Abstract: : Purpose: To investigate the mechanism for light regulation of phosducin (pdc) phosphorylation. Methods: Intact bovine retinal rod outer segments (IROS) were incubated under varied illumination conditions, with or without regulators for protein kinases and/or protein phosphatases. The steady state levels and the signal patterns of total pdc and the Serine73-phosphorylated pdc (pdc-pS73) after 1 and 2D gel electrophoreses were monitored by Western blot analyses with anti-pdc-pan and anti-pdc-pS73, respectively. Results: The immunochemical signals for total pdc and pdc-pS73 in dark-adapted IROS were detected as multiple bands/spots. Semi-quantitative Western blot showed that pdc-pS73 constitutes 15-35% of total pdc. At 0ºC, the pdc-pS73 spots were localized mostly to 33-34 kDa and pH 4.7-4.9, a region for mono- and diphosphorylated pdc. At 30ºC these spots showed time-dependent migration to 34.5-35 kDa and pH 4.5-4.7, a region for pdc with 2 to 4 phosphates. Exposure to light at 30ºC ...
... is a putative modulator of heterotrimeric G proteins. The protein shares extensive amino acid sequence homology with phosducin, a phosphoprotein expressed in retina and pineal gland. Both phosducin-like protein and phosphoducin have been shown to regulate G-protein signaling by binding to the beta-gamma subunits of G proteins. [provided by RefSeq, Jul 2008 ...
Xiaojing Ma, Weill Cornell Medicine, 1300 York Avenue, New York, NY 10021. Phone: 212-746-4404; Fax: 212-746-4423; E-mail: xim2002{at}med.cornell.edu; Xin-Gang Cui, Gongli Hospital, Second Military Medical University, 219 Miaopu Road, Shanghai 200135, China. Phone: 8602-1588-58730; Fax: 8602-1338-2163; E-mail: cuixingang{at}smmu.edu.cn; and Ying-Hao Sun, sunyinghaoch{at}yeah.net ...
Humrich J، Bermel C، Bünemann M، وآخرون. (2005). "Phosducin-like protein regulates G-protein betagamma folding by interaction with tailless complex polypeptide-1alpha: dephosphorylation or splicing of PhLP turns the switch toward regulation of Gbetagamma folding.". J. Biol. Chem. 280 (20): 20042-50. PMID 15745879. doi:10.1074/jbc.M409233200. ...
Research Summary. My laboratory has developed powerful genomics methods to map how protein regulators control gene expression programs in healthy and diseased cells. These methods were first used in yeast to discover how gene expression programs are controlled by transcription factors, chromatin regulators, and signaling pathways. Among the key concepts that emerged from these studies is that transcription factors that control cell state typically regulate their own gene expression, that chromatin modifications span active genes in a spatially specific manner, and that signaling pathways have terminal kinases that generally occupy the set of genes they regulate.. These concepts and genome-wide methods have been used recently to map the transcriptional regulatory networks that control pluripotency and self-renewal in human embryonic stem cells, to determine how epigenetic regulators contribute to the embryonic cell state, and to reveal initiation and elongation control mechanisms in mammalian ...
This interactive module explores the phases, checkpoints, and protein regulators of the cell cycle. The module also shows how mutations in genes that encode cell cycle regulators can lead to the development of cancer.. ...
Koprowski P., Grajkowski W., Kubalski A. (2007) The MscS cytoplasmic domain and its conformational changes on the channel gating. Curr. Topics in Membranes, vol. 58. Mechanosensitive ion channels. Part A. Owen P. Hamill, ed., 295-309.. Pomorski P., Krzemiński. P., Wasik A., Wierzbicka K., Barańska J., Kłopocka W. (2007) Actin dynamics in Amoeba proteus motility. Protoplasma 231: 31-41. Fabczak H., Fabczak S. (2006) Photosensory transduction in unicellular eukaryotes. A comparison between ciliate protists and photoreceptor cells of higher organisms (Invited review). J.Photochem. Photobiol. 83: 163-171 Sobierajska K., Fabczak H., Fabczak S. (2005) Alterations of ciliate phosducin phosphorylation in Blepharisma japonicum cells. J. Phorochem. Photobiol. B. 79: 135-143. Grajkowski W., Kubalski A., Koprowski P. (2005) Surface changes of the mechanosensitive channel MscS upon its activation, inactivation and closing. Biophys. J. 88: 3050-3059 ...
Our research is centered on G proteins and G protein-coupled receptors (GPCRs). GPCRs are the target of nearly half of all pharmaceuticals, as well as light, taste, odors, hormones and neurotransmitters. Generally speaking, persistent stimulation of G proteins lead to desensitization. Familiar examples include desensitization to light, odors and chemical stimulants such as caffeine.. Receptors, G proteins, and effector MAP kinases are conserved in evolution and are even found in the simplest eukaryotes such as yeast. We have been conducting large-scale genomic and proteomic analysis in yeast to identify mutants with altered signaling and desensitization properties. These mutants are then characterized biochemically in yeast as well as in animal cells using homologous components. This approach led to the identification in yeast of a family of desensitization factors called RGS proteins (Regulator of G protein signaling). RGS proteins inactivate G proteins by accelerating their intrinsic GTPase ...
The etiology of hypertension in most cases is idiopathic. Current dogma states that all forms of hypertension arise from renal abnormalities of sodium handling. We have hypothesized that a primary abnormality of vascular smooth muscle cell (SMC) contraction can cause hypertension. To directly test this hypothesis we created and characterized inducible, SMC-specific RGS2 knockout mice (RGS2-SMC-KO) harboring a SMC-specific deletion of the PKGIα target the GTPase activating protein Regulator of G-protein Signaling 2. In these studies, SMC-specific deletion of RGS2 was induced at 7 weeks of age and Cre-negative mice treated identically as the RGS2-SMC-KO were used as controls. Telemetric blood pressure (BP) studies in ambulatory mice demonstrated that BP was significantly higher in RGS2-SMC-KO vs. control mice (135 ± 5 vs. 122 ± 7 mmHg, respectively. P,0.01), with no change in heart rate. Ex vivo aortic rings of RGS2-SMC-KO mice were hypercontractile in response to phenylephrine (PE) (138 ± 6 ...
Immune response plays a fundamental role in protecting the organism from infections; however, dysregulation often occurs and can be detrimental for the organism, leading to a variety of immune-mediated diseases. Recently our understanding of the molecular and cellular networks regulating the immune response, and, in particular, adaptive immunity, has improved dramatically. For many years, much of the focus has been on the study of protein regulators; nevertheless, recent evidence points to a fundamental role for specific classes of noncoding RNAs (ncRNAs) in regulating development, activation and homeostasis of the immune system. Although microRNAs (miRNAs) are the most comprehensive and well-studied, a number of reports suggest the exciting possibility that long ncRNAs (lncRNAs) could mediate host response and immune function. Finally, evidence is also accumulating that suggests a role for miRNAs and other small ncRNAs in autocrine, paracrine and exocrine signaling events, thus highlighting an
Figure 2. Identification of phosphorylated phosducin in cytosolic proteins after 2-dimensional separation. The cytosolic fraction phosphorylated in the presence of (γ-32P)ATP and 0.1 mM 8-Br-cyclic GMP was subjected to 2-D electrophoresis, and the separated proteins were transferred to PVDF membrane and probed with an anti-phosducin antibody. Panel A: Western blot; Panel B: Corresponding autoradiogram. Arrows identify phosducin. On the left of Panel A are shown standard proteins with their molecular weights marked in kDa. The abscissa shows the approximate pH of the first dimension separation. The reported pI of phosducin is 4.5 [19].. ...
Increasing evidence suggests that RGS proteins play an integral role in G-protein signaling. To date, however, information regarding the functional roles of RGS proteins is limited primarily to in vitro biochemical assays and heterologous overexpression experiments. Thus, the role played by natively expressed RGS proteins in G-protein signaling remains unclear. A reason for this void in our knowledge is the lack of experimental tools for uncoupling endogenous RGS proteins from G-protein signaling pathways. Thus, the development of the better tools represents a major challenge for understanding the physiological roles subserved by RGS proteins. In this regard, several potential strategies are apparent. First, genetically ablating specific RGS proteins, either acutely (e.g., antisense techniques) or stably (e.g., knock-out mice), might lend insight into endogenous RGS protein functions. However, ,20 mammalian RGS proteins have been identified so far, and it is likely that even single cells contain ...
Ahlers, K. E., Stewart, A., Yang, J., Koland, J. G. & Fisher, R. A. (2017). Molecular Cloning of a Novel 69 kDa Brain-Specific Isoform of Regulator of G Protein Signaling 6 (RGS6). (Vols. 31). (1), pp. Supplement 936.9. The FASEB Journal.. Perschbacher, K. J., Sandgren, J. A., Carrillo-Saenz, L., Witcher, P. C., Pearson, N. A., Santillan, D. A., Devor, E. J., Pierce, G. L., Santillan, M. K., Fisher, R. A., Gibson-Corley, K. N. & Grobe, J. L. (2017). Reduced Placental Regulator of G-Protein Signaling-2 (RGS2) and Preeclampsia. (Vols. 31). (1), pp. Supplement 692.3. The FASEB Journal.. Fisher, R. A. (2016). A High Throughput Screening Campaign for Inhibitors of RGS6 and RGS17. (Vols. 30). pp. 1190.11. FASEB J.. Chakravarti, B., Yang, J., Luo, Z. & Ahlers, K. E. (2016). Contribution of NADPH Oxidase (Nox)-derived Reactive Oxygen Species (ROS) to Doxorubicin-induced Cardiomyopathy Mediated by Regulator of G protein Signaling 6 (RGS6). (Vols. 30). pp. 939.3. FASEB J.. Luo, Z., Ahlers, K. E., Yang, ...
Figure 5. Estimation of rhodopsin and phosducin in retinal homogenate and purified nuclear preparation. Proteins of the retinal homogenate and purified nuclear preparation were subjected to electrophoresis, transferred to PVDF membrane and probed with anti-phosducin (left panel) or anti-rhodopsin (right panel) antibody. A: 100 ng of phosducin standard; B: 100 ng of rhodopsin standard. Numbers under lanes represent the amount of total protein loaded, in μg.. ...
G protein signaling pathways in the retina are critically involved in reception and transduction of visual stimuli. The physiological operation of these pathway...
Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding ...
Signal transduction by G protein coupled receptors (GPCRs) in the cardiovascular, nervous and visual systems is the focus of our research. GPCRs are the largest and most important class of receptors in humans because they are the targets of more than half of all therapeutic agents, as well as many drugs of abuse.. Our research focuses on RGS proteins, a large family we discovered that function as novel regulators, effectors and integrators in GPCR signaling pathways. Indeed, RGS proteins have important roles in hypertension, heart failure, anxiety, schizophrenia, vision and drug addiction. Accordingly, RGS proteins provide a promising new class of drug targets.. Currently our goals are to elucidate the mechanistic and physiological functions of RGS proteins in the cardiovascular, nervous and visual systems through biochemical, cell biological, genetic and physiologic studies of knockout and transgenic mice. We are working to: 1) determine the mechanisms whereby RGS proteins participate in ...
The regulators of heterotrimeric guanosine triphosphate (GTP)-binding protein (G protein) signaling (RGS proteins) were named for their ability to act as GTP-activating proteins (GAPs) for G proteins and, thus, limit the signal generated by G protein-coupled receptors (GPCRs). In addition to this characteristic biochemical trait, RGS proteins constitute a large family of structurally diverse proteins with variable sequence motifs that permit additional specific interactions. RGS proteins may also serve as a bridge from GPCRs to receptor tyrosine kinases or transmembrane channels, allowing signals from GPCRs to regulate signaling through other types of receptors, and vice versa.. ...