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Rb-Ras class paper

Rb-Ras class paper - Neurosci 17 509—512(1994 5 Akaike N...

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Unformatted text preview: Neurosci. 17, 509—512 (1994). 5. Akaike, N., Krishtal, O. A. 8r Maruyama, T. Protoneinduced sodium current in frog isolated dorsal root ganglion cells. I, Neurapliysinl. 63, 8057813 (1990). 6. Canessa, C. M., Horisberger, l. D. & Rossier, B. C. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361, 467—470 (1993). 7. Canessa, C. M. et a]. Amilorideesensitive epithelial Na' channel is made of three homologous subunits. Nature 367, 4637467 (1994), 8. Lingueglia, 13., Voilley, N., Waldmann, R., Lazdunski, M, & Barby, P. Expression cloning of an epithelial amiloridersensitive Na' channel. A new channel type with homologies to Caeuorlmltditis elegans degenerins. FEBS Lett. 318, 95799 (1993). 9. Lingueglia, E. et a1. Different homologous subunits of the amiloride-sensitive Na differently regulated by aldosterone. I. Biol. Chem. 269, 13736713739 (1994). 10. Lingueglia, E., Champigny, (1., Lazdunski, M. St Barbry, P. Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel. Nature 378, 7307733 (1995). 11.Waldmann, Rs Champigny, G., Bassilana, F., Voilley, N. 8r Lazdunski, M. Molecular cloning and functional expression of a novel amiloride-sensitive Na channel. 1. Biol. Chem. 270, 2741 1727414 (1995), 12. Driscoll, M. & Chalfie, M. The mac-4 gene is a member ofa family of Cacnorlrahdilis elegaus genes that can mutate to induce neuronal degeneration. Nature 349, 5887593 (1991), 13, Huang. M. 8: Chalfie, M. Gene interactions affecting meclmnosensory transduction in Caumrlmluiim elegans. Nature 367, 4677470 (1994). 14. Waldmann, R., Champigny, G, Voilley, N., Lauritzen, 1. 3r Lazdunski, M. The mammalian degencrin MDEG, an amiloride-sensitive cation channel activated by mutations causing neurodegeneration in Cuenorhalrditis elegnns. I. Biol. Chem. 271, 10433—10434 (1996). 15. Kovalchuk Yu, N., Krishtal, O. A. & Nowycky, M. C. 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Clotting and expression ofa novel human brain Na' channel. I. Biol. Chem, 271, 7879—7882 (1996). 21. Akaike, N. & Ueno, S. Proton»induced current in neuronal cells. Prog. i’V’EtH'UltiUL 43, 7}A83 (I994). 22. Krishtal, O. A,, Osipchuk, Y. V., Shelest, T. N. & Smirnoff, S. V. Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices. Bruin Res. 436, 352—356 (1987). 23. Chesler, M. & Kaila, K. Modulation opr by neuronal activ , lire/ids Neurosci. 15, 39(17402 (1992). 24. Bevan, S. 8( Yeats, l. Protons activate a cation conductance in a subepopulation of rat dorsal root ganglion neurones. I. Physiol. 433, 1457161 (1991). 25. Lewis, C. et a]. Coexpression ofP2XZ and P2X3 receptor subunits can account for ATPegated currents in sensory neurons. Nature 377, 432—435 (1995). 26. Barnard, E. A. The transmitter-gated channels: a range of receptor types and structures. Trends Pharmacol. Sci. 17, 3057309 (1996). 27. Okada, Y., Miyamoto, T. & Sato, T, Activation of a cation conductance by acetic acid in taste cells isolated from the bullfrog. I. Exp. Biol. 187, 19732 (1994). 28. Liu, 1., Schrank, B. & VVaterson, R. Interaction between a putative mechanosensory membrane channel and a collagen. Science 273, 361—364 (1996). 29. VValdmann, R, Champigny, G. & Lazdunski, M. Functional degenerin-containing chimeras identify residues essential for amiloride-sensitive Na‘ channel function. I, Biol. Chem. 270, 11735—11737 (1995). 30. Renard, S., Lingueglia, E., Voilley, N., Lazdunski, M. & Barbry, P. Biochemical analysis of the membrane topology ofthe amiloridevsensitive Na * channel. I. Biol. Chem. 269, 12981712986 (1994). channel are Acknowledgements. This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the Association Francaise contre les Myopathies (AFM). We thank l. R. de Weille and E. Lingueglia for discussion, G. Jarretou, M. lodar and N. Leroudicr for technical assistance, Y. Benhamou for secretarial assistance, and F. Aguila for help with artwork. Correspondence and requests for materials should be addressed to M1. (eemail: [email protected]). Ras signalling linked to the cell-cycle machinery by the retinoblastoma protein Daniel S. Peepern‘, Todd M. Uptom, Mohamed H. Ladhat, Elizabeth Neumam, Juan Zalvide*, René Bernardst, James A. DeCaprim & Mark E. Ewem * The Dana—Father Cancer Institute and the Harvard Medical School, 44 Binncy Street, Boston, Massachusetts 02115, USA T The Netherlands Cancer Institute, Department of Molecular Carcinogenesis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands The Ras prom-oncogene is a central component of mitogenic signal-transduction pathways, and is essential for cells both to leave a quiescent state (G0) and to pass through the Gl/S transi— tion of the cell cycle”. The mechanism by which Ras signalling regulates cell-cycle progression is unclear, however. Here we NATURE l VOL 386i 13 MARCH 1997 letters to nature report that the retinoblastoma tumour-suppressor protein (Rb), a regulator of G1 exit7, functionally links Ras to passage through the G1 phase. Inactivation of Ras in cycling cells caused a decline , in cyclin D1 protein levels, accumulation of the hypophosphoryl- ated, growth-suppressive form of Rb, and G1 arrest. When Rb was disrupted either genetically or biochemically, cells failed to arrest in G1 following Ras inactivation. In contrast, inactivation of Ras in quiescent cells prevented growth-factor induction of both immediate-early gene transcription and exit from G0 in an Rb- independent manner. These data suggest that Rb is an essential j Gl-specific mediator that links Rats—dependent mitogenic signal- ling to cell-cycle regulation. The function of Ras was neutralized to examine whether the resulting inhibition of proliferation requires Rb function. By inhi- biting rather than activating Ras signalling, we could identify target proteins that are critical for, rather than proteins whose function correlates with, Ras—dependent effects on the cell cycle. Asynchro— nous primary Rh“+ and Rh’” mouse embryo fibroblasts (MEFs) were microinjected with monoclonal antibody Y13—259, which specifically neutralizes Ras4’“, and DNA replication was monitored by incorporation of S—bromodeoxyuridine (BrdU). Neutralization of Ras led to efficient inhibition of DNA synthesis in the RV” MEFs (Fig. 1a). In contrast, inactivation of Ras led to a sevenfold smaller inhibition of DNA synthesis in lef' MEFs, a difference similar to that previously reported for microinjection of plasmids encoding p16, an inhibitor of Cdk4 and Cdk6, into both cell typesg‘w. Ras antibody also inhibited the proliferation of immortalized 3T3 derivatives of the Rh+l+ and Rh” 7 MEFs in an Rb—dependent manner (Fig. 1b). Similar results were obtained for injections ofGl cells after release from a serum—starved state (not shown). These results suggest that the status of Rh can dictate whether cells stop cell—cycle progression in response to inactivation of Ras. As an independent measure to evaluate the role of Rb in Ras- dependent cell-cycle progression, we co—transfected an expression vector for a dominant interfering Ras mutant, RasAsm (refs 11, 12), with a CD20 cell—surface marker and analysed the cell-cycle profile of the transfected population by two—colour flow cytometry. Expres— sion of RasAsnl7 caused a significant arrest in the G1 phase of the cell— cycle in NIH 3T3 cells (Fig. 2a). In contrast, expression of RasAsnl7 failed to cause G1 arrest in three independent R177” 3T3 clones (Fig. 2a, b), although it was expressed to comparable levels in NIH 3T3 and Rb ’ T cells (Fig. 2a). Even higher levels of RasA5nl7 (obtained using stronger promoters) failed to stop R1747 3T3 cells from proliferating (data not shown). In agreement with these results, expression of RasAsnl7 also led to Rb—dependent inhibition of prolifera- tion in long-term growth—suppression assays (data not shown). To extend these observations to another cell type, we compared the effect of Ras inactivation in the Rh” ’ myoblast cell line CC42 with that in the Rb—positive myoblast cell line C2C12. Expression of RasAsnl7 caused significant G1 arrest in C2C12 cells, but not in CC42 cells (Fig. 2c). Similarly, microinjection of neutralizing Ras anti— bodies resulted in an approximately ninefold greater inhibition of DNA synthesis in C2C12 cells than in CC42 cells (data not shown), providing further support for the results described above. We then attempted to exclude the possibility that R197” cells might fail to respond to proliferation—restraining signals in general. 1 Consistent with previous reportsg’lo’lj’”, p16 caused G1 arrest in an i Rb—dependent manner (Fig. 2a). In contrast, both a dominant— negative Cdk3 mutant (Cdk3—dn) and the Cdk inhibitor p27, both of which cause cell—cycle arrest in an Rb—independent manner”, induced G1 arrest as efficiently in R1947 3T3 cells as in NIH 3T3 cells (Fig. 2a). These data indicate that the Rb requirement for induction of G1 arrest is specific for RatsAsnl7 and p16. , To further exclude the possibility that an Rb—independent mechanism might be responsible for the failure of RbT/T cells to stop proliferating in response to Ras inactivation, we performed two types ofexperiments. First, when Rb function was restored in Rb’ / T 177 letters to nature cells, the ability of RasAsnl7 to induce G1 arrest as a function of exogenous Rb expression, and vice versa, was monitored. RasAsnl7 and Rb, when expressed alone, caused only a small increase in the proportion of G1 cells (Fig. 3). In contrast, in both experimental settings, coexpression of RasAsnl7 and Rb induced G1 arrest in a coo erative, rather than additive, fashion. Second, reversal of a Ra snl7-induced G1 arrest by the adenovirus E1A protein was largely dependent on its Rb— f—amily binding domains (data not shown). Thus either transient inactivation or restoration of Rb can determine the response to downregulated Ras activity. It is therefore unlikely that REF/— fibroblasts have acquired an adaptive mutation that renders them unresponsive to Ras signalling. The above results suggest that, in a cycling asynchronous population of cells, inactivation of Ras causes G1 arrest in an Rb—dependent Hb‘/' MEFS Fib‘” MEFS DNA ,, 100 100 75 75 50 50 25 25 0 0 (<% s3 a“ e6 a“ BrdU 5' Nuclei incorporating BrdU (0/0) Figure 1 Microinjection of Res-neutralizing antibody inhibits cell—cycle progres- sion in an Rb—dependent manner. a, Rae-neutralizing antibody inhibits DNA synthesis in Hot” MEFs but notRb ’/’ MEFS. Ras monoclonal antibody (Y13-259) was microinjected intothe cytoplasm of asynchronous cultures of primary MEFs derived from Rbt/t (left) or Rb’” (right) mouse embryo littermates (nuclei of injected cells are indicated by arrows). Top. staining for immunoglobulin-positive, injected cells, Middle, identical field stained for 5-bromodeoxyuridine (BrdU). Bottom, nuclear counterstaining with DAPI. b, Percentage of BrdU-incorporating cells after microinjection with Ras antibody (filled bars)or control lgG (open bars). As well as primary MEFs (left), immortalized (3T3) fibroblasts derived from Rb“t and Rb’” mouse embryos, and NIH 3T3 cells were used (right). At least 400 microinjected cells were assayed per cell type and antibody used. The results show mean + s.e. for at least four independent experiments. 178 manner. Previous work suggests that Ras activity is required for immediate—early gene activation and exit from a quiescent (G0) state4’8’”. We therefore examined whether Rb is involved in either of these two processes. RasASI117 completely blocked growth—factor induction of immediate—early gene transcription in Rb“ T 3T3 cells (Fig. 43), as in NIH 3T3 cells”. Moreover, inactivation of Ras prevented growth factor—stimulated cell—cycle re—entry of quiescent Rb"‘ 3T3 cells (Fig. 4b, c). These results are consistent with the observation that primary R17" 7 MEFs and R17" _ 3T3 cells can be arrested by serum deprivation1617 (Fig. 4). Furthermore, these data indicate that, in Rb ’ T 3T3 cells, Ras signalling can be blocked by either RasAsnwor Ras antibody. More importantly they suggest that Rb IS a critical component of Ras—dependent signalling during G1 but not G0. Our results suggest that inactivation of Ras prevents G1 exit by '5 I? «‘5 a ,\ 6.» Q10 9&5 g 30 r—i rfi E, 25 ' 5 ._ , Asn17 S 20 Has “Jfias Q 0 E 15 (D ‘ ' _ 15iNK4a E 10 “ - p (D 8 5 - e I . 0—PZ7K'P‘ :5 0 g _. _ '\ e 9e .1!" Q? xé‘ 6:». ‘- Cdks-dn v ex: 3+ ,{l- 49" 9" o 61. b c g; 30 g 30 5 25 g 25 E ‘5 a 20 a 20 8 o .— 15 ‘1 15 0 5 .E 10 .E 10 ‘D :1) U) co 5 ‘0 5 §I g o ‘ E o '0 /\ 29° Yeé‘ 9 <29 <29 Figure 2 Expression of RasASW causes G1 cell-cycle arrest in an Rb-dependent manner. a, Left, NlH 3T3 cells (filled bars) and Rb’“ 3T3 cells (open bars) were transfected with control plasmid, or with plasmids as indicated, together with a plasmid encoding the CD20 cell—surface marker. The transfected cell population was identified by staining with fluorescein isothiocyanate (FlTC)—conjugated anti- CDZO antibody and DNA content was monitored by staining with propidium iodide and two—colourflow cytometw. The absolute changes in the percentage of cells in G1 compared to control transfections are shown with the mean + s. e from at least three independent experiments. Right, ectopic expression of Rae/“”17, p16, p27 and Cdl<3- dn-HA in NIH 8T3 and Rb ’ 3T3 cells, as visualized by immunoprecipitation, SDS—PAGE and autoradiography; — and + indicate control (vector) and expression plasmid transfections, respectively. b, As a, pen‘ormed with two other independently derived immortalized Rb”’ 3T8 cell populations (hatched and open bars), relative to NIH 3T3 cells (filled bar), A representative experiment is shown. c, As a, performed with the Rb»positive myoblast cell line 02012 (filled bars) and the Rb ’/" myoblast cell line 0042 (open bars). NATUREIVOL 386i 13 MARCH 1997 modulating Rb activity. To test this, we determined whether expression of RasAsnl7 affects the phosphorylation status of Rb. When expressed ectopically in NIH 3T3 cells, Rb migrated as a doublet, suggesting that both hypophosphorylated (growth—sup— pressive) and hyperphosphorylated (inactive) Rb species were present in roughly equimolar amounts (Fig. 5a). In contrast, when coexpressed with RasAS‘m, Rb accumulated almost exclusively in its hypophosphorylated form, suggesting that inhibition of Ras activity leads to inhibition of Rb phosphorylation. The accumulation of cyclin D proteins, the regulatory subunits of Cdk4 and Cdk6 that phosphorylate Rb, is regulated in part by mitogens”. This finding, together with the above results, prompted us to examine whether Ras inactivation decreases the accumulation of cyclin D1 protein. Indeed, a significant reduction in cyclin D1 a b A 20 30 o\° .5 15 E 3 20 8 10 Q (g 5 -— 10 (D 8 o E O E .5 O o 1 2 0 e 12 Rb (Hg) RasAsn17 (H9) Figure 3 Reintroduction of Rb into Rb ’/ fibroblasts rescues induction of Gt arrest by Ras inactivation. a. Individual cell populations of Rb 3T3 cells were transfected with a constant amount of plasmids expressing Rae/“W and CD20, together with an increasing amount of a plasmid encoding Rb. Aftertransfection, cells were processed and the absolute change in the percentage of cells in Gt. relative to a control transfection, was determined by FACS analysis. Data were corrected for cell-cycle effects caused by expression of Rb alone; mean + s.e. for four independent experiments. b, As a, except Rb plasmid was held constant and RasASW expression plasmid was added in increasing amounts Cell-cycle effects caused by expression of RasASW. in the absence of Rb, were corrected for; mean + s.e. for two independent experiments. a b A b 15 9% 50 EA % 40 $2 03 [D g 10 O‘) a; E 30 e E a 513 E B '95 5 9 2° L O L3 o m g 10 co '6 o - 5 o |___i I_.l g EGF/PDGF: - + c so .3 50 E a 40 o + (/3 30 E 9 20 '03 0 1o 0 I l—i %i l—l Treatment : - PDGF TPA FBS +ins. NATUREIVOL 386i 13 MARCH 1997 letters to nature protein levels was observed when RasAsr117 was expressed (Fig. 5b, left). This effect was specific for cyclin D1, as Ras inactivation did not affect the expression of either Cdk4 or tubulin. Moreover, a Cde—dn-induced G1 arrest did not result in a reduction of cyclin D1. Importantly, expression of RasAsnl7 in Rb” T 3T3 cells also led to a decline in cyclin D1 (Fig. 5b, right). Because proliferating RbT/T 3T3 cells do not undergo G1 arrest when Ras is inactivated (Figs 1, 2 and 3), this result uncouples downregulation of cyclin D1 by RasAsnl7 from any cell—cycle effects, and indicates that the signalling pathway between Ras and cyclin D1 in REIT/7 3T3 cells is intact. To examine whether it is the decline in cyclin D1 that causes inhibition of Rb phosphorylation when Ras is inactivated, we monitored the effect of individual G1 kinase subunits on Rb phosphorylation in this setting. Coexpression with either Cdk4 or Cde alone did not have a significant effect on the inhibition of Rb phosphorylation by RasAsnl7 (Fig. 5a), consistent with the observa- tion that Cdk4 levels are not altered by RasASHI7 expression (Fig. 5b). In contrast, coexpression of cyclins D1, D2 or E rescued the inhibition of Rb phosphorylation by RasASW. This suggests that Ras inactivation causes downregulation of cyclin D1, which in turn leads to the accumulation of hypophosphorylated and active Rb, preventing G1 exit. We then prevented the downregulation of cyclin D1 to determine whether it is an essential step during the induction of G1 arrest by Ras inactivation. Both cyclin D1—Cdk4 and cyclin D2—Cdk4, as well as activated Rasvam, completely overcame RasAsnn—induced G1 arrest, either in the absence or presence of nocodazole (Fig. 5c, d). This suggests that downregulation of cyclin D1 is required for RasAsnl7 to induce cell-cycle arrest. These observations are consistent with previous reports on the regulation of cyclin D1 transcriptionlg‘zo, that induction of cyclin D1 by oncogenic Ras may contribute to transformation”? and that p16 blocks Ras plus Myc—induced transformation“. Cyclin E—Cde was less effective than cyclin D—Cdk4 in reversing the G1 arrest induced by Rats/““7. A possible explanation for this is the suggested coordinate action of cyclin D— and E—dependent kinases in the complete phosphorylation and inactivation of szs. Without initial phosphorylation of Rb by cyclin D kinases, cyclin E—Cde may be capable of only partly phosphorylating (and inactivating) Rb. However, this may be sufficient to cause a shift of Rb on a denaturing gel (Fig. 5a). Figure 4 Inhibition of Ras activity blocks G0 exit in an Rb—independent manner. a. FiasAW expression inhibits growth factor-induced serum response element (SRE) activity in Rb / 3T3 cells. Rb /’ 3T3 cells were transfected with RasAW expression plasmid (filled bars) or empty vector (open bars), together with an SRE-luciferase reporter plasmid. Then, 24h after transfection, cells were serum starved (0.1% FBS) for 72 h. At this time. no growth f...
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