities, which coincided with increased protection against apoptosis. Conversely, forced expression of the dominant negative form of TRAF2 or GCK in late-stage melanoma cells reduced NF-kappaB activity and decreased Fas expression, resulting in a lower degree of UV-induced, Fas-mediated cell death. Our results illustrate a mechanism in which protection from, or promotion of, UV-induced melanoma cell death depends on the nature of the apoptotic cascade (TNF or Fas) and on the availability of TRAF2/GCK, whose expression increases during melanoma progression. Oncogene (2000) 19, 933 - 942.


Subject(s)
Apoptosis/radiation effects , Melanoma/metabolism , Melanoma/pathology , Protein Serine-Threonine Kinases/physiology , Proteins/physiology , Radiation Tolerance , Ultraviolet Rays , Germinal Center/enzymology , Germinal Center/radiation effects , Germinal Center Kinases , Humans , Melanoma/enzymology , NF-kappa B/physiology , Neoplasm Staging , Protein Biosynthesis , Signal Transduction/radiation effects , TNF Receptor-Associated Factor 2 , Tumor Cells, Cultured
14.
J Biol Chem ; 275(10): 7365-72, 2000 Mar 10.
Article in English | MEDLINE | ID: mdl-10702309

ABSTRACT

Atrial natriuretic peptide (ANP) inhibits the proliferation of many cells, in part through interfering with signal transduction enacted by G protein-coupled growth factor receptors. Signaling interactions between ANP and the G protein-coupled growth factor receptor ligand, endothelin-3 (ET-3), regulate astrocyte proliferation at a very proximal but undefined point. Here, we find that ANP inhibits the ability of ET-3 to activate Galpha(q) and Galpha(i) in these cells. ANP stimulated the translocation of endogenous regulators of G protein-signaling (RGS) proteins 3 and 4 from the cytosol to the cell membrane, and enhanced their association with Galpha(q) and Galpha(i). ANP effects were significantly blocked by HS-142-1, an inhibitor of guanylate cyclase activation, or by ET-3. KT5823, an inhibitor of cyclic GMP-dependent protein kinase (PKG) reversed the RGS translocation induced by ANP; conversely, expression of an active catalytic subunit of PKG-I, or 8-bromo-cyclic GMP stimulated RGS translocation. ANP caused the phosphorylation of both RGS proteins in a PKG-dependent fashion, and the expressed PKG (in the absence of ANP) also stimulated RGS phosphorylation. A novel cross-talk between PKG and RGS proteins is stimulated by ANP and leads to the increased translocation and association of RGS proteins with Galpha. The rapid inactivation of G proteins provides a mechanism by which ANP inhibits downstream signaling to the cell proliferation program.


Subject(s)
Atrial Natriuretic Factor/pharmacology , Cyclic GMP-Dependent Protein Kinases/physiology , GTP-Binding Proteins/antagonists & inhibitors , RGS Proteins/physiology , Biological Transport , Endothelin-3/pharmacology , GTP Phosphohydrolases/drug effects , Phosphorylation
15.
J Immunol ; 164(4): 1829-38, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10657631

ABSTRACT

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.


Subject(s)
B-Lymphocytes/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Proteins/physiology , RGS Proteins , Signal Transduction/immunology , Animals , B-Lymphocytes/immunology , COS Cells , Down-Regulation/immunology , GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors , GTPase-Activating Proteins/physiology , Humans , Jurkat Cells , K562 Cells , Protein Binding/immunology , Protein Biosynthesis , Rats , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/physiology , Tumor Cells, Cultured
16.
Blood ; 95(3): 776-82, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10648385

ABSTRACT

STE20-related kinases play significant regulatory roles in a range of cellular responses to environmental stimuli. GCKR (also referred to as KHS1) is a serine/threonine protein kinase that has an STE20-like protein kinase domain and that stimulates the stress-activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. GCKR has a large C-terminal regulatory domain that provides sites for interactions with other proteins. Adaptor proteins mediate the interactions between signaling molecules. In this study we showed that the adaptor proteins Crk and CrkL associated with GCKR. When Crk-I, Crk-II, or CrkL was transiently expressed in HEK 293T cells along with GCKR, each coimmunoprecipitated with GCKR. Furthermore, in the Bcr-Abl transformed cell line, K562 endogenous GCKR and CrkL coimmunoprecipitated, indicating a constitutive association. Detection of the CrkL-GCKR interaction required the SH3 domains of CrkL and 2 regions in GCKR-1 between amino acids 387 and 395 that contains a consensus SH3 binding motif and the other between amino acids 599 and 696. Crk or CrkL overexpression increased GCKR catalytic activity. A dominant negative form of Ras abolished Crk- or CrkL-induced GCKR activation, suggesting a dependence on Ras activation for their activation of GCKR. Finally, we showed impairment of the known ability of CrkL to activate the SAPK pathway by a catalytically inactive form of GCKR or by a GCKR antisense construct. Thus, GCKR associates with other proteins through interactions mediated by SH2/SH3 adaptor proteins, which can lead to GCKR and SAPK activation.


Subject(s)
Adaptor Proteins, Signal Transducing , Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins/physiology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/physiology , Signal Transduction/physiology , Cell Line , Enzyme Activation , Gene Expression Regulation , Genes, Dominant , Genes, ras , Humans , JNK Mitogen-Activated Protein Kinases , K562 Cells , Macromolecular Substances , Nuclear Proteins/genetics , Oligonucleotides, Antisense/pharmacology , Peptide Fragments/pharmacology , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-crk , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/physiology , Recombinant Fusion Proteins/physiology , Transfection , src Homology Domains
17.
Mol Cell Biol ; 19(10): 6665-72, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10490605

ABSTRACT

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are mediators of many members of the TNF receptor superfamily and can activate both the nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK; also known as c-Jun N-terminal kinase) signal transduction pathways. We previously described the involvement of a TRAF-interacting molecule, TRAF-associated NF-kappaB activator (TANK), in TRAF2-mediated NF-kappaB activation. Here we show that TANK synergized with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually formed weak interactions with germinal center kinase (GCK)-related kinase (GCKR). However, when coexpressed, they formed a strong complex with GCKR, thereby providing a potential mechanism for TRAF and TANK synergy in GCKR-mediated SAPK activation, which is important in TNF family receptor signaling. Our results also suggest that TANK can form potential intermolecular as well as intramolecular interactions between its amino terminus and carboxyl terminus. This study suggests that TANK is a regulatory molecule controlling the threshold of NF-kappaB and SAPK activities in response to activation of TNF receptors. In addition, CD40 activated endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions.


Subject(s)
Adaptor Proteins, Signal Transducing , Lymphocytes/metabolism , MAP Kinase Kinase 4 , MAP Kinase Kinase Kinase 1 , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , B-Lymphocytes/metabolism , CD40 Antigens/metabolism , Enzyme Activation , Germinal Center/cytology , Germinal Center Kinases , Humans , MAP Kinase Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Palatine Tonsil/cytology , Peptide Fragments/metabolism , Proteins/genetics , T-Lymphocytes/metabolism , TNF Receptor-Associated Factor 2 , TNF Receptor-Associated Factor 5 , TNF Receptor-Associated Factor 6
18.
Neuron ; 23(4): 675-87, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10482235

ABSTRACT

Sonic hedgehog (Shh) specifies the identity of both motor neurons (MNs) and interneurons with morphogen-like activity. Here, we present evidence that the homeodomain factor HB9 is critical for distinguishing MN and interneuron identity in the mouse. Presumptive MN progenitors and postmitotic MNs express HB9, whereas interneurons never express this factor. This pattern resembles a composite of the avian homologs MNR2 and HB9. In mice lacking Hb9, the genetic profile of MNs is significantly altered, particularly by upregulation of Chx10, a gene normally restricted to a class of ventral interneurons. This aberrant gene expression is accompanied by topological disorganization of motor columns, loss of the phrenic and abducens nerves, and intercostal nerve pathfinding defects. Thus, MNs actively suppress interneuron genetic programs to establish their identity.


Subject(s)
Gene Expression Regulation, Developmental/genetics , Genes, Homeobox/genetics , Hedgehog Proteins/physiology , Homeodomain Proteins/biosynthesis , Interneurons/physiology , Motor Neurons/physiology , Transcription Factors/biosynthesis , Xenopus Proteins , Animals , Axons/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Chick Embryo , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/genetics , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Transgenic , Mitosis/physiology , Repressor Proteins/genetics , Stem Cells/physiology , Transcription Factors/genetics , Xenopus laevis/physiology
19.
J Immunol ; 163(6): 3279-85, 1999 Sep 15.
Article in English | MEDLINE | ID: mdl-10477597

ABSTRACT

TNF-induced activation of stress activated protein kinases (SAPKs, Jun NH2-terminal kinases) requires TNF receptor associated factor 2 (TRAF2). TRAF2 is a potent activator of a 95-kDa serine/threonine kinase termed germinal center kinase related (GCKR, also referred to as KHS1), which signals activation of the SAPK pathway. Consistent with a role for GCKR in TNF- induced SAPK activation, a kinase-inactive mutant of GCKR is a dominant negative inhibitor of TRAF2-induced SAPK activation. Here we show that TRAF2 interacts with GCKR. This interaction depended upon the TRAF domain of TRAF2 and the C-terminal 150 aa of GCKR. The full activation of GCKR by TRAF2 required the TRAF2 RING finger domain. TNF treatment of a T cell line, Jurkat, increased both GCRK and SAPK activity and enhanced the coimmunoprecipitation of GCKR with TRAF2. Similar results were found with the B cell line HS-Sultan. These findings are consistent with a model whereby TNF signaling results in the recruitment and activation of GCKR by TRAF2, which leads to SAPK activation.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Germinal Center/enzymology , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/metabolism , Proteins/physiology , Receptors, Tumor Necrosis Factor/physiology , Signal Transduction/immunology , Tumor Necrosis Factor-alpha/physiology , Amino Acid Sequence , Catalytic Domain/immunology , Cell Line , Enzyme Activation/genetics , Enzyme Activation/immunology , Germinal Center Kinases , Humans , JNK Mitogen-Activated Protein Kinases , Jurkat Cells , Molecular Sequence Data , NF-kappa B/metabolism , Protein Biosynthesis , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/chemistry , Proteins/genetics , Proteins/metabolism , Receptors, Tumor Necrosis Factor/genetics , Sequence Deletion , Signal Transduction/genetics , TNF Receptor-Associated Factor 2 , Tumor Necrosis Factor-alpha/genetics
20.
Nat Genet ; 23(1): 71-5, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10471502

ABSTRACT

In most mammals the pancreas develops from the foregut endoderm as ventral and dorsal buds. These buds fuse and develop into a complex organ composed of endocrine, exocrine and ductal components. This developmental process depends upon an integrated network of transcription factors. Gene targeting experiments have revealed critical roles for Pdx1, Isl1, Pax4, Pax6 and Nkx2-2 (refs 3,4,5,6,7, 8,9,10). The homeobox gene HLXB9 (encoding HB9) is prominently expressed in adult human pancreas, although its role in pancreas development and function is unknown. To facilitate its study, we isolated the mouse HLXB9 orthologue, Hlxb9. During mouse development, the dorsal and ventral pancreatic buds and mature beta-cells in the islets of Langerhans express Hlxb9. In mice homologous for a null mutation of Hlxb9, the dorsal lobe of the pancreas fails to develop. The remnant Hlxb9-/- pancreas has small islets of Langerhans with reduced numbers of insulin-producing beta-cells. Hlxb9-/- beta-cells express low levels of the glucose transporter Glut2 and homeodomain factor Nkx 6-1. Thus, Hlxb9 is key to normal pancreas development and function.


Subject(s)
Homeodomain Proteins/genetics , Islets of Langerhans/abnormalities , Nerve Tissue Proteins , Pancreas/abnormalities , Transcription Factors/genetics , Animals , DNA-Binding Proteins/metabolism , Eye Proteins , Forkhead Transcription Factors , Genotype , Glucagon/metabolism , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/metabolism , Humans , Immunohistochemistry , Insulin/metabolism , Islets of Langerhans/embryology , Islets of Langerhans/metabolism , LIM-Homeodomain Proteins , Mice , Models, Genetic , Molecular Sequence Data , Motor Neurons/metabolism , Nuclear Proteins , PAX6 Transcription Factor , Paired Box Transcription Factors , Pancreas/embryology , Pancreas/metabolism , Pancreatic Polypeptide/metabolism , Repressor Proteins , Somatostatin/metabolism , Time Factors , Trans-Activators/metabolism , Transcription Factors/metabolism , Zebrafish Proteins
SELECTION OF CITATIONS
SEARCH DETAIL
...