scholarly journals Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

2004 ◽  
Vol 279 (44) ◽  
pp. 46242-46252 ◽  
Author(s):  
Larry E. Wagner ◽  
Wen-Hong Li ◽  
Suresh K. Joseph ◽  
David I. Yule
Keyword(s):  
Splice Variants ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Igm Antibody ◽  
Temporal Properties ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Spatio Temporal ◽  
In Cells

Regulation of Ca2+release through inositol 1,4,5-trisphosphate receptors (InsP3R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca2+signals. In this study, regulation of Ca2+release by phosphorylation of type 1 InsP3R (InsP3R-1) was investigated by constructing “phosphomimetic” charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP3R-1 splice variants. Ca2+release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP3R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP3R-1, InsP3-induced Ca2+release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca2+release through the S2- S1589E/S1755E InsP3R-1 was enhanced ∼8-fold over wild type and ∼50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP3R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca2+release was enhanced ∼3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca2+release. The sensitivity of Ca2+release in cells expressing S2+ S1755E InsP3R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP3R-1. In contrast, mutation of S2+ S1589E InsP3R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP3R-1 resulted in robust Ca2+oscillations when cells were stimulated with concentrations of α-IgM antibody that were threshold for stimulation in S2- wild type InsP3R-1-expressing cells. However, at higher concentrations of α-IgM antibody, Ca2+oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP3R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.

scholarly journals PDGF receptor-β modulates metanephric mesenchyme chemotaxis induced by PDGF AA

2009 ◽  
Vol 296 (2) ◽  
pp. F406-F417 ◽  
Author(s):  
Jill M. Ricono ◽  
Brent Wagner ◽  
Yves Gorin ◽  
Mazen Arar ◽  
Andrius Kazlauskas ◽  
...  
Keyword(s):  
Cell Migration ◽  
Kidney Development ◽  
Receptor Activation ◽  
Ros Generation ◽  
Pdgf Receptor ◽  
Metanephric Mesenchyme ◽  
Wild Type ◽  
Intracellular Ca ◽  
Β Receptor ◽  
In Cells

PDGF B chain or PDGF receptor (PDGFR)-β-deficient (−/−) mice lack mesangial cells. To study responses of α- and β-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and −/− mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for −/− MMCs. The mechanism by which PDGFR-β inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular Ca2+ and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of −/− MMCs with the wild-type β-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of Ca2+ signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular Ca2+ concentration, suggesting that ROS act as upstream mediators of Ca2+ in suppressing PDGF AA-induced migration. These data indicate that ROS and Ca2+ generated by active PDGFR-β play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs. During kidney development, PDGFR β-mediated ROS generation and Ca2+ influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme.

scholarly journals Regions of the Herpes Simplex Virus Type 1 Latency-Associated Transcript That Protect Cells from Apoptosis In Vitro and Protect Neuronal Cells In Vivo

2002 ◽  
Vol 76 (2) ◽  
pp. 717-729 ◽  
Author(s):  
Maryam Ahmed ◽  
Martin Lock ◽  
Cathie G. Miller ◽  
Nigel W. Fraser
Keyword(s):  
Herpes Simplex ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Exon 1 ◽  
Induced Apoptosis ◽  
Simplex Virus ◽  
In Cells

ABSTRACT Recent studies have suggested that the latency-associated transcript (LAT) region of herpes simplex virus type 1 (HSV-1) is effective at blocking virus-induced apoptosis both in vitro and in the trigeminal ganglia of acutely infected rabbits (Inman et al., J. Virol. 75:3636–3646, 2001; Perng et al., Science 287:1500–1503, 2000). By transfecting cells with a construct expressing the Pst-Mlu segment of the LAT, encompassing the LAT exon 1, the stable 2.0-kb intron, and 5′ part of exon 2, we confirmed that this region was able to diminish the onset of programmed cell death initiated by anti-Fas and camptothecin treatment. In addition, caspase 8-induced apoptosis was specifically inhibited in cells expressing the Pst-Mlu LAT fragment. To further delineate the minimal region of LAT that is necessary for this antiapoptotic function, LAT mutants were used in our cotransfection assays. In HeLa cells, the plasmids lacking exon sequences were the least effective at blocking apoptosis. However, similar to previous work (Inman et al., op. cit.), our data also indicated that the 5′ end of the stable 2.0-kb LAT intron appeared to contribute to the promotion of cell survival. Furthermore, cells productively infected with the 17N/H LAT mutant virus, a virus deleted in the LAT promoter, exon 1, and about half of the intron, exhibited a greater degree of DNA fragmentation than cells infected with wild-type HSV-1. These data support the finding that the exon 1 and 2.0-kb intron region of the LAT transcription unit display an antiapoptotic function both in transfected cells and in the context of the virus infection in vitro. In trigeminal ganglia of mice acutely infected with the wild-type virus, 17, and 17ΔSty, a virus lacking most of exon 1, apoptosis was not detected in cells that were positive for virus particles. However, dual staining was observed in cells from mice infected with 17N/H virus, indicating that the LAT antiapoptotic function demonstrated in cells transfected by LAT-expressing constructs may also play a role in protecting cells from virus-induced apoptosis during acute viral infection in vivo.

scholarly journals Opposing Effects of a Tyrosine-Based Sorting Motif and a PDZ-Binding Motif Regulate Human T-Lymphotropic Virus Type 1 Envelope Trafficking

2010 ◽  
Vol 84 (14) ◽  
pp. 6995-7004 ◽  
Author(s):  
Anna Ilinskaya ◽  
Gisela Heidecker ◽  
David Derse
Keyword(s):  
Virus Type ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Degradation Pathway ◽  
Target Cells ◽  
Binding Motif ◽  
Wild Type ◽  
T Lymphotropic Virus ◽  
In Cells

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein mediates binding of the virus to its receptor on the surface of target cells and subsequent fusion of virus and cell membranes. To better understand the mechanisms that control HTLV-1 Env trafficking and activity, we have examined two protein-protein interaction motifs in the cytoplasmic domain of Env. One is the sequence YSLI, which matches the consensus YXXΦ motifs that are known to interact with various adaptor protein complexes; the other is the sequence ESSL at the C terminus of Env, which matches the consensus PDZ-binding motif. We show here that mutations that destroy the YXXΦ motif increased Env expression on the cell surface and increased cell-cell fusion activity. In contrast, mutation of the PDZ-binding motif greatly diminished Env expression in cells, which could be restored to wild-type levels either by mutating the YXXΦ motif or by silencing AP2 and AP3, suggesting that interactions with PDZ proteins oppose an Env degradation pathway mediated by AP2 and AP3. Silencing of the PDZ protein hDlg1 did not affect Env expression, suggesting that hDlg1 is not a binding partner for Env. Substitution of the YSLI sequence in HTLV-1 Env with YXXΦ elements from other cell or virus membrane-spanning proteins resulted in alterations in Env accumulation in cells, incorporation into virions, and virion infectivity. Env variants containing YXXΦ motifs that are predicted to have high-affinity interaction with AP2 accumulated to lower steady-state levels. Interestingly, mutations that destroy the YXXΦ motif resulted in viruses that were not infectious by cell-free or cell-associated routes of infection. Unlike YXXΦ, the function of the PDZ-binding motif manifests itself only in the producer cells; AP2 silencing restored the incorporation of PDZ-deficient Env into virus-like particles (VLPs) and the infectivity of these VLPs to wild-type levels.

scholarly journals Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+ signals, and vasoconstriction in cerebral arteries

2008 ◽  
Vol 295 (5) ◽  
pp. C1376-C1384 ◽  
Author(s):  
Guiling Zhao ◽  
Adebowale Adebiyi ◽  
Eva Blaskova ◽  
Qi Xi ◽  
Jonathan H. Jaggar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cerebral Artery ◽  
Purinergic Receptor ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Physiological Functions ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca

Inositol 1,4,5-trisphosphate receptors (IP3Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP3R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP3R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP3R blockers, reduced Ca2+ wave activation and global intracellular Ca2+ ([Ca2+]i) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP3R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP3R1 being the most abundant isoform at 82% of total IP3R message. IP3R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca2+ wave frequency and global [Ca2+]i but abolished UTP-induced Ca2+ wave activation and reduced the UTP-induced global [Ca2+]i elevation by ∼61%. Antibodies targeting IP3R1 and IP3R1 knockdown reduced UTP-induced nonselective cation current ( Icat) activation. IP3R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca2+ by ∼45%. These data indicate that IP3R1 is the predominant IP3R isoform expressed in rat cerebral artery smooth muscle cells. IP3R1 stimulation contributes to UTP-induced Icat activation, Ca2+ wave generation, global [Ca2+]i elevation, and vasoconstriction. In addition, IP3R1 activation constricts cerebral arteries in the absence of SR Ca2+ release by stimulating plasma membrane Icat.

scholarly journals Protein kinase C-mediated desensitization of the neurokinin 1 receptor

2001 ◽  
Vol 280 (5) ◽  
pp. C1097-C1106 ◽  
Author(s):  
Olivier Déry ◽  
Kathryn A. Defea ◽  
Nigel W. Bunnett
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Neurokinin 1 Receptor ◽  
Kinase C ◽  
Naturally Occurring ◽  
Neurokinin 1 ◽  
In Cells

An understanding of the mechanisms that regulate signaling by the substance P (SP) or neurokinin 1 receptor (NK1-R) is of interest because of their role in inflammation and pain. By using activators and inhibitors of protein kinase C (PKC) and NK1-R mutations of potential PKC phosphorylation sites, we determined the role of PKC in desensitization of responses to SP. Activation of PKC abolished SP-induced Ca2+ mobilization in cells that express wild-type NK1-R. This did not occur in cells expressing a COOH-terminally truncated NK1-R (NK1-Rδ324), which may correspond to a naturally occurring variant, or a point mutant lacking eight potential PKC phosphorylation sites within the COOH tail (NK1-R Ser-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392, Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, the t ½ of SP-induced Ca2+mobilization was seven- and twofold greater in cells expressing NK1-Rδ324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in the t ½ of SP-induced Ca2+mobilization. Neither inhibition of PKC nor receptor mutation affected desensitization of Ca2+ mobilization to repeated challenge with SP or SP-induced endocytosis of the NK1-R. Thus PKC regulates SP-induced Ca2+ mobilization by full-length NK1-R and does not regulate a naturally occurring truncated variant. PKC does not mediate desensitization to repeated stimulation or endocytosis of the NK1-R.

scholarly journals Functional Ca2+ Channels between Channel Clusters are Necessary for the Propagation of IP3R-Mediated Ca2+ Waves

2019 ◽  
Vol 24 (2) ◽  
pp. 61
Author(s):  
Estefanía Piegari ◽  
Silvina Ponce Dawson
Keyword(s):  
Signal Propagation ◽  
Cell Types ◽  
Quantitative Comparison ◽  
Preliminary Conclusion ◽  
Qualitative Comparison ◽  
Excitable System ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Spatio Temporal ◽  
Ca2 Channels

The specificity and universality of intracellular Ca 2 + signals rely on the variety of spatio-temporal patterns that the Ca 2 + concentration can display. Ca 2 + release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP 3 Rs) is key for this variety. The opening probability of IP 3 Rs depends on the cytosolic Ca 2 + concentration. All of the dynamics are then well described by an excitable system in which the signal propagation depends on the ability of the Ca 2 + released through one IP 3 R to induce the opening of other IP 3 Rs. In most cell types, IP 3 Rs are organized in clusters, i.e., the cytosol is a “patchy” excitable system in which the signals can remain localized (i.e., involving the release through one or more IP 3 Rs in a cluster), or become global depending on the efficiency of the Ca 2 + -mediated coupling between clusters. The spatial range over which the signals propagate determines the responses that the cell eventually produces. This points to the importance of understanding the mechanisms that make the propagation possible. Our previous qualitative comparison between experiments and numerical simulations seemed to indicate that Ca 2 + release not only occurs within the close vicinity of the clearly identifiable release sites (IP 3 R clusters) but that there are also functional IP 3 Rs in between them. In this paper, we present a quantitative comparison between experiments and models that corroborate this preliminary conclusion. This result has implications on how the Ca 2 + -mediated coupling between clusters works and how it can eventually be disrupted by the different Ca 2 + trapping mechanisms.

scholarly journals ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs

2009 ◽  
Vol 284 (24) ◽  
pp. 16156-16163 ◽  
Author(s):  
Matthew J. Betzenhauser ◽  
Larry E. Wagner ◽  
Hyung Seo Park ◽  
David I. Yule
Keyword(s):  
Single Channel ◽  
Mutational Analysis ◽  
Atp Binding ◽  
Wild Type ◽  
Open Probability ◽  
Permeabilized Cells ◽  
Atp Binding Site ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

Elevated resting [Ca2+]i in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR

2007 ◽  
Vol 292 (5) ◽  
pp. C1591-C1598 ◽  
Author(s):  
Tianzhong Yang ◽  
Eric Esteve ◽  
Isaac N. Pessah ◽  
Tadeusz F. Molinski ◽  
Paul D. Allen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Muscle Relaxants ◽  
Wild Type ◽  
Inhalation Anesthetics ◽  
Structural Conformation ◽  
Intracellular Ca ◽  
Percent Decrease

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca2+ microelectrodes in myotubes expressing wild-type RyR1 ( WTRyR1) or RyR1 with one of four common MH mutations ( MHRyR1), we measured resting intracellular Ca2+ concentration ([Ca2+]i). Changes in resting [Ca2+]i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca2+]i than those expressing WTRyR1. Exposure of myotubes expressing MHRyR1s to ryanodine (500 μM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 μM) had no effects on their resting [Ca2+]i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca2+]i was significantly reduced ( P < 0.01). Interestingly, the percent decrease in resting [Ca2+]i in myotubes expressing MHRyR1s was significantly greater than that for WTRyR1. From these data, we propose that the high resting myoplasmic [Ca2+]i in MHRyR1 expressing myotubes is due in part to a related structural conformation of MHRyR1s that favors “passive” calcium leak from the sarcoplasmic reticulum.

scholarly journals The Protein Tyrosine Kinase p56lck Is Required for Triggering NF-κB Activation upon Interaction of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein gp120 with Cell Surface CD4

1998 ◽  
Vol 72 (7) ◽  
pp. 6207-6214 ◽  
Author(s):  
Laurence Briant ◽  
Véronique Robert-Hebmann ◽  
Claire Acquaviva ◽  
Annegret Pelchen-Matthews ◽  
Mark Marsh ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Translocation ◽  
Mutant Form ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
In Cells

ABSTRACT We have previously shown that NF-κB nuclear translocation can be observed upon human immunodeficiency virus type 1 (HIV-1) binding to cells expressing the wild-type CD4 molecule, but not in cells expressing a truncated form of CD4 that lacks the cytoplasmic domain (M. Benkirane, K.-T. Jeang, and C. Devaux, EMBO J. 13:5559–5569, 1994). This result indicated that the signaling cascade which controls HIV-1-induced NF-κB activation requires the integrity of the CD4 cytoplasmic tail and suggested the involvement of a second protein that binds to this portion of the molecule. Here we investigate the putative role of p56 lck as a possible cellular intermediate in this signal transduction pathway. Using human cervical carcinoma HeLa cells stably expressing CD4, p56 lck , or both molecules, we provide direct evidence that expression of CD4 and p56 lck is required for HIV-1-induced NF-κB translocation. Moreover, the fact that HIV-1 stimulation did not induce nuclear translocation of NF-κB in cells expressing a mutant form of CD4 at position 420 (C420A) and the wild-type p56 lck indicates the requirement for a functional CD4-p56 lck complex.

scholarly journals Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase

2010 ◽  
Vol 299 (6) ◽  
pp. C1363-C1369 ◽  
Author(s):  
Fei Han ◽  
Julie Bossuyt ◽  
Jody L. Martin ◽  
Sanda Despa ◽  
Donald M. Bers
Keyword(s):  
Cardiac Myocytes ◽  
Double Mutant ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Pump Rate ◽  
Necessary And Sufficient ◽  
Pkc Activation ◽  
Major Target ◽  
In Cells

Phospholemman (PLM) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits Na+-K+-ATPase (NKA), mainly by reducing its affinity for internal Na+. The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Ser68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-α1 and we measured the effect of PKA and PKC activation on NKA-mediated intracellular Na+ concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal Na+ and had no significant effect on the maximum pump rate ( Vmax). PKA activation with forskolin (20 μM) restored NKA Na+ affinity in cells expressing WT but not AA PLM and did not affect Vmax in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA Na+ affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Ser63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition.

Sign in / Sign up

Export Citation Format

Share Document