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Martin J. Lohse - One of the best experts on this subject based on the ideXlab platform.

  • sumo 1 controls the protein stability and the biological function of Phosducin
    GBM Fall meeting Hamburg 2007, 2007
    Co-Authors: Christoph Klenk, Jan Humrich, Ursula Quitterer, Martin J. Lohse
    Abstract:

    Phosducin regulates Gβγ-stimulated signaling by binding to Gβγ subunits of heterotrimeric G-proteins. Control of Phosducin activity by phosphorylation is well established. However, little is known about other mechanisms that may control Phosducin activity. Here we report that Phosducin is regulated at the posttranslational level by modification with the small ubiquitin-related modifier, SUMO. We demonstrate modification with SUMO for Phosducin in vitro expressed in cells and for native Phosducin purified from retina and the heart. A consensus motif for SUMOylation was identified in Phosducin at amino acid positions 32–35. Mutation of the conserved lysine 33 to arginine in this motif abolished SUMOylation of Phosducin, indicating that SUMO is attached to lysine 33 of Phosducin. In transfected cells the steady-state levels of the K33R mutant protein were much lower compared with wild-type Phosducin. The investigation of the stability of wild-type Phosducin and of PhosducinK33R showed a decreased protein stability of the SUMOylation-deficient mutant. The decreased protein stability correlated with increased ubiquitinylation of the SUMOylation-deficient mutant. These findings indicate that SUMOylation protects Phosducin from proteasomal degradation. SUMOylation of Phosducin decreased its ability to bind Gβγ. PhlP, a closely related member of the Phosducin family, was not a target for SUMOylation, but its SUMOylation can be achieved by a single amino acid insertion in the conserved N terminus of PhlP. Together, these findings show that Phosducin is a previously unrecognized target of SUMO modification and that SUMOylation controls Phosducin stability in cells as well as its functional properties.

  • effects of two gbetagamma binding proteins n terminally truncated Phosducin and beta adrenergic receptor kinase c terminus betaarkct in heart failure
    Gene Therapy, 2003
    Co-Authors: K L Laugwitz, Jan Humrich, K Pinkernell, I Pragst, C Baumgartner, E Hoffmann, K Rosport, Gotz Munch, Alessandra Moretti, Martin J. Lohse
    Abstract:

    Myocardial overexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct) has been shown to result in a positive inotropic effect or an improvement of survival in heart failure. However, it is not clear whether this beneficial effect is mainly because of dominant-negative inhibition of betaARK1, and a consecutive resensitization of beta-adrenergic receptors (betaAR), or rather due to inhibition of other Gbetagamma-mediated effects. In this study, we tested whether overexpression of N-terminally truncated Phosducin (nt-del-Phosducin), another Gbetagamma-binding protein that does not resensitize betaARs owing to simultaneous inhibition of GDP release from Galpha subunits, shows the same effects as betaARKct. Adenoviral gene transfer was used to express nt-del-Phosducin and betaARKct in isolated ventricular cardiomyocytes and in myocardium of rabbits, which suffered from heart failure because of rapid ventricular pacing. BetaAR-stimulated cAMP formation was increased by betaARKct, but not by nt-del-Phosducin, whereas both proteins inhibited Gbetagamma-mediated effects. Both transgenes also increased contractility of normal and failing isolated cardiomyocytes and improved contractility in rabbits with heart failure after gene transfer in vivo. In conclusion, overexpression of nt-del-Phosducin enhances the contractility of cardiomyocytes to the same extent as betaARKct, suggesting that the effects of betaARKct might be owing to inhibition of Gbetagamma rather than to betaAR resensitization.

  • regulation of Phosducin like protein by casein kinase 2 and n terminal splicing
    Journal of Biological Chemistry, 2003
    Co-Authors: Jan Humrich, Ursula Quitterer, Christina M Bermel, Tobias Grubel, Martin J. Lohse
    Abstract:

    Phosducin-like protein (PhLP) is a member of the Phosducin family of G-protein βγ-regulators and exists in two splice variants. The long isoform PhLPLand the short isoform PhLPS differ by the presence or absence of an 83-amino acid N terminus. In isolated biochemical assay systems, PhLPL is the more potent Gβγ-inhibitor, whereas the functional role of PhLPS is still unclear. We now report that in intact HEK 293 cells, PhLPS inhibited Gβγ-induced inositol phosphate generation with ∼20-fold greater potency than PhLPL. Radiolabeling of transfected HEK 293 cells with [32P] revealed that PhLPL is constitutively phosphorylated, whereas PhLPS is not. Because PhLPL has several consensus sites for the constitutively active kinase casein kinase 2 (CK2) in its N terminus, we tested the phosphorylation of the recombinant proteins by either HEK cell cytosol in the presence or absence of kinase inhibitors or by purified CK2. PhLPL was a good CK2 substrate, whereas PhLPS and Phosducin were not. Progressive truncation and serine/threonine to alanine mutations of the PhLPL N terminus identified a serine/threonine cluster (Ser-18/Thr-19/Ser-20) within a small N-terminal region of PhLPL (amino acids 5–28) as the site in which PhLPL function was modified in HEK 293 cells. In native tissue, PhLPL also seems to be regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLPL were detected in mouse brain and adrenal gland. Moreover, the alternatively spliced isoform PhLPS was also found in adrenal tissue. Therefore, the physiological control of G-protein regulation by PhLP seems to involve phosphorylation by CK2 and alternative splicing of the regulator.

  • phosphorylation of Phosducin and Phosducin like protein by g protein coupled receptor kinase 2
    Journal of Biological Chemistry, 2000
    Co-Authors: Ana Ruizgomez, Martin J. Lohse, Jan Humrich, Ursula Quitterer, Cristina Murga, Federico Mayor
    Abstract:

    Abstract G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and Phosducin-like protein (PhLP) are cytosolic proteins that bind Gβγ subunits and act as regulators of G-protein signaling. In this report, we identify Phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified Phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 ± 0.1 and 0.83 ± 0.09 mol of Pi/mol of protein, respectively). The phosphorylation reactions exhibit apparent K m values in the range of 40–100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of Phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of Phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both Phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with Gβγ subunits. Phosphorylation of Phosducin by GRK2 markedly reduces its Gβγ binding ability, suggesting that GRK2 may modulate the activity of the Phosducin protein family by disrupting this interaction. The identification of Phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

  • quantification of the tissue levels and function of the g protein regulator Phosducin like protein phlp
    Naunyn-schmiedebergs Archives of Pharmacology, 2000
    Co-Authors: Stefan Schroder, Martin J. Lohse
    Abstract:

    Phosducin-like protein is a protein with widespread expression that has been shown to be capable of inhibiting G-protein function in vitro. However, it is not clear whether it is expressed in sufficient amounts to actually exert such functions in vivo. Here we quantify the expression of the short and the long splice variants of Phosducin-like protein, PhlPs and PhlPl. Western blots of various rat tissues showed that PhlPl was by far the dominant splice variant; its levels were 1.5–2 pmol/mg cytosolic protein in brain, liver and kidney, and about 0.5 pmol/mg cytosolic protein in lung, heart and skeletal muscle. These values correspond to concentrations of 150–200 nM and 50 nM, respectively. The levels of PhlPs were about 20-fold lower. Recombinant Phosducin, PhlPl and PhlPs inhibited the interaction between G-protein α- und βγ-subunits with IC50-values of 6 nM, 6 nM and 90 nM, respectively, as determined by Gβγ-dependent ADP-ribosylation of Gαi1 by pertussis-toxin. Thus, tissue concentrations of PhlPl are clearly sufficient to affect G-protein function in vivo, while the expression levels and the Gβγ-affinity of PhlPs are most likely too low to have significant inhibitory effects on Gβγ (G-protein βγ-subunits).

Barry M. Willardson - One of the best experts on this subject based on the ideXlab platform.

  • function of Phosducin like proteins in g protein signaling and chaperone assisted protein folding
    Cellular Signalling, 2007
    Co-Authors: Barry M. Willardson, Alyson C Howlett
    Abstract:

    Members of the Phosducin gene family were initially proposed to act as down-regulators of G protein signaling by binding G protein βγ dimers (Gβγ) and inhibiting their ability to interact with G protein α subunits (Gα) and effectors. However, recent findings have overturned this hypothesis by showing that most members of the Phosducin family act as cochaperones with the cytosolic chaperonin complex (CCT) to assist in the folding of a variety of proteins from their nascent polypeptides. In fact rather than inhibiting G protein pathways, Phosducin-like protein 1 (PhLP1) has been shown to be essential for G protein signaling by catalyzing the folding and assembly of the Gβγ dimer. PhLP2 and PhLP3 have no role in G protein signaling, but they appear to assist in the folding of proteins essential in regulating cell cycle progression as well as actin and tubulin. Phosducin itself is the only family member that does not participate with CCT in protein folding, but it is believed to have a specific role in visual signal transduction to chaperone Gβγ subunits as they translocate to and from the outer and inner segments of photoreceptor cells during light-adaptation.

  • mechanism of assembly of g protein βγ subunits by protein kinase ck2 phosphorylated Phosducin like protein and the cytosolic chaperonin complex
    Journal of Biological Chemistry, 2006
    Co-Authors: Georgi L. Lukov, Craig D Thulin, Christine M Baker, Paul J Ludtke, Michael D Carter, Ryan A Hackett, Barry M. Willardson
    Abstract:

    Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein βγ subunit complex (Gβγ) that has been recently shown to catalyze the formation of the Gβγ dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gβγ dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gβγ-assembly process. The results reported here delineate a mechanism for Gβγ assembly in which a stable ternary complex is formed between PhLP, the nascent Gβ subunit, and CCT that does not include Gγ. PhLP phosphorylation permits the release of a PhLP·Gβ intermediate from CCT, allowing Gγ to associate with Gβ in this intermediate complex. Subsequent interaction of Gβγ with membranes releases PhLP for another round of assembly.

  • Phosducin like protein acts as a molecular chaperone for g protein βγ dimer assembly
    The EMBO Journal, 2005
    Co-Authors: Georgi L. Lukov, Joseph N Mclaughlin, Heidi E Hamm, Barry M. Willardson
    Abstract:

    Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein βγ subunit dimer (Gβγ). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gβγ expression and G protein signaling. This inhibition was caused by an inability of nascent Gβγ to form dimers. Phosphorylation of PhLP at serines 18–20 by protein kinase CK2 was required for Gβγ formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gβ in the absence of Gγ, and S18–20 phosphorylation was required for Gγ to associate with the PhLP-Gβ complex. Once Gγ bound, PhLP was released. These results suggest a mechanism for Gβγ assembly in which PhLP stabilizes the nascent Gβ polypeptide until Gγ can associate, resulting in membrane binding of Gβγ and release of PhLP to catalyze another round of assembly.

  • Role of the isoprenyl pocket of the G protein beta gamma subunit complex in the binding of Phosducin and Phosducin-like protein.
    Biochemistry, 2004
    Co-Authors: Georgi L. Lukov, James C. Garrison, Chang-seon Myung, William E. Mcintire, Jianyin Shao, S. Scott Zimmerman, Barry M. Willardson
    Abstract:

    Phosducin (Pdc) and Phosducin-like protein (PhLP) regulate G protein-mediated signaling by binding to the betagamma subunit complex of heterotrimeric G proteins (Gbetagamma) and removing the dimer from cell membranes. The binding of Pdc induces a conformational change in the beta-propeller structure of Gbetagamma, creating a pocket between blades 6 and 7. It has been proposed that the isoprenyl group of Gbetagamma inserts into this pocket, stabilizing the Pdc.Gbetagamma structure and decreasing the affinity of the complex for the lipid bilayer. To test this hypothesis, the binding of Pdc and PhLP to several Gbetagamma dimers containing variants of the beta or gamma subunit was measured. These variants included modifications of the isoprenyl group (gamma), residues involved in the conformational change (beta), and residues lining the proposed prenyl pocket (beta). Switching prenyl groups from farnesyl to geranylgeranyl or vice versa had little effect on binding. However, alanine substitution of one residue in the beta subunit involved in the conformational change (W332) decreased binding 5-fold. Alanine substitution of certain residues within the prenyl pocket caused only minor decreases in binding, while a lysine substitution of T329 within the pocket inhibited binding 10-fold. Molecular modeling of the binding energy of the Pdc.Gbeta(1)gamma(2) complex required insertion of the geranylgeranyl group into the prenyl pocket in order to accurately predict the effects of prenyl pocket amino acid substitutions. Finally, a dimer containing a gamma subunit with no prenyl group (gamma(2)-C68S) decreased binding by nearly 20-fold. These results support the structural model in which the prenyl group escapes contact with the aqueous milieu by inserting into the prenyl pocket and stabilizing the Pdc-binding conformation of Gbetagamma.

  • ubiquitylation of the transducin βγ subunit complex regulation by Phosducin
    Journal of Biological Chemistry, 2002
    Co-Authors: Martin S Obin, Rehwa H. Lee, Vadim Y Arshavsky, Barry M. Willardson, Rachelle Gaudet, Bruce Y Lee, Gretchen Meinke, A Bohm, Johnathan A Hopp, Allen Taylor
    Abstract:

    G proteins (Gαβγ) are essential signaling molecules, which dissociate into Gα and Gβγ upon activation by heptahelical membrane receptors. We have identified the βγ subunit complex of the photoreceptor-specific G protein, transducin (T), as a target of the ubiquitin-proteasome pathway. Ubiquitylated species of the transducin γ-subunit (Tγ) but not the α- or β-subunits were assembled de novo in bovine photoreceptor preparations. In addition, Tγ was exclusively ubiquitylated when Tβγ was dissociated from Tα. Ubiquitylation of Tβγ on Tγ was selectively catalyzed by human ubiquitin-conjugating enzymes UbcH5 and UbcH7 and was coincident with degradation of the entire Tβγ subunit complex in vitroby a mechanism requiring ATP and the proteasome. We also show that Tβγ association with Phosducin, a photoreceptor-specific protein of unknown physiological function, blocks Tβγ ubiquitylation and subsequent degradation. Phosphorylation of Phosducin by Ca2+/calmodulin-dependent protein kinase II, which inhibits Phosducin-Tβγ complex formation, completely restored Tβγ ubiquitylation and degradation. We conclude that Tβγ is a substrate of the ubiquitin-proteasome pathway and suggest that Phosducin serves to protect Tβγ following the light-dependent dissociation of Tαβγ.

Cheryl M Craft - One of the best experts on this subject based on the ideXlab platform.

  • light driven translocation of the protein phosphatase 2a complex regulates light dark dephosphorylation of Phosducin and rhodopsin
    Biochemistry, 2002
    Co-Authors: Bruce M Brown, Richard N. Lolley, Xuemei Zhu, Brian L Carlson, Cheryl M Craft
    Abstract:

    In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), Phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B' subunit, B56 epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B' subunit (sequenced to be B56 alpha) or with the B alpha regulatory subunit have no Phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56 epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the Phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (approximately 20 ng/mouse retina) and 9 times more of the C subunit (approximately 4 ng/mouse retina) than of the B56 epsilon subunit (approximately 0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56 epsilon subunit, greatly prefers phosphorylated Phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic Phosducin and membrane-bound rhodopsin, by the same enzyme.

  • modulation of crx transactivation activity by Phosducin isoforms
    Molecular and Cellular Biology, 2000
    Co-Authors: Xuemei Zhu, Cheryl M Craft
    Abstract:

    Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) βγ subunits (Gβγ), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gβγ. A retina- and pineal gland-specific transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using PhLOP1 as the bait. Direct protein-protein interactions between Phd or PhLOP1 and CRX were demonstrated using a β-galactosidase quantitative assay in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine if the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase reporter construct containing a CRX consensus element (GATTAA) was cotransfected into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or PhLOP1. Phd and PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast, PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential protein interacting partners, we identified immunoreactive proteins for Phd and CRX in retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and PhLOP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreactive peptides are seen in light-adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isoforms could inhibit CRX transcriptional activation.

  • The Carboxyl Terminal Domain of Phosducin Functions as a Transcriptional Activator
    Biochemical and biophysical research communications, 2000
    Co-Authors: Xuemei Zhu, Cheryl M Craft
    Abstract:

    Abstract In previous work, we identified a set of Phosducin (Phd) isoforms with unknown function including the Phosducin (Phd)-like orphan protein 1 (PhLOP1), an amino terminal truncated isoform of the retinal Phd lacking the Gβγ binding domain. To investigate the potential biological function of PhLOP1, PhLOP1 was fused at its amino terminus with the DNA binding domain (BD) of the yeast transcriptional factor, GAL4, and used as bait in a yeast two-hybrid screen. Two potential functional protein partners were identified during the screen: SUG1, a subunit of the 26S proteasome and a putative transcriptional mediator, and CRX, a retina- and pineal-specific transcription factor. Upon localizing the interacting domain of PhLOP1 with one of the new partners, SUG1, we found that a domain of 40 amino acids at the carboxyl terminus of Phd and PhLOP1 had intrinsic transcriptional activation activity in yeast. The transactivation activity was further confirmed in mammalian cells. This region contains an acidic domain that has been shown to be involved in the function of several transcriptional activators. In addition, we showed that Phd is cytoplasmic while PhLOP1 is localized predominantly to the nucleus when fused to an enhanced green fluorescent protein (EGFP) and transiently expressed in transfected cells, suggesting that PhLOP1 may play a distinct functional role in transcriptional regulation independent of the known Phd interaction/regulation of Gβγ transduction.

  • Phosducin like protein 1 phlp1 and Phosducin like orphan protein 1 phlop1
    1999
    Co-Authors: Cheryl M Craft, Xuemei Zhu, Jaji Murage
    Abstract:

    Phosducin (Phd) is a soluble phosphoprotein, selectively binding to the beta-gamma (βγ) complex of G-proteins, playing a role in intracellular signaling processes. We have molecularly identified 3 Phd isoforms expressed in the human retina. Based on their ability or inability to interact in our assay system with Gβγ proteins, we named them Phosducin-Like Protein 1 (PhLP1) or Phosducin-Like Orphan Proteins (PhLOP1 & PhLOP2), respectively. To further study these Phd isoforms and their potential involvement in normal and abnormal visual processes through genetic alterations, we identified and characterized an 85 kb P1-PhLP human genomic clone that contains PhLPl and PhLOP1. With fluorescence in situ hybridization (FISH) to human metaphase chromosomes, this P1-PhLP genomic clone was mapped to human chromosome 1q25.3. Previous work identified a gene locus for Phosducin (PDC) to 1q25-q31.1 by somatic cell hybridization and in situ hybridization. Within the P1-PhLP genomic clone, the previously characterized complete PDC gene was identified; furthermore, the 5’-flanking region and a potential promoter region of PhLOP1 was identified between Exon 2 and Exon 3 of the PDC gene. Initial transfection experiments with luciferase activity reporter PhLOP1 constructs, ranging in size from 386 to 852 basepairs (bp), suggest that no tissue specific retinal promoter activity is contained in this 5’-flanking region of the PhLOP1 when compared to the high retinal specific promoter activity observed for a 123bp construct for Interphotoreceptor Retinoid-Binding Protein (IRBP). Alternatively, a represser element is still present and preventing promoter activity in the PhLOP1 constructs. These results correspond to the low levels of mRNA of the PhLOP1 observed in the retina compared to Phd. These data suggest that both PhLP1 and PhLOP1 are created through alternative splicing of the Phd gene and that a single PDC gene at 1q25.3 is responsible for these three retinal isoforms.

  • phlps and phlops in the Phosducin family of g beta gamma binding proteins
    Biochemistry, 1998
    Co-Authors: Cheryl M Craft, Vladlen Z Slepak, Xinyi Zhanpoe, Xuemei Zhu, Bruce M Brown, Richard N. Lolley
    Abstract:

    In this study, we identify new isoforms of the retinal Phosducin and investigate the expression of the Phosducin family, showing that an isoform, PhLP1, has sequence homology with Phd and Gbeta gamma binding capability, whereas two isoforms (Phosducin-like orphan proteins, PhLOPs) share sequence homology with Phd but fail to bind Gbeta gamma. Original identification of PhLP1 and the PhLOPs was from a human retina cDNA library, using a PCR product for library hybridization screening that contained a predicted functional epitope domain. The screen identified Phd and three related, but distinct, recombinants (PhLP1, PhLOP1, and PhLOP2). By RT-PCR, all isoforms are expressed in either retina or forskolin-stimulated Y79 retinoblastoma cells; however, the new isoforms are below the level of detection on Northern blot analysis. The predicted amino acid translation of each homologue revealed major differences, arising from either splice variants or gene duplication of Phd. To test the functional interaction of all Phosducin isoforms with Gbeta gamma in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified retinal transducin Gbeta gamma was verified for GST-Phd and demonstrated for GST-PhLP1; however, neither GST-PhLOP1 nor GST-PhLOP2 bound Gbeta gamma. Comparable results were observed when the GST-Phosducin fusion proteins selectively sequestered Gbeta gammas from retinal extracts or when functional Gbeta gamma interactions were assessed using surface plasmon resonance technology. Phosducin and its isoforms are widely distributed in body tissues where they may participate in signal transduction pathways. Phd and PhLP1 possess an 11-amino acid conserved epitope domain (TGPKGVINDWR) that controls the high-affinity binding of Gbeta gamma; these isoforms are implicated in the G-protein signaling pathway. The Phosducin-like orphan proteins (PhLOPs) fail to bind Gbeta gamma, suggesting that the PhLOP isoforms may participate in still unidentified signaling pathways.

Mark W Bitensky - One of the best experts on this subject based on the ideXlab platform.

  • subcellular localization of Phosducin in rod photoreceptors
    Visual Neuroscience, 2005
    Co-Authors: Jing Chen, Tatsuro Yoshida, Koichi Nakano, Mark W Bitensky
    Abstract:

    Phosducin (Pd) is a 28-kD phosphoprotein whose expression in retina appears limited to photoreceptor cells. Pd binds to the beta,gamma subunits of transducin (Gt). Their binding affinity is markedly diminished by Pd phosphorylation. While Pd has long been regarded as a candidate for the regulation of Gt, the molecular details of Pd function remain unclear. This gap in understanding is due in part to a lack of precise information concerning the total amount and subcellular localization of rod Pd. While earlier studies suggested that Pd was a rod outer segment (ROS) protein, recent findings have demonstrated that Pd is distributed throughout the rod. In this report, the subcellular distribution and amounts of rat Pd are quantified with immunogold electron microscopy. After light or dark adaptation, retinal tissues were fixed in situ and prepared for ultrathin sectioning and immunogold labeling. Pd concentrations were analyzed over the entire length of the rod. The highest Pd labeling densities were found in the rod synapse. Less intense Pd staining was observed in the ellipsoid and myoid regions, while minimal labeling densities were found in the ROS and the rod nucleus. In contrast with rod Gt, no evidence was found for light-dependent movement of Pd between inner and outer segments. There is a relative paucity of Pd in the ROS as compared with the large amounts of Gt found there. This does not support the earlier idea that Pd could modulate Gt activity by controlling its concentration. On the other hand, the presence of Pd in the nucleus is consistent with its possible role as a regulator of transcription. The functions of Pd in the ellipsoid and myoid regions remain unclear. The highest concentration of Pd was found at the rod synapse, consistent with a suggested role for Pd in the regulation of synaptic function.

  • rethinking the role of Phosducin light regulated binding of Phosducin to 14 3 3 in rod inner segments
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Koichi Nakano, Tatsuro Yoshida, Jing Chen, George E Tarr, Julia M Flynn, Mark W Bitensky
    Abstract:

    Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity in the rod outer segment through interaction with the heterotrimeric G protein transducin. But, based on histochemistry and Western blot analysis, Pd is found almost entirely in the inner segment in both light and dark, most abundantly near the rod synapse. We report a second small protein, 14-3-3, in the rod with a similar distribution. By immunoprecipitation, phospho-Pd is found to interact with 14-3-3 in material from dark-adapted retina, and this interaction is markedly diminished by light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds to inner segment G protein(s) in the light. From these results and reported functions of 14-3-3, we have constructed a hypothesis for the regulation of light sensitivity at the level of rod synapse. By dissociating the Pd/14-3-3 complex, light enables both proteins to function in this role.

  • regulation of g protein activation in retinal rods by Phosducin
    1997
    Co-Authors: Barry M. Willardson, Tatsuro Yoshida, Jon F Wilkins, Mark W Bitensky
    Abstract:

    G-proteins shuttle signaling information between a vast array of seven transmembrane helical receptors to intracellular effectors (1). Effectors determine the concentration of important second messenger molecules in the cell such as cyclic nucleotides, inositol phosphates and Ca2+. It is the concentration of these second messengers that determines the ultimate response of the cell to a stimulus, whether it be cell growth, differentiation or death, neuronal signaling, the rate of heart muscle contraction, or a number of other responses.

  • regulation of the kinetics of Phosducin phosphorylation in retinal rods
    Journal of Biological Chemistry, 1996
    Co-Authors: Jon F Wilkins, Mark W Bitensky, Barry M. Willardson
    Abstract:

    Phosducin (Pd) is a widely expressed phosphoprotein that regulates G-protein (G) signaling. Unphosphorylated Pd binds to Gbetagamma subunits and blocks their interaction with Galpha. This binding sequesters Gbetagamma and inhibits both receptor-mediated activation of Galpha and direct interactions between Gbetagamma and effector enzymes. When phosphorylated by cAMP-dependent protein kinase, Pd does not affect these functions of Gbetagamma. To further understand the role of Pd in regulating G-protein signaling in retinal rod photoreceptor cells, we have measured the abundance of Pd in rods and examined factors that control the rate of Pd phosphorylation. Pd is expressed at a copy number comparable to that for the rod G-protein, transducin (Gt). The ratio of rhodopsin (Rho) to Pd is 15. 5 +/- 3.5 to 1. The rate of Pd phosphorylation in rod outer segment preparations was dependent on [cAMP]. K1/2 for cAMP was 0.56 +/- 0. 09 microM, and the maximal rate of phosphorylation was approximately 500 pmol PO4 incorporated/min/nmol Rho. In the presence of Gtbetagamma this rate was decreased approximately 50-fold. From these data, one can estimate a t1/2 of approximately 3 min for the rephosphorylation of Pd in rods during the recovery period after a light response. This relatively slow rephosphorylation of the Pd.Gtbetagamma complex may provide a period of molecular memory in which sensitivity to further light stimuli is reduced as a result of sequestration of Gtbetagamma by Pd.

  • the phosphorylation state of Phosducin determines its ability to block transducin subunit interactions and inhibit transducin binding to activated rhodopsin
    Journal of Biological Chemistry, 1994
    Co-Authors: Tatsuro Yoshida, Barry M. Willardson, Jon F Wilkins, Grant J Jensen, B D Thornton, Mark W Bitensky
    Abstract:

    Heterotrimeric GTP-binding proteins (G-proteins) serve many different signal transduction pathways. Phosducin, a 28-kDa phosphoprotein, is expressed in a variety of mammalian cell types and blocks activation of several classes of G-proteins. Phosphorylation of Phosducin by cyclic AMP-dependent protein kinase prevents Phosducin-mediated inhibition of G-protein GTPase activity (Bauer, P. H., Muller, S., Puzicha, M., Pippig, S., Obermaier, B., Helmreich, E. J. M., and Lohse, M. J. (1992) Nature 358, 73-76). In retinal rods, Phosducin inhibits transducin (Gt) activation by binding its beta gamma subunits. While rod Phosducin is phosphorylated in the dark and dephosphorylated after illumination (Lee, R.-H., Brown, B. M., and Lolley, R. N. (1984) Biochemistry 23, 1972-1977), the significance of these reactions is still unclear. The data presented here permit a more precise characterization of Phosducin function and the consequences of its phosphorylation. DephosphoPhosducin blocked binding of the Gt alpha 1 subunit to activated rhodopsin in the presence of stoichiometric amounts of Gt beta gamma, whereas phosphoPhosducin did not. Surprisingly, the binding affinity of phosphoPhosducin for Gt beta gamma was not significantly reduced compared with the binding affinity of dephosphoPhosducin. However, the association of Phosducin with Gt beta gamma in a size exclusion column matrix was dependent on the phosphorylation state of Phosducin. Moreover, the ability of Phosducin to compete with Gt alpha for binding to Gt beta gamma was also dependent on the phosphorylation state of Phosducin. No interaction was found between Phosducin and Gt alpha. These data indicate that Phosducin decreases rod responsiveness by binding to the beta gamma subunits of Gt and preventing their interaction with Gt alpha, thereby inhibiting Gt alpha activation by the activated receptor. Moreover, phosphorylation of Phosducin blocks its ability to compete with Gt alpha for binding to Gt beta gamma.

Richard N. Lolley - One of the best experts on this subject based on the ideXlab platform.

  • light driven translocation of the protein phosphatase 2a complex regulates light dark dephosphorylation of Phosducin and rhodopsin
    Biochemistry, 2002
    Co-Authors: Bruce M Brown, Richard N. Lolley, Xuemei Zhu, Brian L Carlson, Cheryl M Craft
    Abstract:

    In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), Phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B' subunit, B56 epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B' subunit (sequenced to be B56 alpha) or with the B alpha regulatory subunit have no Phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56 epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the Phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (approximately 20 ng/mouse retina) and 9 times more of the C subunit (approximately 4 ng/mouse retina) than of the B56 epsilon subunit (approximately 0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56 epsilon subunit, greatly prefers phosphorylated Phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic Phosducin and membrane-bound rhodopsin, by the same enzyme.

  • phlps and phlops in the Phosducin family of g beta gamma binding proteins
    Biochemistry, 1998
    Co-Authors: Cheryl M Craft, Vladlen Z Slepak, Xinyi Zhanpoe, Xuemei Zhu, Bruce M Brown, Richard N. Lolley
    Abstract:

    In this study, we identify new isoforms of the retinal Phosducin and investigate the expression of the Phosducin family, showing that an isoform, PhLP1, has sequence homology with Phd and Gbeta gamma binding capability, whereas two isoforms (Phosducin-like orphan proteins, PhLOPs) share sequence homology with Phd but fail to bind Gbeta gamma. Original identification of PhLP1 and the PhLOPs was from a human retina cDNA library, using a PCR product for library hybridization screening that contained a predicted functional epitope domain. The screen identified Phd and three related, but distinct, recombinants (PhLP1, PhLOP1, and PhLOP2). By RT-PCR, all isoforms are expressed in either retina or forskolin-stimulated Y79 retinoblastoma cells; however, the new isoforms are below the level of detection on Northern blot analysis. The predicted amino acid translation of each homologue revealed major differences, arising from either splice variants or gene duplication of Phd. To test the functional interaction of all Phosducin isoforms with Gbeta gamma in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified retinal transducin Gbeta gamma was verified for GST-Phd and demonstrated for GST-PhLP1; however, neither GST-PhLOP1 nor GST-PhLOP2 bound Gbeta gamma. Comparable results were observed when the GST-Phosducin fusion proteins selectively sequestered Gbeta gammas from retinal extracts or when functional Gbeta gamma interactions were assessed using surface plasmon resonance technology. Phosducin and its isoforms are widely distributed in body tissues where they may participate in signal transduction pathways. Phd and PhLP1 possess an 11-amino acid conserved epitope domain (TGPKGVINDWR) that controls the high-affinity binding of Gbeta gamma; these isoforms are implicated in the G-protein signaling pathway. The Phosducin-like orphan proteins (PhLOPs) fail to bind Gbeta gamma, suggesting that the PhLOP isoforms may participate in still unidentified signaling pathways.

  • Regulation of retinal cGMP cascade by Phosducin in bovine rod photoreceptor cells. Interaction of Phosducin and transducin.
    The Journal of biological chemistry, 1992
    Co-Authors: R H Lee, T D Ting, B S Lieberman, D E Tobias, Richard N. Lolley
    Abstract:

    Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of Phosducin-T beta gamma and the possible regulatory role of Phosducin on the cGMP cascade. Addition of Phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of Phosducin. The inhibitory effects of Phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that Phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The Phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of Phosducin. This result implies that T beta gamma may undergo a conformational change upon Phosducin binding which leads to the release of T alpha. Since Phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, Phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, Phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.

  • induction of experimental autoimmune uveitis by the retinal photoreceptor cell protein Phosducin
    Current Eye Research, 1992
    Co-Authors: Harminder S Dua, Richard N. Lolley, Rehwa H. Lee, Jeffrey A Barrett, Michael Abrams, John V Forrester, Larry A Donoso
    Abstract:

    Experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) are CD4+ T cell mediated inflammatory diseases of the retina and uveal tract of the eye and the pineal gland respectively. They can be induced in experimental animals by immunization with several well characterized retinal autoantigens. We induced a mild to moderate EAU and EAP in Lewis rats by immunization with Phosducin, a 33K retinal phosphoprotein which is involved in the phototransduction of vision. In contrast to the severe EAU induced by other retinal antigens like S-antigen (SAg) or interstitial retinoid binding protein (IRBP), the clinical disease was late in onset, low grade in severity and predominantly affected the posterior segment of the eye. Our study demonstrates that another photoreceptor cell protein, Phosducin, is capable of eliciting EAU and EAP.

  • rat pineal gland Phosducin cdna isolation nucleotide sequence and chromosomal assignment in the mouse
    Genomics, 1991
    Co-Authors: Cheryl M Craft, Richard N. Lolley, Rehwa H. Lee, Michael F Seldin
    Abstract:

    The pineal gland contains a soluble phosphoprotein, Phosducin, which is homologous to that of retinal photoreceptors. Phosducin has been shown to bind the beta, gamma subunits of the retinal G-protein transducin. Retinal Phosducin has been cloned and now we report a rat pineal cDNA encoding Phosducin. A 1217-nucleotide cDNA was isolated from a rat pineal library by DNA-DNA hybridization with a polymerase chain reaction-amplified cDNA of bovine retina mRNA for Phosducin. Northern blot analysis demonstrates that the mRNA for Phosducin is approximately 1.3 kb in both rat pineal and rat retina. The translated mRNA from rat pineal encodes a protein with 246 amino acids, compared to the 245 amino acids of bovine retina Phosducin. The predicted molecular weight of rat pineal Phosducin is 28,201. Immunoblot analysis with affinity-purified antibodies against bovine retina Phosducin identify a single immunoreactive protein of approximately 33 kDa in both rat retina and rat pineal. The amino acid sequence of rat pineal Phosducin is homologous to that of bovine retina Phosducin, revealing 89% identity and another 5.7% similarity. Both rat pineal and bovine retina Phosducins are acidic proteins with pIs of 4.3 and 4.5, respectively. The translated protein lacks hydrophobic domains that would suggest an integral membrane protein. Rat pineal Phosducin has a single consensus phosphorylation domain for protein kinase A that is nearly identical to that of retinal Phosducin, which is phosphorylated by protein kinase A in situ. Rat Phosducin also contains three potential phosphorylation domains for protein kinase C and nine for casein kinase II as well as a predicted site for N-glycosylation. The cDNA encoding Phosducin was used to localize the gene within a linkage group to a large segment of mouse chromosome 1 in a conserved region with the long arm of human chromosome 1 with a panel of DNA samples from an interspecific cross. In keeping with a proposed role of retinal Phosducin in down-regulation of the photo-transduction cascade, a modulatory role in signal transduction is proposed for pineal Phosducin.