G protein-coupled receptor kinase 5 regulates beta 1-adrenergic receptor association with PSD-95.

We previously reported that the beta(1)-adrenergic receptor (beta(1)AR) associates with PSD-95 through a PDZ domain-mediated interaction, by which PSD-95 modulates beta(1)AR function and facilitates the physical association of beta(1)AR with other synaptic proteins such as N-methyl-d-aspartate receptors. Here we demonstrate that beta(1)AR association with PSD-95 is regulated by G protein-coupled receptor kinase 5 (GRK5). When beta(1)AR and PSD-95 were coexpressed with either GRK2 or GRK5 in COS-7 cells, GRK5 alone dramatically decreased the association of beta(1)AR with PSD-95, although GRK2 and GRK5 both could be co-immunoprecipitated with beta(1)AR and both could enhance receptor phosphorylation in vivo. Increasing expression of GRK5 in the cells led to further decreased beta(1)AR association with PSD-95. Stimulation with the beta(1)AR agonist isoproterenol further decreased PSD-95 binding to beta(1)AR. In addition, GRK5 protein kinase activity was required for this regulatory effect since a kinase-inactive GRK5 mutant had no effect on PSD-95 binding to beta(1)AR. Moreover, the regulatory effect of GRK5 on beta(1)AR association with PSD-95 was observed only when GRK5 was expressed together with the receptor, but not when GRK5 was coexpressed with PSD-95. Thus, we propose that GRK5 regulates beta(1)AR association with PSD-95 through phosphorylation of beta(1)AR. Regulation of protein association through receptor phosphorylation may be a general mechanism used by G protein-coupled receptors that associate via PDZ domain-mediated protein/protein interactions.     

Molecular mechanisms regulating the differential association of kainate receptor subunits with SAP90/PSD-95 and SAP97

Recent studies have demonstrated that kainate receptors are associated with members of the SAP90/PSD-95 family (synapse-associated proteins (SAPs)) in neurons and that SAP90 can cluster and modify the electrophysiological properties of GluR6/KA2 kainate receptors when co-expressed in transfected cells. In vivo, SAP90 tightly binds kainate receptor subunits, while SAP97 is only weakly associated, suggesting that this glutamate receptor differentially associates with SAP90/PSD-95 family members. Here, green fluorescent protein (GFP)-tagged chimeras and deletion mutants of SAP97 and SAP90 were employed to define the molecular mechanism underlying their differential association with kainate receptors. Our results show that a weak interaction between GluR6 and the PDZ1 domain of SAP97 can account for the weak association of GluR6 with the full-length SAP97 observed in vivo. Expression studies in HEK293 cells and in vitro binding studies further show that although the individual Src homology 3 and guanylate kinase domains in SAP97 can interact with the C-terminal tail of KA2 subunit, specific intramolecular interactions in SAP97 (e.g. the SAP97 N terminus (S97N) binding to the Src homology 3 domain) interfere with KA2 binding to the full-length molecule. Because receptor subunits are known to segregate to different parts of the neuron, our results imply that differential association of kainate receptors with SAP family proteins may be one mechanism of subcellular localization.     

AKAP79-mediated targeting of the cyclic AMP-dependent protein kinase to the beta1-adrenergic receptor promotes recycling and functional resensitization of the receptor

Resensitization of G protein-coupled receptors (GPCR) following prolonged agonist exposure is critical for restoring the responsiveness of the receptor to subsequent challenges by agonist. The 3'-5' cyclic AMP-dependent protein kinase (PKA) and serine 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) were both necessary for efficient recycling and resensitization of the agonist-internalized beta(1)-AR (Gardner, L. A., Delos Santos, N. M., Matta, S. G., Whitt, M. A., and Bahouth, S. W. (2004) J. Biol. Chem. 279, 21135-21143). Because PKA is compartmentalized near target substrates by interacting with protein kinase A anchoring proteins (AKAPs), the present study was undertaken to identify the AKAP involved in PKA-mediated phosphorylation of the beta(1)-AR and in its recycling and resensitization. Here, we report that Ht-31 peptide-mediated disruption of PKA/AKAP interactions prevented the recycling and functional resensitization of heterologously expressed beta(1)-AR in HEK-293 cells and endogenously expressed beta(1)-AR in SK-N-MC cells and neonatal rat cortical neurons. Whereas several endogenous AKAPs were identified in HEK-293 cells, small interfering RNA-mediated down-regulation of AKAP79 prevented the recycling of the beta(1)-AR in this cell line. Co-immunoprecipitations and fluorescence resonance energy transfer (FRET) microscopy experiments in HEK-293 cells revealed that the beta(1)-AR, AKAP79, and PKA form a ternary complex at the carboxyl terminus of the beta(1)-AR. This complex was involved in PKA-mediated phosphorylation of the third intracellular loop of the beta(1)-AR because disruption of PKA/AKAP interactions or small interfering RNA-mediated down-regulation of AKAP79 both inhibited this response. Thus, AKAP79 provides PKA to phosphorylate the beta(1)-AR and thereby dictate the recycling and resensitization itineraries of the beta(1)-AR.     

beta 1-adrenergic receptor association with the synaptic scaffolding protein membrane-associated guanylate kinase inverted-2 (MAGI-2). Differential regulation of receptor internalization by MAGI-2 and PSD-95

The beta1-adrenergic receptor (beta1AR) is known to be localized to synapses and to modulate synaptic plasticity in many brain regions, but the molecular mechanisms determining beta1AR subcellular localization are not fully understood. Using overlay and pull-down techniques, we found that the beta1AR carboxyl terminus associates with MAGI-2 (membrane-associated guanylate kinase inverted-2), a protein also known as S-SCAM (synaptic scaffolding molecule). MAGI-2 is a multidomain scaffolding protein that contains nine potential protein-protein interaction modules, including 6 PDZ domains, 2 WW domains, and a guanylate kinase-like domain. The beta1AR carboxyl terminus binds with high affinity to the first PDZ domain of MAGI-2, with the last few amino acids of the beta1AR carboxyl terminus being the key determinants of the interaction. In cells, the association of full-length beta1AR with MAGI-2 occurs constitutively and is enhanced by agonist stimulation of the receptor, as assessed by both co-immunoprecipitation experiments and immunofluorescence co-localization studies. Agonist-induced internalization of the beta1AR is markedly increased by co-expression with MAGI-2. Strikingly, this result is the opposite of the effect of co-expression with PSD-95, a previously reported binding partner of the beta1AR. Further cellular experiments revealed that MAGI-2 has no effect on beta1AR oligomerization but does promote association of beta1AR with the cytoplasmic signaling protein beta-catenin, a known MAGI-2 binding partner. These data reveal that MAGI-2 is a specific beta1AR binding partner that modulates beta1AR function and facilitates the physical association of the beta1AR with intracellular proteins involved in signal transduction and synaptic regulation.     

Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90. A possible regulatory role in molecular clustering at synaptic sites

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.     

The palmitoylation state of the beta(2)-adrenergic receptor regulates the synergistic action of cyclic AMP-dependent protein kinase and beta-adrenergic receptor kinase involved in its phosphorylation and desensitization

Although palmitoylation of the beta(2)-adrenergic receptor (beta(2)AR), as well as its phosphorylation by the cyclic AMP-dependant protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK), are known to play important roles in agonist-promoted desensitization, their relative contribution and mutual regulatory influences are still poorly understood. In this study, we investigated the role that the carboxyl tail PKA site (Ser(345,346)) of the beta(2)AR plays in its rapid agonist-promoted phosphorylation and desensitization. Mutation of this site (Ala(345,346)beta(2)AR) significantly reduced the rate and extent of the rapid desensitization promoted by sustained treatment with the agonist isoproterenol. The direct contribution of Ser(345,346) in desensitization was then studied by mutating all other putative PKA and beta ARK phosphorylation sites (Ala(261,262)beta ARK(-)beta(2)AR). We found this mutant receptor to be phosphorylated upon receptor activation but not following direct activation of PKA, suggesting a role in receptor-specific (homologous) but not heterologous phosphorylation. However, despite its phosphorylated state, Ala(261,262)beta ARK(-)beta(2)AR did not undergo rapid desensitization upon agonist treatment, indicating that phosphorylation of Ser(345,346) alone is not sufficient to promote desensitization. Taken with the observation that mutation of either Ser(345,346) or of the beta ARK phosphorylation sites prevented both the hyper-phosphorylation and constitutive desensitization of a palmitoylation-less mutant (Gly(341)beta(2)AR), our data suggest a concerted/synergistic action of the two kinases that depends on the palmitoylation state of the receptor. Consistent with this notion, in vitro phosphorylation of Gly(341)beta(2)AR by the catalytic subunit of PKA facilitated further phosphorylation of the receptor by purified beta ARK. Our study therefore allows us to propose a coordinated mechanism by which sequential depalmitoylation, and phosphorylation by PKA and beta ARK lead to the functional uncoupling and desensitization of the ss(2)AR.     

Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase.

Human SK-N-MC neurotumor cells express beta 1- but not beta 2-adrenergic receptors. Following exposure of the cells to isoproterenol, there was no reduction in the maximum response of adenylyl cyclase to the agonist but a 3-fold shift to less sensitivity in the concentration response. This desensitization was very rapid and dose dependent; half-maximal effects occurred at 10 nM isoproterenol. A similar shift was observed when membranes from control cells were incubated with ATP and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). No shift, however, was observed in intact cells exposed to either dibutyryl cyclic AMP or dopamine, which stimulates adenylyl cyclase in these cells through D1 dopamine receptors. To pursue the role of protein kinases in the desensitization process, cells were made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), and exposed to isoproterenol. The PKA inhibitor but not heparin blocked the agonist-mediated desensitization. In contrast, desensitized human tumor cells (HeLa and A431), which express beta 2-adrenergic receptors, exhibited both a shift in concentration response and a reduction in maximum response; the former was blocked by the PKA inhibitor and the latter by heparin. Our results indicated that whereas both human beta 1- and beta 2-adrenergic receptors are susceptible to PKA, only the beta 2 receptors are susceptible to beta ARK. These differences in desensitization may be due to differences in receptor structure as the human beta 1 receptor has fewer potential phosphorylation sites for beta ARK in the carboxyl terminus than the human beta 2 receptor.     

beta 2-adrenergic receptor activates extracellular signal-regulated kinases (ERKs) via the small G protein rap1 and the serine/threonine kinase B-Raf

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. Heterotrimeric G proteins are composed of both alpha and betagamma subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases. In this study, we examined the ability of isoproterenol, an agonist of the beta(2)-adrenergic receptor (beta(2)AR), to stimulate extracellular signal-regulated kinases (ERKs). Using HEK293 cells, which express endogenous beta(2)AR, we show that isoproterenol stimulates ERKs via beta(2)AR. This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Galpha(s) activation of cAMP-dependent protein kinase is required. Interestingly, beta(2)AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms. beta(2)AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf. beta(2)AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling. We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.     

Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen.

TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. Here, we show that the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) of this sequence bound TAR RNA with affinities similar to that of the entire TRBP, thus suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, we determined that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovered TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment revealed that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. We suggest that these proteins are related by virtue of sharing a common RNA-binding moiety.     

Phosphorylation of the Gq/11-coupled m3-muscarinic receptor is involved in receptor activation of the ERK-1/2 mitogen-activated protein kinase pathway

We investigated the role played by agonist-mediated phosphorylation of the G(q/11)-coupled M(3)-muscarinic receptor in the mechanism of activation of the mitogen-activated protein kinase pathway, ERK-1/2, in transfected Chinese hamster ovary cells. A mutant of the M(3)-muscarinic receptor, where residues Lys(370)-Ser(425) of the third intracellular loop had been deleted, showed a reduced ability to activate the ERK-1/2 pathway. This reduction was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. Importantly, the ERK-1/2 responses to both the wild-type M(3)-muscarinic receptor and DeltaLys(370)-Ser(425) receptor mutant were dependent on the activity of protein kinase C. Our results, therefore, indicate the existence of two mechanistic components to the ERK-1/2 response, which appear to act in concert. First, the activation of protein kinase C through the diacylglycerol arm of the phospholipase C signaling pathway and a second component, absent in the DeltaLys(370)-Ser(425) receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the DeltaLys(370)-Ser(425) receptor mutant to activate the ERK-1/2 pathway correlated with an approximately 80% decrease in the ability of the receptor to undergo agonist-mediated phosphorylation. Furthermore, we have previously shown that M(3)-muscarinic receptor phosphorylation can be inhibited by a dominant negative mutant of casein kinase 1alpha and by expression of a peptide corresponding to the third intracellular loop of the M(3)-muscarinic receptor. Expression of these inhibitors of receptor phosphorylation reduced the wild-type M(3)-muscarinic receptor ERK-1/2 response. We conclude that phosphorylation of the M(3)-muscarinic receptor on sites in the third intracellular loop by casein kinase 1alpha contributes to the mechanism of receptor activation of ERK-1/2 by working in concert with the diacylglycerol/PKC arm of the phospholipase C signaling pathway.     

RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells

PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.     

Regulation of adrenergic receptor function by phosphorylation. II. Effects of agonist occupancy on phosphorylation of alpha 1- and beta 2-adrenergic receptors by protein kinase C and the cyclic AMP-dependent protein kinase

The present study was undertaken to determine the ability of protein kinase C and protein kinase A to directly phosphorylate the purified alpha 1- and beta 2-adrenergic receptors (AR). Both the catalytic subunit of protein kinase A and the protein kinase C, purified from bovine heart and pig brain, respectively, are able to phosphorylate the purified alpha 1-AR from DDT1 MF-2 smooth muscle cells. Occupancy of the receptor by an alpha 1 agonist, norepinephrine (100 microM), increases the rate of phosphorylation by protein kinase C but not by protein kinase A. The maximum stoichiometry of phosphorylation obtained is not affected by the agonist and reached 3 mol of PO4/mol of receptor for protein kinase C and 1 mol of PO4/mol of receptor for protein kinase A. The phosphopeptide maps of the trypsinized alpha 1-AR phosphorylated by each kinase differ drastically. The beta 2-AR purified from hamster lungs can also be phosphorylated by the two kinases. In contrast to the alpha 1-AR, the occupancy of the beta 2-AR by the agonist isoproterenol (20 microM) increases the rate of phosphorylation of the beta 2-AR by protein kinase A but not by protein kinase C. The maximum amount of phosphate incorporated into the receptor is not affected in either case by the agonist and reaches 1 mol of PO4/mol of receptor with protein kinase A and 0.4 mol of PO4/mol of receptor with protein kinase C. The phosphopeptide maps of the trypsinized receptor phosphorylated by either kinase reveal similar profiles. Thus, both alpha 1-AR and beta 2-AR are substrates for protein kinase A and protein kinase C. Agonist occupancy of the two receptors facilitates their phosphorylation only by the protein kinase coupled to their own signal transduction pathway. These observations suggest that "feedback" and "cross-system" phosphorylation may represent distinct and differently regulated mechanisms of modulation of receptor function.     

Function of the alpha 1 beta 2 gamma 2S gamma-aminobutyric acid type A receptor is modulated by protein kinase C via multiple phosphorylation sites.

Activation of protein kinase C (PKC) results in down-modulation of the gamma-aminobutyric acid type A (GABAA) receptor. In this study, the recombinant subunit combination alpha 1 beta 2 gamma 2S was expressed in Xenopus oocytes. The resulting channel was shown to be modulated by 2 microM oleoylacetylglycerol or, stereo-specifically, by low concentrations (10 nM) of the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. By site-specific mutagenesis, we altered the serine or threonine residues of consensus phosphorylation sites for PKC in the large, intracellular domain of alpha 1, beta 2, and gamma 2S. Mutant subunits were co-expressed with wild type subunits to yield alpha 1 beta 2 gamma 2S combinations. All of the tested 14 mutations did not affect the level of expression of GABA current. Two of these mutations, Ser-410 in beta 2 and Ser-327 in gamma 2S, resulted in a significant reduction of the effect of the activator of PKC, 4 beta-phorbol 12-myristate 13-acetate, on the GABA current amplitude. Thus, we have identified two single serine residues, Ser-410 in the subunit beta 2 and Ser-327 in gamma 2S, as phosphorylation sites of a PKC endogenous to Xenopus oocytes. Co-expression of the mutant subunits suggests that phosphorylation of both sites is required for a full, PKC-mediated down-regulation of GABA currents.     

Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)     

G-protein-coupled receptor stimulation of the p42/p44 mitogen-activated protein kinase pathway is attenuated by lipid phosphate phosphatases 1, 1a, and 2 in human embryonic kidney 293 cells

Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid phosphate phosphatases (1, 1a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid phosphate phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. In this study, we show that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid, all of which bind to G(i/o)-coupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid phosphate phosphatases 1, 1a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid phosphate phosphatase activity. These findings were supported by results showing that lipid phosphate phosphatases 1, 1a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G(i/o)-coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid phosphate phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid phosphate phosphatases 1, 1a, and 2 may function to perturb G-protein-coupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors.     

Regulation of the phosphatidylinositol 3-kinase,Akt/protein kinase B,FRAP/mammalian target of rapamycin,and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the thyroid gland

Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85alpha regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85alpha regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo.     

Inhibition of T-cell receptor signal transduction and viral expression by the linker for activation of T cells-interacting p12(I) protein of human T-cell leukemia/lymphoma virus type 1

The p12(I) protein of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is a small oncoprotein that increases calcium release following protein kinase C activation by phorbol myristate acetate, and importantly, this effect is linker for activation of T cells (LAT) independent. Here, we demonstrate that p12(I) inhibits the phosphorylation of LAT, Vav, and phospholipase C-gamma 1 and decreases NFAT (nuclear factor of activated T cells) activation upon engagement of the T-cell receptor (TCR) with anti-CD3 antibody. Furthermore, we demonstrate that p12(I) localizes to membrane lipid rafts and, upon engagement of the TCR, relocalizes to the interface between T cells and antigen-presenting cells, defined as the immunological synapse. A p12(I) knockout molecular clone of HTLV-1 expresses more virus upon antigen stimulation than the isogenic wild type, suggesting that, by decreasing T-cell responsiveness, p12(I) curtails viral expression. Thus, p12(I) has contrasting effects on TCR signaling: it down-regulates TCR in a LAT-dependent manner on one hand, and on the other, it increases calcium release in a LAT-independent manner. The negative regulation of T-cell activation by p12(I) may have evolved to minimize immune recognition of infected CD4(+) T cells, to impair the function of infected cytotoxic CD8(+) T cells, and to favor viral persistence in the infected host.     

In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.