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First published online 12 September 2007
doi: 10.1242/jcs.011254

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RGS19 regulates Wnt–-catenin signaling through inactivation of G_o

Department of Pharmacology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA

View larger version (45K): [in this window] [in a new window]   Fig. 1. RGS protein expression in F9 cells: analysis at the mRNA level. (A) RT-PCR was performed on cDNA generated from RNA isolated from wild-type F9 cells. Primers specific for each RGS cDNA were utilized in the PCR reaction. PCR products were separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide. RGS3, 5, 16, 10, 12, 14, 20, 17, 19, 9, 11, axin and conductin are expressed at the mRNA level in F9 cells. The lane labeled `No cDNA' shows that the PCR reaction is not contaminated with exogenous DNA. bp, base pairs; MK, marker ladder. (B) Members of each RGS subfamily are expressed in F9 cells (R4, R12, RZ, R7, RA). All RGS proteins examined for mRNA expression are listed. Italic-bold type indicates expression in F9 cells.

View larger version (33K): [in this window] [in a new window]   Fig. 2. RGS protein overexpression screen. (A) F9 cells transiently transfected with vectors containing Fz1, M50 and the indicated RGS proteins were treated with or without Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The results show that expression of RGS19 alone can significantly attenuate Wnt3a-induced gene transcription by more than 50%. *P<0.05 for the difference between wild-type control cells and those expressing RGS19. Con, control. The data shown are mean values (±s.e.) of at least three separate experiments. (B) Lysates were collected from F9 cells individually transiently transfected with an HA-tagged RGS protein. Each sample was subject to SDS-PAGE on an 11% acrylamide gel, transferred to nitrocellulose and probed with an antibody against HA. (C) Western blot analysis was used to determine the presence of RGS19 at the protein level in F9 cells. Wild-type lysates were separated by centrifugation into crude membrane (Mem) and cytosolic (Cyto) fractions. Each sample was subject to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against RGS19. In agreement with previous reports, one band is seen in the cytosolic fraction and two in the membrane fraction. Each fraction was treated with alkaline phosphatase (AP), then subjected to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against RGS19. P-RGS19, phospho-RGS19.

View larger version (21K): [in this window] [in a new window]   Fig. 3. RGS19 overexpression attenuates Wnt3a-induced Lef-Tcf reporter activation. (A) F9 cells transiently transfected with vectors containing Fz1, M50 and RGS19 were treated with Wnt3a for the indicated lengths of time, then collected and subjected to luciferase assay. Overexpression of RGS19 (+ RGS19) blocked Wnt3a-induced reporter activation in a time-dependent manner. *P<0.05 for the difference between wild-type, Wnt3a-treated cells and those expressing RGS19 and Wnt3a-treated for the indicated time points. RLU, relative luciferase units. (B) F9 cells transiently transfected with vectors containing Fz1, M50 and the indicated amounts of RGS19 were treated with or without Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The results show that increasing levels of RGS19 can block the Wnt3a-induced reporter activation. *P<0.05 for the difference between wild-type, Wnt3a-treated cells and those expressing RGS19 and Wnt3a-treated for the indicated time points. **P<0.05 for the difference between wild-type, untreated cells and those expressing RGS19 for the individual time points. Lower panel: immunoblotting (IB) western blot analysis was used to measure the expression of transiently transfected HA-tagged RGS19. Lysates were collected from cells transfected with the indicated amounts of HA-RGS19, then subjected to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against HA. The expression of actin was used as a loading control. The results displayed are data from single experiments, representative of more than three independent tests. (C) F9 cells transiently transfected with vectors containing Fz1, M50 and the indicated amounts of RGS17 or RGS19 were treated with or without Wnt3a for 8 hours, then collected and subjected to luciferase assay. RGS19 suppressed Wnt3a signaling in a dose-dependent manner, whereas overexpression of RGS17 had little effect. Results are displayed as the difference in reporter activation upon Wnt3a stimulation (means ± s.e.) as compared with untransfected control and are representative of three independent experiments. *P<0.05 for the difference between wild-type cells and those expressing RGS19 at each concentration. (D) IB western blot analysis was used to measure the expression of transiently transfected HA-tagged RGS19 and RGS17. Lysates were collected from cells transfected with the indicated amounts of HA-RGS19 and HA-RGS17, then subjected to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against HA.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Constitutively active Go rescues inhibition of Wnt3a action by RGS19. (A) F9 cells transiently transfected with vectors containing Fz1, M50 and/or RGS19, Q205L Go, Q209L G11 and Q209L Gq were treated with or without Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The results show that expression of Q205L Go (left panel), but not Q209L G11 (right panel) or Q209L Gq (lower panel), can rescue the inhibitory effect of RGS19 on Wnt3a-mediated transcription. Results are displayed as the difference in reporter activation upon Wnt3a stimulation (means ± s.e.) as compared with an untransfected control and are representative of three independent experiments. *P<0.05 for the difference between wild-type cells and those expressing either RGS19 or RGS19 and Q209L G11 or Q209L Gq. RLU, relative luciferase units. (B) Western blot analysis was used to measure the expression of transiently transfected vectors encoding Q205L Go, Q209L G11 and Q209L Gq. Lysates were collected from cells transfected with vectors encoding Q205L Go, Q209L G11 and Q209L Gq, then subjected to SDS-PAGE on an 11% acrylamide gel, transferred to nitrocellulose and probed with an antibody against each G-protein.

View larger version (19K): [in this window] [in a new window]   Fig. 5. RGS19 overexpression attenuates -catenin stabilization that results from Wnt3a stimulation. (A) F9 cells transiently transfected with Fz1 and either empty vector or RGS19 were treated with Wnt3a for the indicated lengths of time. Lysates were collected and treated with Con A to separate cytosolic from membrane-associated -catenin. Cleared samples were subjected to SDS-PAGE on an 11% acrylamide gel, transferred to nitrocellulose and probed with an antibody against -catenin. The increase in cytosolic -catenin that is seen in response to Wnt3a is attenuated by overexpression of RGS19 (+ RGS19). The results displayed are from a single experiment representative of more than three independent tests. IB, immunoblotting. (B) Bands from multiple experiments were quantified by densitometry. The data shown are mean values (±s.e.) of at least three separate experiments. *P<0.05 for the difference between wild-type cells and those expressing RGS19 for the indicated time points.

View larger version (25K): [in this window] [in a new window]   Fig. 6. RGS19 attenuates Wnt–-catenin signaling upstream of Dvl. (A) F9 cells transiently transfected with vectors encoding Fz1 and RGS19 were treated with or without Wnt3a for the indicated lengths of time. Lysates were collected and separated by SDS-PAGE on a 10% acrylamide gel, transferred to nitrocellulose and probed with an antibody against Dvl3. The increase in phosphorylated Dvl3 (P-Dvl3) that is seen in response to Wnt3a can be attenuated by overexpression of RGS19. (B) F9 cells transiently transfected with vectors encoding Fz1 and RGS19 were treated with or without Wnt3a for the indicated lengths of time. Lysates were collected, treated with alkaline phosphatase for 1 hour, separated by SDS-PAGE on a 10% acrylamide gel, transferred to nitrocellulose and probed with an antibody against Dvl3. The absence of the slowest migrating band indicates that this band is a phosphorylated isoform of Dvl3. Min, minutes. (C) F9 cells transiently transfected with vectors encoding Fz1 and Dvl3 or RGS19 were collected and subjected to the luciferase assay. The increase in gene transcription stimulated by Dvl3 cannot be attenuated by RGS19. The results displayed are from a single experiment representative of more than three independent tests. The data shown in C are mean values (±s.e.) of at least three separate experiments. *P<0.05 for the difference between wild-type cells and those expressing Dvl3 or Dvl3 and RGS19.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Overexpression of RGS19 protein attenuates Wnt3a-induced formation of PE. (A) F9 cells transiently transfected with vectors encoding Fz1, or Fz1 and RGS19, were treated with or without Wnt3a or retinoic acid (RA) for 5 days. Lysates were collected and separated by SDS-PAGE on an 11% acrylamide gel, transferred to nitrocellulose and probed with an antibody against cytokeratin endo A (TROMA), a specific marker for PE formation. Cells expressing only Fz1 were able to differentiate upon Wnt3a stimulation, whereas those containing both Fz1 and RGS19 were not able. RGS19 was unable to block PE formation induced by RA treatment. The results displayed are from a single experiment representative of more than three independent tests. (B) Bands from multiple experiments were quantified by densitometry. Results are displayed as the `fold change' in cytokeratin endo A expression as compared with untransfected control. The data shown are mean values (±s.e.) of at least three separate experiments. *P<0.05 for the difference between untreated cells and those treated with either Wnt3a or RA. (C) F9 cells transiently transfected with RGS17, RGS19 or an empty vector were treated with or without Wnt3a for 4 days. Cells were stained with an antibody against cytokeratin endo A and imaged by indirect immunofluorescence. Wnt3a treatment induced cytokeratin endo A expression in cells transfected with the empty vector or one encoding RGS17. Overexpression of RGS19 blocked cytokeratin endo A expression. IF, immunofluorescence; OE, overexpression; PC, phase contrast.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Knockdown of RGS19 protein attenuates Wnt3a-induced Lef-Tcf reporter activation. (A) F9 cells transiently transfected with an expression vector harboring siRNA sequences targeting RGS19 (or a control) and encoding Fz1 and M50 were treated with or without Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The results show that specific knockdown of RGS19 decreases Wnt3a-mediated gene transcription by more than 50%. *P<0.05 for the difference between cells transfected with the control siRNA vector and those transfected with the siRNA vector targeting the expression of RGS19. (B) Western blot analysis was used to measure the effect of siRNA treatment on RGS19 protein levels. Lysates were collected from cells transfected with each siRNA vector, then subjected to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against RGS19. The siRNA targeting RGS19 was able to lower RGS19 protein expression to less than 10% that of control (Con) cells. The expression of actin was used as a loading control. The results displayed are data from single experiments, representative of more than three independent tests. (C) F9 cells transiently transfected with an expression vector harboring siRNA sequences targeting the expression of RGS19 (or a control) were treated with Wnt3a for the indicated lengths of time. Lysates were collected and treated with Con A to separate cytosolic from membrane-associated -catenin. Cleared samples were subjected to SDS-PAGE on an 11% acrylamide gel, transferred to nitrocellulose and probed with an antibody against -catenin. The increase in cytosolic -catenin that is seen in response to Wnt3a is attenuated by knockdown of RGS19. (D) F9 cells transiently transfected with vectors encoding Fz1, M50, the indicated amounts of RGS19 and an expression vector harboring siRNA sequences targeting the expression of RGS19 were treated with Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The data shown are mean values (±s.e.) of at least three separate experiments. *P<0.05 for the difference between cells transfected with the control siRNA vector and those transfected with the siRNA vector targeting expression of RGS19 alone or in combination with 0.5 µg or 1 µg of RGS19.

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