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Rapamycin Inhibition of the G1 to S Transition Is Mediated by Effects on Cyclin D1 mRNA and Protein Stability

Rapamycin Inhibition of the G1 to S Transition Is Mediated by Effects on Cyclin D1 mRNA and... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 23, Issue of June 5, pp. 14424 –14429, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Rapamycin Inhibition of the G to S Transition Is Mediated by Effects on Cyclin D1 mRNA and Protein Stability* (Received for publication, January 22, 1998, and in revised form, March 13, 1998) Said Hashemolhosseini‡, Yoshikuni Nagamine§, Simon J. Morley¶ i, Sylvane Desrivie ` res‡**, Luka Mercep‡, and Stefano Ferrari‡ ‡‡ From the ‡Institute for Experimental Cancer Research, Tumor Biology Center, P. O. Box 1120, 79011 Freiburg, Germany, §Friedrich Miescher Institute, P. O. Box 2543, 4002 Basel, Switzerland, and ¶Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom The immunosuppressant rapamycin has been shown activity and regulates the activation of the S6 ribosomal pro- S6k /S transition in several cell previously to inhibit the G tein kinase p70 in vivo; this requires both the FRAP kinase phase of the cell cycle. This types by prolonging the G domain and the N-terminal domains of FRAP (4). As a result of S6k process appears to be controlled, in part, by the rapa- this interaction, rapamycin causes rapid inactivation of p70 mycin-sensitive FK506-binding protein-rapamycin-asso- and dephosphorylation of S6 in vivo (reviewed in Ref. 1). Al- S6k ) pathway and the ciated protein-p70 S6 kinase (p70 though this has only a small effect on the overall rate of cyclin-dependent kinases (Cdk). We now show that in translation (5– 8), it greatly inhibits translation of mRNAs serum-stimulated NIH 3T3 cells, rapamycin treatment containing a 59-terminal oligopyrimidine tract (59-TOP) proxi- delays the accumulation of cyclin D1 mRNA during pro- mal to the cap structure (reviewed in Ref. 9). The importance of . Rapamycin also appears to affect gression through G S6k p70 is shown by the finding that microinjection of quiescent stability of the transcript. The combined transcriptional S6k fibroblasts with a polyclonal antibody against p70 abolished and post-transcriptional effects of the drug ultimately the serum-induced entry into S phase (10). result in decreased levels of cyclin D1 protein. More- S6k Rapamycin selectively inhibits the activation of p70 , pre- over, degradation of newly synthesized cyclin D1 pro- vents cyclin-dependent kinase activation, retinoblastoma pro- tein is accelerated by rapamycin, a process prevented by tein (pRb) phosphorylation, and G progression (1, 11–17). In- inclusion of the proteasome inhibitor, N-acetyl-Leu-Leu- norleucinal. The overall effect of rapamycin on cyclin hibition of cell cycle progression may be mediated via inhibition D1 leads, in turn, to impaired formation of active com- of protein synthesis or via effects on the Cdks. Regulation of plexes with Cdk4, a process which triggers retargeting Cdk activity, which is crucial for the orderly initiation and Kip1 inhibitor to cyclin E/Cdk2. In view of this of the p27 progression of the cell division cycle, involves modulation of the novel experimental evidence, we discuss a possible level of synthesis of cyclins and Cdk inhibitors (18, 19). After mechanism for the rapamycin-induced cell cycle arrest stimulation, cells sequentially synthesize the G cyclins D and /S transition. at the G E, which are rate-limiting for entry into S phase (Ref. 19 and references therein). D-type cyclins (D1, D2, and D3), expressed in a lineage-dependent fashion, are induced in response to Rapamycin, initially characterized as an inhibitor of G cell growth factor stimulation (20). In fibroblasts, cyclin D1 is both cycle progression, has been utilized to unravel a growth factor- necessary and rate-limiting for G progression (21) and its stimulated signaling pathway leading to the preferential trans- overexpression has been implicated in the development of a lation of a specific subset of mRNAs. The drug forms a stable wide range of tumors (reviewed in Refs. 22–24). It forms ho- complex with the immunophilin FK506-binding protein, which loenzymes with Cdk4 and Cdk6 whose activity triggers transi- binds to a family of kinases, FK506-binding protein rapamycin- tion through a G checkpoint by phosphorylation and func- 1 1 associated protein (FRAP) in human cells, the target of rapa- tional inactivation of pRb (18, 19, 25). In turn, pRb might help mycin in yeast (reviewed in Refs. 1 and 2). FRAP is a member to positively regulate cyclin D1 transcription, thus establishing of phosphatidylinositol kinase-related kinases, which impinge the existence of a regulatory loop between the two gene prod- upon cellular events as diverse as cell cycle regulation in re- ucts (26). Although abnormalities of D-type cyclin expression sponse to stress and DNA recombination (reviewed in Refs. 2 can be associated with increased levels of mRNA, there is also and 3). Although poorly understood, FRAP has intrinsic kinase evidence for enhanced stability of cyclin D protein in human sarcoma cells (27). Little is known about the mechanisms reg- ulating the translation of cyclin D mRNA, although there are * This study was supported by the DFG (German Research Council) Grant FE 387/2-1 (to S. F.). The costs of publication of this article were indications for control at the level of nucleocytoplasmic trans- defrayed in part by the payment of page charges. This article must port in cells artificially overexpressing eIF-4E (28, 29). therefore be hereby marked “advertisement” in accordance with 18 On the other hand, Cdk inhibitors act stoichiometrically and U.S.C. Section 1734 solely to indicate this fact. i Senior Research Fellow of The Wellcome Trust. oscillations in their levels can have a profound effect on cell Kip1 ** Present address: Biocentre, Dept. of Biochemistry, University of proliferation (30 –35). The Cdk inhibitor p27 is present at Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland. high levels in quiescent cells and down-regulated by mitogenic ‡‡ To whom correspondence should be addressed: Dept. of Oncology, stimulation (17, 32, 36); the latter process being blocked by Novartis Pharma Ltd., Klybeckstr. 141, 4002 Basel, Switzerland. Tel.: Kip1 41-61-696-1715; Fax: 41-61-696-3835; E-mail: [email protected]. rapamycin (36). The level of p27 protein appears to be The abbreviations used are: FRAP, FK506-binding protein rapamy- modulated in part through translational control and in part via cin-associated protein; LLnL, N-acetyl-Leu-Leu-norleucinal; 59-TOP, Kip1 protein stability (37, 38). However, the importance of p27 59-terminal oligopyrimidine; pRb, retinoblastoma protein; Cdk, cyclin- regulation in the antiproliferative effect of rapamycin is a mat- dependent kinase; PAGE, polyacrylamide gel electrophoresis; GST, glu- tathione S-transferase. ter of some debate (34, 39). 14424 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Cyclin D1 Regulation by Rapamycin 14425 S6k fusion protein as substrate. GST-Rb phosphorylation was analyzed Inhibition of FRAP-p70 signaling by rapamycin or wort- 1–79 by SDS-PAGE and visualized by autoradiography. A Cdk2 activity mannin (1, 40 – 42) also blocks the phosphorylation of two ad- S6k assay was performed as described (45) using 50 mM p70 pep- 408 – 427 ditional proteins (4E-BP1 and 4E-BP2), which interact with tide as substrate. Incorporation of [ P]phosphate onto the peptide initiation factor eIF-4E and inhibit cap structure-dependent substrate was determined by spotting 20-ml aliquots on P81 paper translation. Phosphorylation of 4E-BP1 disrupts its interaction (Whatman) (45). For Western blotting, recovered proteins were eluted with eIF-4E, liberating eIF-4E to interact with a conserved with Laemmli sample buffer and resolved by SDS-PAGE. Northern Blotting—Small scale RNA preparation was performed as hydrophobic region of eIF-4G. Formation of this complex facil- described (46), and its quality assessed by examining the 18 S/28 S itates the binding of mRNA to the ribosome and promotes ratio. Oligo-primed labeling of mouse cyclin D1 was carried out follow- translation initiation (reviewed in Refs. 7 and 43). However, ing standard procedures, and membranes were incubated with 2 3 10 rapamycin does not prevent the phosphorylation of eIF-4E in cpm of the probe. Signals were revealed and quantified using a Phos- primary T cells (5), Xenopus oocytes (5), Chinese hamster ovary phorImager (Molecular Dynamics). cells in response to insulin (44), or NIH 3T3 cells in response to RESULTS serum (8). Cyclin D1 mRNA Levels Are Affected by Rapamycin—Ex- We have examined the role of rapamycin-sensitive pathways pression of cyclin D1 mRNA is down-regulated in mitogen- in the accumulation of cyclin D1 after serum-stimulation of deprived cells and induced upon stimulation of fibroblasts with quiescent NIH 3T3 cells. Our results demonstrate that rapa- serum, epidermal growth factor, platelet-derived growth factor mycin can delay the serum-stimulated accumulation of cyclin and basic fibroblast growth factor (47, 48). To dissect the mech- D1 mRNA. Although cyclin D1 mRNA contains a putative anism by which rapamycin regulates cyclin D1 expression, we polypyrimidine tract in its 59 untranslated region, rapamycin have analyzed the transcriptional activation of cyclin D1 did not affect the rate of synthesis of D1 protein. Rather, the mRNA. NIH 3T3 cells were arrested in G by serum starvation drug promoted degradation of newly synthesized cyclin D1, for 36 h and restimulated with 10% (v/v) serum in the presence resulting in decreased Cdk4 kinase activity, decreased phos- Kip1 or absence of rapamycin. Total RNA was purified at different phorylation of pRb, and increased association of p27 with times of activation and the level of cyclin D1 mRNA was esti- cyclin E/Cdk2. mated by Northern blot analysis. Hybridization of the products EXPERIMENTAL PROCEDURES with a mouse specific cDNA probe showed that serum in- Materials—Unless specified, chemicals were from Merck or Calbio- creased cyclin D1 mRNA levels and treatment of cells with chem. Media for cell culture (Dulbecco’s modified Eagle’s medium and rapamycin caused a delay in the accumulation as well as a fetal bovine serum) were from BioWhittaker, Inc. Radioactive isotopes decrease in the absolute amount of cyclin D1 transcript (Fig. were purchased by NEN Life Science Products. The glutathione S- 1A). To distinguish between effects of rapamycin on transcrip- transferase pRb (GST-Rb ) fusion construct was kindly provided by 1–79 Dr. L. Meijer (Roscoff, France). tion and the stability of the mRNA, quiescent NIH 3T3 fibro- Cell Culture and in Vivo Labeling—NIH 3T3 fibroblasts were seeded blasts were stimulated as described above and treated either in and maintained in Dulbecco’s modified Eagle’s medium containing 10% the presence or the absence of actinomycin D. The results fetal bovine serum and penicillin/streptomycin (100 units/ml and 100 obtained indicate that the stability of cyclin D1 mRNA is mg/ml, respectively). Cells at approximately 60 –70% confluency were clearly affected by rapamycin (Fig. 1B). arrested in G by serum deprivation for 36 h in Dulbecco’s modified Rapamycin Inhibits Serum-stimulated Cyclin D1 Protein Ex- Eagle’s medium containing 0.5% fetal bovine serum and re-stimulated to enter the cell cycle by adding 10% (v/v) serum for the times indicated pression—Examination of the 59-untranslated region (59-UTR) in the figure legends. In vivo labeling of cells was carried out employing of the human cyclin D1 gene suggested the presence of a puta- 100 mCi/ml of a [ S]methionine/cysteine-labeling mix. To examine the tive polypyrimidine tract at the start site of transcription (49). rate of cyclin D1 synthesis, cells were pulsed for the last 30 min before This element, comprising a cytidine residue at the cap site harvesting in Dulbecco’s modified Eagle’s medium containing 10% dia- followed by 4 uninterrupted pyrimidines, is similar to se- lyzed fetal bovine serum. To determine the protein’s half-life, cells were quences demonstrated to confer translational control in a cell pulsed for2hinthe medium described above, 2 h after re-stimulation and chased for the next4hinthe presence of an excess of unlabeled type- and sequence context-dependent manner (9, 50). To ad- amino acids. dress the question of whether D-type cyclin mRNAs are under Western Blotting, Immunoprecipitations, and Protein Kinase Assay— such selective translation control, we examined the role of the Cell extracts were prepared at the indicated times by Dounce homoge- S6k FRAP-p70 signaling pathway on the expression of cyclin D1 nization in ice cold Buffer A (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 20 protein. Fig. 2A shows that serum increased the expression of mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM sodium pyrophosphate, 30 cyclin D1 protein within 4 – 6 h following addition. This was mM p-nitrophenyl phosphate, 1 mM benzamidine, 0.1 mM phenylmeth- ylsulfonyl fluoride, 1% Nonidet P-40). Extracts were centrifuged at greatly reduced and delayed by treatment of cells with rapa- 18,000 3 g for 10 min at 4 °C. Protein concentration was determined mycin, in a manner similar to that reported for ribosomal S6k with a BCA protein assay kit (Pierce). To analyze extracts, equal proteins in T lymphocytes (51). Under these conditions, p70 amounts of protein (5 mg) were resolved by SDS-PAGE and transferred activity was completely inhibited (see Ref. 45 and below). To to polyvinylidene difluoride membrane (Millipore). The resulting mem- Kip1 exclude the possibility that the observed drop in cyclin D1 branes were probed with anti-cyclin D1, Cdk4, or p27 polyclonal protein was nonspecific, the effect of rapamycin on the expres- antibody (Santa Cruz Biotechnology), a polyclonal serum specific for S6k p70 , or with a monoclonal antibody to pRb (Pharmingen) and re- sion of Cdk4 was also examined. Fig. 2B shows that rapamycin vealed using the ECL system (Amersham Pharmacia Biotech). To an- did not influence the level of expression of this protein. Similar S6k alyze proteins present in the cyclin-Cdk complexes and monitor kinase results were obtained for p70 (see below). Pulse-labeling activity, mouse polyclonal antisera to Cdk2 (Upstate Biotechnology, experiments with [ S]methionine/cysteine, indicated that at Inc.) or Cdk4 (Santa Cruz Biotechnology) were used. Extractions and each time point examined, the initial rate of cyclin D1 synthesis immunoprecipitations were carried out in Buffer B (25 mM Tris-HCl, pH was unchanged by the presence of rapamycin (data not shown). 7.5, 60 mM b-glycerophosphate, 15 mM MgCl ,15mM EGTA, 0.1 mM S6k NaF, 15 mM p-nitrophenyl phosphate, 1 mM dithiothreitol, 0.1 mM Taken together, these data indicate that the p70 signaling phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40) using equal pathway is not required for enhanced synthesis of cyclin D1 amounts of total protein (300 mg). Antibodies were immobilized on and that the putative 59-TOP sequence in the mRNA may not protein A-Sepharose beads, and the resin washed with 3 3 1mlof have a role in vivo. ice-cold Buffer B and 1 ml of ice-cold Buffer C (50 mM Tris-HCl, pH 7.5, When rapamycin was added to cells 4 h after serum-stimu- 10 mM MgCl ,1mM dithiothreitol. For kinase assays, the resin was lation, only a partial decrease in the accumulation of cyclin D1 resuspended in a total volume of 25 ml of Buffer C, containing 5 mM [ P]ATP (specific activity, 50 mCi/nmol), and 10 mg of GST-Rb protein was observed (35% versus 55%) (Fig. 2C). These data 1–79 14426 Cyclin D1 Regulation by Rapamycin FIG.2. Rapamycin prevents the serum-stimulated increase of cyclin D1 protein during G progression. Serum-starved NIH 3T3 fibroblasts were stimulated with complete medium in the presence or absence of 20 nM rapamycin. Cell extracts were prepared at the times indicated and subjected to SDS-PAGE. The level of expression of cyclin D1 (A) and Cdk4 (B) were visualized by Western blotting employing specific polyclonal antisera, as described under “Experimental Proce- dures.” C, serum-starved NIH 3T3 fibroblasts were stimulated with complete medium for 4 h before the addition of vehicle or rapamycin. The level of expression of cyclin D1 was determined 8 h post-stimulation and quantified with a Bio-Rad GS-700 densitometer. The values are given as optical density/mm , 8.16 6 0.28 (left lane); 5.34 6 0.21 (right lane). Absence or presence of rapamycin is denoted by 2 and 1, respectively. FIG.3. Rapamycin accelerates the turnover of cyclin D1. Se- rum-starved cells were incubated in the absence or presence of rapa- FIG.1. Cyclin D1 mRNA levels are affected by rapamycin. Se- mycin, as described in the legend to Fig. 2. Cells were pulsed with a rum-starved NIH 3T3 fibroblasts were stimulated with complete me- 35 [ S]methionine/cysteine labeling mix and chased as described under dium in the absence or presence of 20 nM rapamycin (rap)(A). Total “Experimental Procedures.” Total protein was extracted, immunopre- RNA was extracted at the indicated time points and analyzed by North- cipitated with a cyclin D1-specific antiserum and resolved by SDS- ern blot hybridization employing a mouse-specific cDNA probe to cyclin PAGE. Signals were revealed and quantified using a PhosphorImager. D1 as described under “Experimental Procedures” (upper panel). Before Rap, rapamycin; CycD1, cyclin D1. hybridization the membrane was stained with methylene blue to con- firm equal loading of each sample (lower panel). In B, cells were treated administration of rapamycin to cells in culture has been attrib- as in A, with the exception that 2 mg/ml actinomycin D was added 1 h uted to a number of distinct events. Among those is an overall post-stimulation. Total RNA was extracted and analyzed as in A. Open inhibition of protein synthesis due to decreased ribosome as- and closed circles denote presence or absence of rapamycin, respec- tively. Signals were revealed and quantified using a PhosphorImager sembly (51), the lack of formation of active cyclin E-Cdk2 com- (Molecular Dynamics). Kip1 plexes (14) and the prevention of p27 elimination from the cyclin E-Cdk2 complex (36). To assess whether the rapamycin- are in agreement with earlier reports (13, 52) that cells already induced degradation of cyclin D1 has any physiological effect on in G are less responsive to the inhibitory effects of rapamycin. Cdk4 activity and G progression in these cells, we have as- 1 1 Rapamycin Stimulates the Rate of Degradation of Cyclin D1 sayed Cdk4 activity using a GST-Rb fusion protein as sub- 1–79 Protein—The data above indicate that rapamycin affected cy- strate. The increase of Cdk4 activity that occurred within 4 – 6 clin D1 at both the transcriptional and post-transcriptional h after serum stimulation was prevented by the addition of level. To examine other possible effects of rapamycin that may rapamycin to the cells (Fig. 5A). In addition, we analyzed the influence the expression of cyclin D1 protein, we have consid- phosphorylation status of endogenous pRb by separation of cell ered the rate of cyclin D1 polypeptide degradation. To directly extracts by SDS-PAGE and Western blotting with serum spe- compare half-lives, quiescent NIH 3T3 cells were stimulated in cific for pRb protein. As indicated by the characteristic shift in the presence or absence of rapamycin, pulsed with [ S]methi- mobility upon phosphorylation and inactivation, pRb phospho- onine/cysteine labeling mix and chased in the presence of an rylation was increased after serum stimulation of cells (Fig. excess of cold methionine/cysteine. The data presented in Fig. 3 5B). This mobility shift was prevented by rapamycin, suggest- show that the turnover rate of cyclin D1 was clearly accelerated ing a lack of pRb phosphorylation in these cells and indicative upon treatment of cells with rapamycin. To further substanti- of a decrease in Cdk4 activity. Western blot analysis of immu- ate this finding, we carried out studies with the proteasome noprecipitated Cdk4 indicated that inhibition of Cdk4 activity inhibitor, N-acetyl-Leu-Leu-norleucinal (LLnL). As described was not caused by a decrease in Cdk4 protein levels (Fig. 5C) above, rapamycin decreased the accumulation of cyclin D1 pro- but was because of lack of the kinase regulatory partner cyclin tein. However, this was prevented by the simultaneous addi- D1 (Fig. 6A). Kip1 tion of LLnL (Fig. 4A). This compound did not influence pro- Reduced Levels of Cyclin D1 Facilitate Retargeting of p27 S6k Kip1 teins with a longer half-life, such as Cdk4 (Fig. 4B) and p70 to Cdk2—The inhibitor protein p27 has been found to as- (Fig. 4C). To rule out possible effects of LLnL on cyclin D1, sociate with both Cdk4 and Cdk2 via the regulatory partner, which might be independent of inhibition of the proteasome, cyclins D and E, respectively (19, 23). The distribution of Kip1 the compound was administered to cells either alone or in p27 among different cyclin-kinase complexes has been pre- combination with rapamycin and the levels of cyclin D1 were dicted to maintain a correct balance between their active forms examined 8 h post-stimulation. Fig. 4D shows that LLnL af- (19, 53, 54). To determine whether inhibition of cyclin D1 Kip1 fected solely protein degradation. accumulation in these cells affected the association of p27 Reduced Levels of Cyclin D1 Affect Cdk4 Activity and pRb with Cdk4 or Cdk2, cells were stimulated with serum in the Phosphorylation—The cause of the G /S block observed after presence or absence of rapamycin and Cdk4 or Cdk2 complexes 1 Cyclin D1 Regulation by Rapamycin 14427 Kip1 FIG.6. Effect of rapamycin on p27 association with G Cdks. A, extracts were prepared from cells stimulated in the presence or absence of rapamycin for 8 h before immunoprecipitation of Cdk4, as described. Recovered proteins were resolved by SDS-PAGE and the level of complexed cyclin D1 (left panel) and Cdk4 (right panel) were visualized by Western blotting. B, extracts obtained from cells 8 or 12 h after stimulation were subjected to immunoprecipitation with antisera specific to Cdk4 (right panel) or Cdk2 (left panel). Recovered proteins Kip1 were resolved on SDS-PAGE and the amount of p27 protein present in either complex was assayed by Western blotting. FIG.4. The proteasome inhibitor, LLnL, rescues cyclin D1 deg- radation induced by rapamycin. Serum-starved NIH 3T3 fibro- TABLE I blasts were treated as described in the legend to Fig. 2, except that the Effect of rapamycin on Cdk2 activity proteasome inhibitor LLnL (50 mM) was added together with rapamycin Extracts obtained from synchronized NIH 3T3 cells at 10 h post- at the time of restimulation. Extracts were prepared at the times stimulation were immunoprecipitated with a Cdk2-specific antiserum indicated and subjected to SDS-PAGE and immunoblot analysis, em- and assayed for kinase activity as described under “Experimental Pro- S6k ploying antisera specific to cyclin D1 (A), Cdk4 (B), or p70 (C). D, cedures.” Data represent the mean value of three independent experi- extracts obtained from synchronized NIH 3T3 cells 8 h post-stimulation ments. in the presence or absence of 20 nM rapamycin, 50 mM LLnL, or a combination of the two were resolved and probed with an antiserum Addition cpm incorporated 6 S.D. specific to cyclin D1. None 7204 6 281 Rapamycin 936 6 26 increase in the synthesis of RNA and protein and culminating in a doubling of protein mass prior to DNA synthesis and cell division (reviewed in Refs. 43 and 55). Physiological regulation of protein synthesis is almost always exerted at the level of polypeptide chain initiation, mediated in part by the availabil- ity of eIF-4E to participate in the initiation process and the phosphorylation of ribosomal protein S6 (reviewed in Ref. 43). Here we set out to investigate the contribution of rapamycin- sensitive signaling pathways in conveying external growth sig- FIG.5. Rapamycin-induced cyclin D1 degradation results in a nals to the cell cycle regulatory machinery. Recent studies decreased Cdk4 activity and pRb phosphorylation. Serum- showed that regulated translation functions in modulating the starved NIH 3T3 fibroblasts were treated as described in the legend to activity of Cdks in mammalian cells (2, 38, 53). One possible Fig. 2, and extracts were prepared at the times indicated. A, cell target for regulation is cyclin D1, whose synthesis is induced extracts containing equal amounts of protein were subject to immuno- precipitation with serum specific to Cdk4, as described under “Experi- during the delayed early response to growth factor addition to mental Procedures.” Cdk4 activity was assayed in immunocomplex cells. The D-type cyclins are the first cyclins synthesized during assay using GST-Rb fusion protein as substrate followed by SDS- 1–79 the cell cycle and complex with Cdk4 and Cdk6. As such, they PAGE and autoradiography. B, phosphorylation of endogenous pRb was are detected in mid G and function as pRb kinases, overcom- visualized after separation of cellular proteins by SDS-PAGE and West- ern blotting. The slower and faster migrating bands correspond to ing the growth-suppressive effects of pRb and thus allow pro- hyper- and hypophosphorylated pRb, respectively. C, cell extracts were gression through G /S (18, 21, 47, 56). It has been postulated fractionated as described above and the level of Cdk4 protein was that this class of cyclins represents the link between extracel- determined by Western blotting. lular signals and the cell cycle machinery (20). Cyclin D1 ac- tivity appears to be responsible for activation of cyclin E and recovered by immunoprecipitation. The antisera employed cyclin A, both partnered by Cdk2, which are active at the G /S were highly specific and showed no cross-reactivity with other boundary and during S phase, respectively (57– 61). Cdks (data not shown). Treatment of cells with rapamycin Kip1 We observed that cyclin D1 mRNA and protein (Figs. 1A and resulted in a decrease in the association of p27 with Cdk4 2A) were increased after a lag period of 2– 4 h after stimulation (Fig. 6B). Conversely, complexes of Cdk2 and cyclin E appeared Kip1 of quiescent NIH 3T3 fibroblasts to proliferate. Rapamycin to be enriched in p27 upon treatment of cells with rapamy- treatment significantly affected the level of cyclin D1 tran- cin (Fig. 6B). Accordingly, rapamycin caused an almost 8-fold script, apparently causing a delay in its accumulation. This can inhibition of Cdk2 activity (Table I). These data suggest that, be attributed to effects on either transcription or stability of the apart from any direct effect on phosphorylation of pRb, the mRNA. The former possibility could not be assessed because of decrease in cyclin D1 protein levels facilitates retargeting of Kip1 low template activity. With regard to the latter, inspection of p27 to cyclin E-Cdk2 complexes and in turn inhibition of the 39-UTR of cyclin D1 mRNA did not reveal the presence of the associated kinase activity. This further promotes the G /S AU-rich sequences, which account for rapid degradation of block in the cell cycle. interleukin-3 mRNA by a rapamycin-sensitive mechanism (62). DISCUSSION However, measurement of cyclin D1 mRNA stability showed a Treatment of quiescent cells with growth factors or hor- substantial effect of rapamycin on the transcript’s half-life (Fig. mones induces re-entry into the cell cycle, accompanied by an 1B). This indicates that novel rapamycin-sensitive pathways 14428 Cyclin D1 Regulation by Rapamycin immunoprecipitation experiments and L. Meijer (Roscoff, France) for are possibly involved in regulation of cyclin D1 mRNA levels. the generous supply of GST-Rb . We are also indebted to Dr. P. 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Rapamycin Inhibition of the G1 to S Transition Is Mediated by Effects on Cyclin D1 mRNA and Protein Stability

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 23, Issue of June 5, pp. 14424 –14429, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Rapamycin Inhibition of the G to S Transition Is Mediated by Effects on Cyclin D1 mRNA and Protein Stability* (Received for publication, January 22, 1998, and in revised form, March 13, 1998) Said Hashemolhosseini‡, Yoshikuni Nagamine§, Simon J. Morley¶ i, Sylvane Desrivie ` res‡**, Luka Mercep‡, and Stefano Ferrari‡ ‡‡ From the ‡Institute for Experimental Cancer Research, Tumor Biology Center, P. O. Box 1120, 79011 Freiburg, Germany, §Friedrich Miescher Institute, P. O. Box 2543, 4002 Basel, Switzerland, and ¶Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom The immunosuppressant rapamycin has been shown activity and regulates the activation of the S6 ribosomal pro- S6k /S transition in several cell previously to inhibit the G tein kinase p70 in vivo; this requires both the FRAP kinase phase of the cell cycle. This types by prolonging the G domain and the N-terminal domains of FRAP (4). As a result of S6k process appears to be controlled, in part, by the rapa- this interaction, rapamycin causes rapid inactivation of p70 mycin-sensitive FK506-binding protein-rapamycin-asso- and dephosphorylation of S6 in vivo (reviewed in Ref. 1). Al- S6k ) pathway and the ciated protein-p70 S6 kinase (p70 though this has only a small effect on the overall rate of cyclin-dependent kinases (Cdk). We now show that in translation (5– 8), it greatly inhibits translation of mRNAs serum-stimulated NIH 3T3 cells, rapamycin treatment containing a 59-terminal oligopyrimidine tract (59-TOP) proxi- delays the accumulation of cyclin D1 mRNA during pro- mal to the cap structure (reviewed in Ref. 9). The importance of . Rapamycin also appears to affect gression through G S6k p70 is shown by the finding that microinjection of quiescent stability of the transcript. The combined transcriptional S6k fibroblasts with a polyclonal antibody against p70 abolished and post-transcriptional effects of the drug ultimately the serum-induced entry into S phase (10). result in decreased levels of cyclin D1 protein. More- S6k Rapamycin selectively inhibits the activation of p70 , pre- over, degradation of newly synthesized cyclin D1 pro- vents cyclin-dependent kinase activation, retinoblastoma pro- tein is accelerated by rapamycin, a process prevented by tein (pRb) phosphorylation, and G progression (1, 11–17). In- inclusion of the proteasome inhibitor, N-acetyl-Leu-Leu- norleucinal. The overall effect of rapamycin on cyclin hibition of cell cycle progression may be mediated via inhibition D1 leads, in turn, to impaired formation of active com- of protein synthesis or via effects on the Cdks. Regulation of plexes with Cdk4, a process which triggers retargeting Cdk activity, which is crucial for the orderly initiation and Kip1 inhibitor to cyclin E/Cdk2. In view of this of the p27 progression of the cell division cycle, involves modulation of the novel experimental evidence, we discuss a possible level of synthesis of cyclins and Cdk inhibitors (18, 19). After mechanism for the rapamycin-induced cell cycle arrest stimulation, cells sequentially synthesize the G cyclins D and /S transition. at the G E, which are rate-limiting for entry into S phase (Ref. 19 and references therein). D-type cyclins (D1, D2, and D3), expressed in a lineage-dependent fashion, are induced in response to Rapamycin, initially characterized as an inhibitor of G cell growth factor stimulation (20). In fibroblasts, cyclin D1 is both cycle progression, has been utilized to unravel a growth factor- necessary and rate-limiting for G progression (21) and its stimulated signaling pathway leading to the preferential trans- overexpression has been implicated in the development of a lation of a specific subset of mRNAs. The drug forms a stable wide range of tumors (reviewed in Refs. 22–24). It forms ho- complex with the immunophilin FK506-binding protein, which loenzymes with Cdk4 and Cdk6 whose activity triggers transi- binds to a family of kinases, FK506-binding protein rapamycin- tion through a G checkpoint by phosphorylation and func- 1 1 associated protein (FRAP) in human cells, the target of rapa- tional inactivation of pRb (18, 19, 25). In turn, pRb might help mycin in yeast (reviewed in Refs. 1 and 2). FRAP is a member to positively regulate cyclin D1 transcription, thus establishing of phosphatidylinositol kinase-related kinases, which impinge the existence of a regulatory loop between the two gene prod- upon cellular events as diverse as cell cycle regulation in re- ucts (26). Although abnormalities of D-type cyclin expression sponse to stress and DNA recombination (reviewed in Refs. 2 can be associated with increased levels of mRNA, there is also and 3). Although poorly understood, FRAP has intrinsic kinase evidence for enhanced stability of cyclin D protein in human sarcoma cells (27). Little is known about the mechanisms reg- ulating the translation of cyclin D mRNA, although there are * This study was supported by the DFG (German Research Council) Grant FE 387/2-1 (to S. F.). The costs of publication of this article were indications for control at the level of nucleocytoplasmic trans- defrayed in part by the payment of page charges. This article must port in cells artificially overexpressing eIF-4E (28, 29). therefore be hereby marked “advertisement” in accordance with 18 On the other hand, Cdk inhibitors act stoichiometrically and U.S.C. Section 1734 solely to indicate this fact. i Senior Research Fellow of The Wellcome Trust. oscillations in their levels can have a profound effect on cell Kip1 ** Present address: Biocentre, Dept. of Biochemistry, University of proliferation (30 –35). The Cdk inhibitor p27 is present at Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland. high levels in quiescent cells and down-regulated by mitogenic ‡‡ To whom correspondence should be addressed: Dept. of Oncology, stimulation (17, 32, 36); the latter process being blocked by Novartis Pharma Ltd., Klybeckstr. 141, 4002 Basel, Switzerland. Tel.: Kip1 41-61-696-1715; Fax: 41-61-696-3835; E-mail: [email protected]. rapamycin (36). The level of p27 protein appears to be The abbreviations used are: FRAP, FK506-binding protein rapamy- modulated in part through translational control and in part via cin-associated protein; LLnL, N-acetyl-Leu-Leu-norleucinal; 59-TOP, Kip1 protein stability (37, 38). However, the importance of p27 59-terminal oligopyrimidine; pRb, retinoblastoma protein; Cdk, cyclin- regulation in the antiproliferative effect of rapamycin is a mat- dependent kinase; PAGE, polyacrylamide gel electrophoresis; GST, glu- tathione S-transferase. ter of some debate (34, 39). 14424 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Cyclin D1 Regulation by Rapamycin 14425 S6k fusion protein as substrate. GST-Rb phosphorylation was analyzed Inhibition of FRAP-p70 signaling by rapamycin or wort- 1–79 by SDS-PAGE and visualized by autoradiography. A Cdk2 activity mannin (1, 40 – 42) also blocks the phosphorylation of two ad- S6k assay was performed as described (45) using 50 mM p70 pep- 408 – 427 ditional proteins (4E-BP1 and 4E-BP2), which interact with tide as substrate. Incorporation of [ P]phosphate onto the peptide initiation factor eIF-4E and inhibit cap structure-dependent substrate was determined by spotting 20-ml aliquots on P81 paper translation. Phosphorylation of 4E-BP1 disrupts its interaction (Whatman) (45). For Western blotting, recovered proteins were eluted with eIF-4E, liberating eIF-4E to interact with a conserved with Laemmli sample buffer and resolved by SDS-PAGE. Northern Blotting—Small scale RNA preparation was performed as hydrophobic region of eIF-4G. Formation of this complex facil- described (46), and its quality assessed by examining the 18 S/28 S itates the binding of mRNA to the ribosome and promotes ratio. Oligo-primed labeling of mouse cyclin D1 was carried out follow- translation initiation (reviewed in Refs. 7 and 43). However, ing standard procedures, and membranes were incubated with 2 3 10 rapamycin does not prevent the phosphorylation of eIF-4E in cpm of the probe. Signals were revealed and quantified using a Phos- primary T cells (5), Xenopus oocytes (5), Chinese hamster ovary phorImager (Molecular Dynamics). cells in response to insulin (44), or NIH 3T3 cells in response to RESULTS serum (8). Cyclin D1 mRNA Levels Are Affected by Rapamycin—Ex- We have examined the role of rapamycin-sensitive pathways pression of cyclin D1 mRNA is down-regulated in mitogen- in the accumulation of cyclin D1 after serum-stimulation of deprived cells and induced upon stimulation of fibroblasts with quiescent NIH 3T3 cells. Our results demonstrate that rapa- serum, epidermal growth factor, platelet-derived growth factor mycin can delay the serum-stimulated accumulation of cyclin and basic fibroblast growth factor (47, 48). To dissect the mech- D1 mRNA. Although cyclin D1 mRNA contains a putative anism by which rapamycin regulates cyclin D1 expression, we polypyrimidine tract in its 59 untranslated region, rapamycin have analyzed the transcriptional activation of cyclin D1 did not affect the rate of synthesis of D1 protein. Rather, the mRNA. NIH 3T3 cells were arrested in G by serum starvation drug promoted degradation of newly synthesized cyclin D1, for 36 h and restimulated with 10% (v/v) serum in the presence resulting in decreased Cdk4 kinase activity, decreased phos- Kip1 or absence of rapamycin. Total RNA was purified at different phorylation of pRb, and increased association of p27 with times of activation and the level of cyclin D1 mRNA was esti- cyclin E/Cdk2. mated by Northern blot analysis. Hybridization of the products EXPERIMENTAL PROCEDURES with a mouse specific cDNA probe showed that serum in- Materials—Unless specified, chemicals were from Merck or Calbio- creased cyclin D1 mRNA levels and treatment of cells with chem. Media for cell culture (Dulbecco’s modified Eagle’s medium and rapamycin caused a delay in the accumulation as well as a fetal bovine serum) were from BioWhittaker, Inc. Radioactive isotopes decrease in the absolute amount of cyclin D1 transcript (Fig. were purchased by NEN Life Science Products. The glutathione S- 1A). To distinguish between effects of rapamycin on transcrip- transferase pRb (GST-Rb ) fusion construct was kindly provided by 1–79 Dr. L. Meijer (Roscoff, France). tion and the stability of the mRNA, quiescent NIH 3T3 fibro- Cell Culture and in Vivo Labeling—NIH 3T3 fibroblasts were seeded blasts were stimulated as described above and treated either in and maintained in Dulbecco’s modified Eagle’s medium containing 10% the presence or the absence of actinomycin D. The results fetal bovine serum and penicillin/streptomycin (100 units/ml and 100 obtained indicate that the stability of cyclin D1 mRNA is mg/ml, respectively). Cells at approximately 60 –70% confluency were clearly affected by rapamycin (Fig. 1B). arrested in G by serum deprivation for 36 h in Dulbecco’s modified Rapamycin Inhibits Serum-stimulated Cyclin D1 Protein Ex- Eagle’s medium containing 0.5% fetal bovine serum and re-stimulated to enter the cell cycle by adding 10% (v/v) serum for the times indicated pression—Examination of the 59-untranslated region (59-UTR) in the figure legends. In vivo labeling of cells was carried out employing of the human cyclin D1 gene suggested the presence of a puta- 100 mCi/ml of a [ S]methionine/cysteine-labeling mix. To examine the tive polypyrimidine tract at the start site of transcription (49). rate of cyclin D1 synthesis, cells were pulsed for the last 30 min before This element, comprising a cytidine residue at the cap site harvesting in Dulbecco’s modified Eagle’s medium containing 10% dia- followed by 4 uninterrupted pyrimidines, is similar to se- lyzed fetal bovine serum. To determine the protein’s half-life, cells were quences demonstrated to confer translational control in a cell pulsed for2hinthe medium described above, 2 h after re-stimulation and chased for the next4hinthe presence of an excess of unlabeled type- and sequence context-dependent manner (9, 50). To ad- amino acids. dress the question of whether D-type cyclin mRNAs are under Western Blotting, Immunoprecipitations, and Protein Kinase Assay— such selective translation control, we examined the role of the Cell extracts were prepared at the indicated times by Dounce homoge- S6k FRAP-p70 signaling pathway on the expression of cyclin D1 nization in ice cold Buffer A (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 20 protein. Fig. 2A shows that serum increased the expression of mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM sodium pyrophosphate, 30 cyclin D1 protein within 4 – 6 h following addition. This was mM p-nitrophenyl phosphate, 1 mM benzamidine, 0.1 mM phenylmeth- ylsulfonyl fluoride, 1% Nonidet P-40). Extracts were centrifuged at greatly reduced and delayed by treatment of cells with rapa- 18,000 3 g for 10 min at 4 °C. Protein concentration was determined mycin, in a manner similar to that reported for ribosomal S6k with a BCA protein assay kit (Pierce). To analyze extracts, equal proteins in T lymphocytes (51). Under these conditions, p70 amounts of protein (5 mg) were resolved by SDS-PAGE and transferred activity was completely inhibited (see Ref. 45 and below). To to polyvinylidene difluoride membrane (Millipore). The resulting mem- Kip1 exclude the possibility that the observed drop in cyclin D1 branes were probed with anti-cyclin D1, Cdk4, or p27 polyclonal protein was nonspecific, the effect of rapamycin on the expres- antibody (Santa Cruz Biotechnology), a polyclonal serum specific for S6k p70 , or with a monoclonal antibody to pRb (Pharmingen) and re- sion of Cdk4 was also examined. Fig. 2B shows that rapamycin vealed using the ECL system (Amersham Pharmacia Biotech). To an- did not influence the level of expression of this protein. Similar S6k alyze proteins present in the cyclin-Cdk complexes and monitor kinase results were obtained for p70 (see below). Pulse-labeling activity, mouse polyclonal antisera to Cdk2 (Upstate Biotechnology, experiments with [ S]methionine/cysteine, indicated that at Inc.) or Cdk4 (Santa Cruz Biotechnology) were used. Extractions and each time point examined, the initial rate of cyclin D1 synthesis immunoprecipitations were carried out in Buffer B (25 mM Tris-HCl, pH was unchanged by the presence of rapamycin (data not shown). 7.5, 60 mM b-glycerophosphate, 15 mM MgCl ,15mM EGTA, 0.1 mM S6k NaF, 15 mM p-nitrophenyl phosphate, 1 mM dithiothreitol, 0.1 mM Taken together, these data indicate that the p70 signaling phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40) using equal pathway is not required for enhanced synthesis of cyclin D1 amounts of total protein (300 mg). Antibodies were immobilized on and that the putative 59-TOP sequence in the mRNA may not protein A-Sepharose beads, and the resin washed with 3 3 1mlof have a role in vivo. ice-cold Buffer B and 1 ml of ice-cold Buffer C (50 mM Tris-HCl, pH 7.5, When rapamycin was added to cells 4 h after serum-stimu- 10 mM MgCl ,1mM dithiothreitol. For kinase assays, the resin was lation, only a partial decrease in the accumulation of cyclin D1 resuspended in a total volume of 25 ml of Buffer C, containing 5 mM [ P]ATP (specific activity, 50 mCi/nmol), and 10 mg of GST-Rb protein was observed (35% versus 55%) (Fig. 2C). These data 1–79 14426 Cyclin D1 Regulation by Rapamycin FIG.2. Rapamycin prevents the serum-stimulated increase of cyclin D1 protein during G progression. Serum-starved NIH 3T3 fibroblasts were stimulated with complete medium in the presence or absence of 20 nM rapamycin. Cell extracts were prepared at the times indicated and subjected to SDS-PAGE. The level of expression of cyclin D1 (A) and Cdk4 (B) were visualized by Western blotting employing specific polyclonal antisera, as described under “Experimental Proce- dures.” C, serum-starved NIH 3T3 fibroblasts were stimulated with complete medium for 4 h before the addition of vehicle or rapamycin. The level of expression of cyclin D1 was determined 8 h post-stimulation and quantified with a Bio-Rad GS-700 densitometer. The values are given as optical density/mm , 8.16 6 0.28 (left lane); 5.34 6 0.21 (right lane). Absence or presence of rapamycin is denoted by 2 and 1, respectively. FIG.3. Rapamycin accelerates the turnover of cyclin D1. Se- rum-starved cells were incubated in the absence or presence of rapa- FIG.1. Cyclin D1 mRNA levels are affected by rapamycin. Se- mycin, as described in the legend to Fig. 2. Cells were pulsed with a rum-starved NIH 3T3 fibroblasts were stimulated with complete me- 35 [ S]methionine/cysteine labeling mix and chased as described under dium in the absence or presence of 20 nM rapamycin (rap)(A). Total “Experimental Procedures.” Total protein was extracted, immunopre- RNA was extracted at the indicated time points and analyzed by North- cipitated with a cyclin D1-specific antiserum and resolved by SDS- ern blot hybridization employing a mouse-specific cDNA probe to cyclin PAGE. Signals were revealed and quantified using a PhosphorImager. D1 as described under “Experimental Procedures” (upper panel). Before Rap, rapamycin; CycD1, cyclin D1. hybridization the membrane was stained with methylene blue to con- firm equal loading of each sample (lower panel). In B, cells were treated administration of rapamycin to cells in culture has been attrib- as in A, with the exception that 2 mg/ml actinomycin D was added 1 h uted to a number of distinct events. Among those is an overall post-stimulation. Total RNA was extracted and analyzed as in A. Open inhibition of protein synthesis due to decreased ribosome as- and closed circles denote presence or absence of rapamycin, respec- tively. Signals were revealed and quantified using a PhosphorImager sembly (51), the lack of formation of active cyclin E-Cdk2 com- (Molecular Dynamics). Kip1 plexes (14) and the prevention of p27 elimination from the cyclin E-Cdk2 complex (36). To assess whether the rapamycin- are in agreement with earlier reports (13, 52) that cells already induced degradation of cyclin D1 has any physiological effect on in G are less responsive to the inhibitory effects of rapamycin. Cdk4 activity and G progression in these cells, we have as- 1 1 Rapamycin Stimulates the Rate of Degradation of Cyclin D1 sayed Cdk4 activity using a GST-Rb fusion protein as sub- 1–79 Protein—The data above indicate that rapamycin affected cy- strate. The increase of Cdk4 activity that occurred within 4 – 6 clin D1 at both the transcriptional and post-transcriptional h after serum stimulation was prevented by the addition of level. To examine other possible effects of rapamycin that may rapamycin to the cells (Fig. 5A). In addition, we analyzed the influence the expression of cyclin D1 protein, we have consid- phosphorylation status of endogenous pRb by separation of cell ered the rate of cyclin D1 polypeptide degradation. To directly extracts by SDS-PAGE and Western blotting with serum spe- compare half-lives, quiescent NIH 3T3 cells were stimulated in cific for pRb protein. As indicated by the characteristic shift in the presence or absence of rapamycin, pulsed with [ S]methi- mobility upon phosphorylation and inactivation, pRb phospho- onine/cysteine labeling mix and chased in the presence of an rylation was increased after serum stimulation of cells (Fig. excess of cold methionine/cysteine. The data presented in Fig. 3 5B). This mobility shift was prevented by rapamycin, suggest- show that the turnover rate of cyclin D1 was clearly accelerated ing a lack of pRb phosphorylation in these cells and indicative upon treatment of cells with rapamycin. To further substanti- of a decrease in Cdk4 activity. Western blot analysis of immu- ate this finding, we carried out studies with the proteasome noprecipitated Cdk4 indicated that inhibition of Cdk4 activity inhibitor, N-acetyl-Leu-Leu-norleucinal (LLnL). As described was not caused by a decrease in Cdk4 protein levels (Fig. 5C) above, rapamycin decreased the accumulation of cyclin D1 pro- but was because of lack of the kinase regulatory partner cyclin tein. However, this was prevented by the simultaneous addi- D1 (Fig. 6A). Kip1 tion of LLnL (Fig. 4A). This compound did not influence pro- Reduced Levels of Cyclin D1 Facilitate Retargeting of p27 S6k Kip1 teins with a longer half-life, such as Cdk4 (Fig. 4B) and p70 to Cdk2—The inhibitor protein p27 has been found to as- (Fig. 4C). To rule out possible effects of LLnL on cyclin D1, sociate with both Cdk4 and Cdk2 via the regulatory partner, which might be independent of inhibition of the proteasome, cyclins D and E, respectively (19, 23). The distribution of Kip1 the compound was administered to cells either alone or in p27 among different cyclin-kinase complexes has been pre- combination with rapamycin and the levels of cyclin D1 were dicted to maintain a correct balance between their active forms examined 8 h post-stimulation. Fig. 4D shows that LLnL af- (19, 53, 54). To determine whether inhibition of cyclin D1 Kip1 fected solely protein degradation. accumulation in these cells affected the association of p27 Reduced Levels of Cyclin D1 Affect Cdk4 Activity and pRb with Cdk4 or Cdk2, cells were stimulated with serum in the Phosphorylation—The cause of the G /S block observed after presence or absence of rapamycin and Cdk4 or Cdk2 complexes 1 Cyclin D1 Regulation by Rapamycin 14427 Kip1 FIG.6. Effect of rapamycin on p27 association with G Cdks. A, extracts were prepared from cells stimulated in the presence or absence of rapamycin for 8 h before immunoprecipitation of Cdk4, as described. Recovered proteins were resolved by SDS-PAGE and the level of complexed cyclin D1 (left panel) and Cdk4 (right panel) were visualized by Western blotting. B, extracts obtained from cells 8 or 12 h after stimulation were subjected to immunoprecipitation with antisera specific to Cdk4 (right panel) or Cdk2 (left panel). Recovered proteins Kip1 were resolved on SDS-PAGE and the amount of p27 protein present in either complex was assayed by Western blotting. FIG.4. The proteasome inhibitor, LLnL, rescues cyclin D1 deg- radation induced by rapamycin. Serum-starved NIH 3T3 fibro- TABLE I blasts were treated as described in the legend to Fig. 2, except that the Effect of rapamycin on Cdk2 activity proteasome inhibitor LLnL (50 mM) was added together with rapamycin Extracts obtained from synchronized NIH 3T3 cells at 10 h post- at the time of restimulation. Extracts were prepared at the times stimulation were immunoprecipitated with a Cdk2-specific antiserum indicated and subjected to SDS-PAGE and immunoblot analysis, em- and assayed for kinase activity as described under “Experimental Pro- S6k ploying antisera specific to cyclin D1 (A), Cdk4 (B), or p70 (C). D, cedures.” Data represent the mean value of three independent experi- extracts obtained from synchronized NIH 3T3 cells 8 h post-stimulation ments. in the presence or absence of 20 nM rapamycin, 50 mM LLnL, or a combination of the two were resolved and probed with an antiserum Addition cpm incorporated 6 S.D. specific to cyclin D1. None 7204 6 281 Rapamycin 936 6 26 increase in the synthesis of RNA and protein and culminating in a doubling of protein mass prior to DNA synthesis and cell division (reviewed in Refs. 43 and 55). Physiological regulation of protein synthesis is almost always exerted at the level of polypeptide chain initiation, mediated in part by the availabil- ity of eIF-4E to participate in the initiation process and the phosphorylation of ribosomal protein S6 (reviewed in Ref. 43). Here we set out to investigate the contribution of rapamycin- sensitive signaling pathways in conveying external growth sig- FIG.5. Rapamycin-induced cyclin D1 degradation results in a nals to the cell cycle regulatory machinery. Recent studies decreased Cdk4 activity and pRb phosphorylation. Serum- showed that regulated translation functions in modulating the starved NIH 3T3 fibroblasts were treated as described in the legend to activity of Cdks in mammalian cells (2, 38, 53). One possible Fig. 2, and extracts were prepared at the times indicated. A, cell target for regulation is cyclin D1, whose synthesis is induced extracts containing equal amounts of protein were subject to immuno- precipitation with serum specific to Cdk4, as described under “Experi- during the delayed early response to growth factor addition to mental Procedures.” Cdk4 activity was assayed in immunocomplex cells. The D-type cyclins are the first cyclins synthesized during assay using GST-Rb fusion protein as substrate followed by SDS- 1–79 the cell cycle and complex with Cdk4 and Cdk6. As such, they PAGE and autoradiography. B, phosphorylation of endogenous pRb was are detected in mid G and function as pRb kinases, overcom- visualized after separation of cellular proteins by SDS-PAGE and West- ern blotting. The slower and faster migrating bands correspond to ing the growth-suppressive effects of pRb and thus allow pro- hyper- and hypophosphorylated pRb, respectively. C, cell extracts were gression through G /S (18, 21, 47, 56). It has been postulated fractionated as described above and the level of Cdk4 protein was that this class of cyclins represents the link between extracel- determined by Western blotting. lular signals and the cell cycle machinery (20). Cyclin D1 ac- tivity appears to be responsible for activation of cyclin E and recovered by immunoprecipitation. The antisera employed cyclin A, both partnered by Cdk2, which are active at the G /S were highly specific and showed no cross-reactivity with other boundary and during S phase, respectively (57– 61). Cdks (data not shown). Treatment of cells with rapamycin Kip1 We observed that cyclin D1 mRNA and protein (Figs. 1A and resulted in a decrease in the association of p27 with Cdk4 2A) were increased after a lag period of 2– 4 h after stimulation (Fig. 6B). Conversely, complexes of Cdk2 and cyclin E appeared Kip1 of quiescent NIH 3T3 fibroblasts to proliferate. Rapamycin to be enriched in p27 upon treatment of cells with rapamy- treatment significantly affected the level of cyclin D1 tran- cin (Fig. 6B). Accordingly, rapamycin caused an almost 8-fold script, apparently causing a delay in its accumulation. This can inhibition of Cdk2 activity (Table I). These data suggest that, be attributed to effects on either transcription or stability of the apart from any direct effect on phosphorylation of pRb, the mRNA. The former possibility could not be assessed because of decrease in cyclin D1 protein levels facilitates retargeting of Kip1 low template activity. With regard to the latter, inspection of p27 to cyclin E-Cdk2 complexes and in turn inhibition of the 39-UTR of cyclin D1 mRNA did not reveal the presence of the associated kinase activity. This further promotes the G /S AU-rich sequences, which account for rapid degradation of block in the cell cycle. interleukin-3 mRNA by a rapamycin-sensitive mechanism (62). DISCUSSION However, measurement of cyclin D1 mRNA stability showed a Treatment of quiescent cells with growth factors or hor- substantial effect of rapamycin on the transcript’s half-life (Fig. mones induces re-entry into the cell cycle, accompanied by an 1B). This indicates that novel rapamycin-sensitive pathways 14428 Cyclin D1 Regulation by Rapamycin immunoprecipitation experiments and L. Meijer (Roscoff, France) for are possibly involved in regulation of cyclin D1 mRNA levels. the generous supply of GST-Rb . We are also indebted to Dr. P. 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