THE JOL~RNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 268, No. 30, Issue of October 25, pp. 22825-22829, 1993 Printed in U.S.A.

FKBP-Rapamycin Inhibits a Cyclin-dependent Kinase Activity and a Cyclin D1-Cdk Association in Early G1 of an Osteosarcoma Cell Line*

(Received for publication, May 26, 1993, and in revised form, July 12, 1993)

Mark W. Albers$§, Richard T. Williams7 11, Eric J. Brown$, Akito TanakaS, Frederick L. Ham**, and Stuart L. Schreiber$ $$ From the $Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, the IDiuision of Orthopedic Surgery, Childrens Hospital Los Angeles, University of Southern California School ofMedicine, Los Angeles, California 90054, and the **Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles, California 90054

Upon entering a cell the natural product rapamycin, like the structurally related immunosuppressant FK506, associates with members of the FKBP family of proteins. One or more of the resulting FKBP-rapamycin com- plexes blocks signaling pathways emanating from some growth factor receptors. Recently, the addition of rapa- mycin was shown to inhibit the phosphorylation and activation of a 70-kDa ribosomal S6 protein kinase, which normally occurs minutes after the activation of certain cytokine and growth factor receptors. We now report that rapamycin can be added 4 to 6 h after the addition of serum growth factors to quiescent human osteosarcoma cells and still arrest these cells in G1. This window of action correlates with the inducible appear- ance of a cyclin-dependent kinase (cdk) activity, and the induction of this activity is inhibited by the addition of rapamycin. Furthermore, p36"YCLin associates with this cdk protein complex in lysates of untreated cells, but does not associate with this cdk protein complex in lysates of rapamycin-treated cells. Together, these stud- ies demonstrate that FKBP-rapamycin can modulate a cyclin-dependent kinase activity and a cyclin D1-cdk as- sociation during early G1 in MG-63 human osteosarcoma cells.

The natural product rapamycin blocks specific signaling events necessary for the initiation of S phase (DNA synthesis) in a number of cell types, such as human and mouse lympho- cytes (1, 21, human and rat hepatocytes (3, 41, and Succharo- myces cerevisiae (5-7). Two lines of evidence suggest that the association of rapamycin with an intracellular FK506 and rapamycin binding protein (FKBP) is necessary for the inhibi- tion of G 1 progression by rapamycin; the actions of rapamycin are reversed by an excess of the structurally related FKBP- ligands FK506 or 506BD (1, 2, 81, and the deletion of the FKBPl2 gene renders S. cereuisiae insensitive to rapamycin (5-7). Recently, rapamycin was shown to block the phospho-

* This work was supported by National Institutes of Health Grant GM-38627 (to S. L. S.), a Biomedical Research Support Grant (to C. H. L. A,), National Science Foundation Grant DCB-9104769 (to F. L. H.), and the John C. Wilson Jr. Endowment (awarded to F. L. H. via Vernon Tolo). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Howard Hughes Medical Institute predoctoral fellow. 11 Affiliate of the University of Queensland, Australia. $$ To whom correspondence should be addressed. Tel.: 617-495-5318

Fax: 617-495-0751.

rylation and activation of the 7O-kDa ribosomal protein S6 kinase pp70S6k, which generally occurs within minutes follow- ing either interleukin-2 stimulation of T lymphocytes (9, lo), insulin stimulation of quiescent hepatocytes (41, or serum stimulation of quiescent fibroblasts (11).

We now report that rapamycin can be added as late as 4 h following serum stimulation of quiescent human osteosarcoma MG-63 cells and still cause G1 arrest. The rapamycin block point can be localized to early G1. p9Ckehs1-, an t i -~34 '~ '~ - , and anti-p33cdk2-precipitable histone H1 kinase activities that nor- mally appear 4 h after serum stimulation are markedly dimin- ished in rapamycin-treated MG-63 cells. Interestingly, cyclin D l coprecipitation correlates with the histone H1 kinase activ- ity of these precipitates; that is, cyclin D l association and the kinase activity are present in extracts from untreated, but not rapamycin-treated, cells. We conclude that critical rapamycin- sensitive signaling events occur at least 4 h following serum stimulation of MG-63 cells and that a cyclin-dependent kinase activity and a cyclin D1-cdk association that appears shortly after 4 h of serum stimulation can be modulated by rapamycin.

EXPERIMENTAL PROCEDURES

The MG-63 human osteosarcoma cell line (ATCC, Rockville, MD) was maintained in RPMI 1640 and 10% FBS' (Life Technologies, Inc.) at 37 "C and 5% COz. Tu render these cells quiescent by serum depriva- tion, 0.8 x lo6 cells were plated in a T-75 flask in RPMI 1640 and 0.5% FBS for 48 h (12). Mimosine (Aldrich) was used at 200 PM final concen- tration to arrest cells in late G1. In preparation for flow cytometric analysis, cells were harvested after 18 h of serum stimulation (Fig. 1B) using trypsin-EDTA (Life Technologies, Inc.), washed in phosphate- buffered saline, fxed in 70% ethanol for 30 min at 4 "C, treated with 40 pg/ml RNase (Sigma) and 50 pg/ml propidium iodide (Sigma) in 0.1% sodium citrate. Flow cytometry was performed on a FACS Star instru- ment (Becton Dickinson, San Jose, CA). Flow cytometric histograms were analyzed by defining borders between GO/Gl and S phase and S phase and G2/M from an asynchronous population (Fig. L4 and using these boundaries in analyses of the experimental samples.

For biochemical experiments, 0.8 x lo6 cells synchronized in a T-75 flask were lysed in 1 ml 50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 2 mM EDTA, 2 m~ EGTA, 25 mM NaF, 0.1 mM Na3V0,, 0.3% Nonidet P-40, 0.2% Triton X-100, 25 mM P-glycerophosphate, 1 pg/ml leupeptin, 1 pg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, for 20 min at 4 "C (13). The lysate was centrifuged at 16,000 x g for 15 min at 4 "C. Protein concentration of the clarified lysate was determined using the Bradford assay (Bio-Rad).

For p9cksh"'-agarose precipitation experiments, extracts of synchro- nized MG-63 cells were prepared 2, 4, 6, and 8 h after the addition of 10% FBS. Each timeltreatment point was cultured in the presence or absence of 5 n~ rapamycin. Quiescent cells were exposed to rapamycin

The abbreviations used are: FBS, fetal bovine serum; PAGE, poly- acrylamide gel electrophoresis; cdk, cyclin-dependent kinase.

22825

22826 Rapamycin Blocks a Cyclin-dependent Kinase Activity for 15 min prior to analysis. The lysates were normalized for protein and precipitated with 20 p1 of p9ck"h"1-agarose (5 mg/ml coupled to AfE-Gel-10 (Bio-Rad) (14)) by rotating at 4 "C for 1 h. The beads were washed three times with 1 ml of lysis buffer a t 4 "C, and 50 pl of 2 x SDS loading buffer was added to each sample of beads prior to boiling. The precipitates were analyzed by SDS-PAGE (10%) followed by immunob- lotting using polyclonal antisera (1:lOOO) raised against a peptide de- rived from the COOH terminus of p3PdC2 (X), polyclonal antisera raised against a peptide derived from the COOH terminus of ~ 3 3 ' ~ ~ ~ (16), polyclonal sera that recognizes cyclin A (121, polyclonal antisera raised against a peptide derived from the COOH terminus of cyclin Dl, which also recognizes cyclin D2 and a 46-kDa cyclin-like protein (13), and polyclonal antibodies (27) and monoclonal antibodies (Pharmingen, San Diego, CA) raised against ~ 5 2 ~ Y ~ " " E, The blots were visualized by incubating with alkaline phosphatase-conjugated antibodies or using

n A

I C

L D

,*,

n I

DNA content

FIG. 1. Rapamycin arrests MG-63 cells in early G1. A, asynchro- nous culture: MG-63 cells. B, S phase entry; MG-63 cells were rendered quiescent by serum deprivation for 48 h, followed by refeeding (10% fetal bovine serum) for 18 h. C , rapamycin blockade in G1; MG-63 cells

followed by refeeding (10% fetal bovine serum) for 18 h in the presence were rendered quiescent by serum deprivation (0.5%) for 48 h (Go),

cells were rendered quiescent by serum deprivation (0.5%) for 48 h (Go), of 5 nM rapamycin. D , FK506 reverses the rapamycin blockade; MG-63

followed by refeeding (10% fetal bovine serum) for 18 h in presence of 5 nM rapamycin and 5 p~ FK506.

chemiluminescence detection system (Amersham Corp.). Recombinant p9Ckshs' was expressed and purified as described previously (17).

Histone H1 kinase assays were performed essentially as described previously (18). [Y-~~PIATP was purchased from Du Pont-New England Nuclear. Briefly, synchronized MG-63 cells were harvested 2, 4, 6, and 8 h after the addition of 10% FBS as well as in late G1. Cells corre- sponding to each t imeheatment point were cultured in the presence or absence of 5 nM rapamycin. Quiescent cells were exposed to rapamycin for 15 min prior to analysis. Approximately lo6 cells were lysed by detergent (13) at the specified time points. Cdks were precipitated with pSckShs1 beads as described above (13); the beads were washed two times with lysis buffer and one time with kinase buffer (18) before incubation with 5 pg of histone H1 (Sigma, type 111-S) and 5 pCi of Iy-32PlATP (1000 cpdpmol) in a total volume of 50 pl for 20 min a t 30 "C. The reaction was terminated by the addition of 2 x SDS sample buffer and boiled. Histone H1 phosphorylation was analyzed by gel electrophoresis followed by autoradiography a t -70 "C using an inten- sifying screen and Kodak XAR5 film. Control experiments using etha- nolamine-capped Affi-Gel-10 resulted in no histone H1 phosphorylation.

For immunoprecipitation experiments, quiescent MG-63 cells were stimulated with serum for either 2 h, 6 h, or 6 h in the presence of rapamycin and then lysed using detergent (13). Each lysate was was precleared with protein A-Sepharose prior to incubation with anti- p34cdc2, a n t i - ~ 3 3 = ~ ~ ~ , or anti-p36Wcli" polyclonal antisera for 1 h a t 4 "C. 50 pl of protein A-agarose (Boehringer Mannheim) was added to the lysates. After a 1-h incubation at 4 "C, each immunoprecipitate was washed three times with the lysis buffer or two times with lysis buffer and one time with kinase buffer. The samples were analyzed by SDS- PAGE and immunoblotting or were assayed for histone H1 kinase ac- tivity as described above.

RESULTS AND DISCUSSION

Rapamycin (5 nM) treatment of MG-63 cells synchronized by serum starvation prevented entry into S phase as determined by flow cytometry (Fig. 1C). A 1000-fold excess of FK506, which alone does not cause G1 arrest in these cells, reversed the rapamycin-induced G1 blockade (Fig. lD), suggesting that rapamycin is not cytotoxic and that one or more FKJ3Ps medi- ates rapamycin's actions (12). Moreover, rapamycin blocked S phase entry when added as late as 4 h following serum stimu- lation of quiescent MG-63 cells (Table I), indicating that the first rapamycin-sensitive signaling events necessary for G1 progression occur after 4 h following serum stimulation of qui- escent MG-63 cells.

To localize the point in the cell cycle where rapamycin arrests MG-63 cells, the levels of specific cyclins were compared in extracts prepared from synchronized MG-63 cells that had been pretreated with either rapamycin or the vehicle and stimulated with serum growth factors. The induction of cyclin D l in early G1 was partially reduced in the rapamycin-treated cells (Fig. %I), whereas the induction of cyclins E and A (normally occur- ing in late G1) (Fig. 2, B and C, respectively) and p46 (Fig. 2A ), a protein normally expressed in mid-G1 and recognized by the anti-p36CYC'i"D1 antiserum (131, were markedly diminished; the

TABLE I Rapamycin added as late as 4 h after serum stimulation still arrests

MG-63 cells in G1 MG-63 cells were rendered quiescent by serum deprivation (0.5%) for

48 h (Go), followed by refeeding (10% fetal bovine serum). Five nM rapamycin was added to separate cultures aRer 0 ,2 ,4 ,6, and 8 h. The cells were harvested 18 h after serum refeeding and analyzed by flow

cells that have entered S phase (mean f S.E.). cytometry. Numbers in the right column of the table represent percent

Time serum added Time rapamycin added Cel's (mean in * .KEJ

h 0

h 78 f 1

%

0 0 15 f 2 0 2 17 f 1 0 4 15 f 2 0 6 27*3 0 8 52 f 4

FIG. 2. Characterization

Rapamycin Blocks a Cyclin-dependent Kinase Activity 22827

of the rapamycin-induced G1 arrest point.

deprivation (0.5% FBS, 48 h). followed by MG-63 cells were synchronized by serum

refeeding in the presence (+) or absence (-) of 5 nM rapamycin. Cells were har- vested in early G1, 6 h after refeeding; late G1, 22 h after refeeding in the pres- ence of 200 p~ mimosine. which block these cells reversibly in late G1 (12); and S phase, 6 h following release from the mimosine blockade. 5 pg of detergent ly- sates were analyzed by Western blotting for expression of cyclin D and p46 (A ) , expression of cyclin E ( B ) , and expression of cyclin A (C). D, histone H1 kinase as- says of p9cknh"'-agarose precipitates from extracts of synchronized MG-63 cells se- rum stimulated for 0 (lanes I and 2). 2 (lanes 3 and 4 ) . 4 (lanes 5 and 6). 6 (lanes 7 and 8). and 8 (lanes 9 and IO) h in the absence (odd numbers) or presence (even numbers) of rapamycin.

69 -

46 - c ClnX

" + ClnD

30 - 22 -

1 2 3 4 5 6 1 2 3 4 5 6

D

levels of these latter proteins were near their basal values. These results suggest that rapamycin arrests MG-63 cells in early G1 (after the induction of cyclin Dl but before the induc- tion of cyclins E and A). To further characterize this arrest point, the temporal properties of histone H1 kinase activity were investigated in the presence and absence of rapamycin. Cdk complexes were precipitated by p9ck"hn1-agarose from ly- sates of synchronized MG-63 cells that had been stimulated with 10% serum for various times. Histone H1 kinase assays of these precipitates revealed a kinase activity in quiescent cells that decreased upon serum stimulation until the activity reap- peared at 6 h (Fig. W, lanes 7 and 9). In the presence of 5 nM rapamycin, the kinase activity in quiescent cells was unper- turbed, but the induction of this kinase activity at 6 and 8 h was markedly diminished (Fig. W, lunes 8 and 10). In contrast, direct addition of 500 nM FKBP12, FKBP25, rapamycin, or both immunophilin-rapamycin complexes to the pgCknhnl afinity- purified kinase complexes present at 6 h did not inhibit their histone H1 kinase activity in vitro, suggesting that these ki- nase complexes are not the direct target of rapamycin.' More- over, immunophilin-rapamycin complexes did not inhibit the growth factor-sensitive proline-directed protein kinase (PDPK) (composed of p58cyc'in A/p34cdc2 and purified to homogeneity from mouse FM3A mammary carcinoma cells (18)).

Since the induction of p9c"h"1-associated kinase activity cor- related with the time window of rapamycin sensitivity as de- termined by flow cytometry, we characterized the protein com- position of p9':kshn1-precipitates prepared from cells that had been pretreated with rapamycin or the vehicle. Using anti- p36'y'"" antiserum, a Western blot of p9ck"hn1-precipitates of MG-63 cell extracts prepared at early G1 time points indicated that p36'y'"" was present in these precipitates; however, the amount of p36'YC"" precipitating with pSCkRhn1 from rapamy-

R. T. Williams, M. W. Albers, F. L. Hall, and S. L. Schreiber, unpub- lished observations.

t ClnE

1 2 3 4 5 6

+ ClnA

- + - + - + - + - +

3 Histone H1 phosphorylatlon

1 2 3 4 5 6 7 8 9 1 0

cin-treated cells was significantly diminished (Fig. 3A ). MG-63 cells do not express cyclin D2 (13). Blotting with anti-~34'~'' and an t i -~33 '~~ ' antibodies indicated that the same amounts of ~34'~'' and ~ 3 3 ~ ~ ~ were associated with p9"'Uh"' a t each time point in the presence or absence of rapamycin, demonstrating that rapamycin does not block the association of p9'"Uh"' with either of these cdks. Cyclin A and cyclin E were present a t barely detectable levels in the lysates and associated with p9C"hs1.3 Their presence may be the result of imperfectly syn- chronized cells.

Studies of the temporal expression of these proteins indi- cated that the level of p36cYc11" peaked between 6 and 8 h (Fig. 3B) in untreated cells, whereas the levels of ~34'"'" and ~ 3 3 ' ~ ~ ' were not significantly altered throughout this period. In the presence of 5 nM rapamycin, the induction of p36'Y'"" ''I

(Fig. 3B) was decreased approximately 50%. whereas the rela- tive levels of ~34'~' ' and ~ 3 3 ' ~ ~ ' were not altered. We note that a form of ~ 3 3 ' ~ ~ ~ with greater electrophoretic mobility that appears in in late G1 is absent in the rapamycin-treated cells. The bands of ~ 3 3 ' ~ ~ ' with greater mobility may result from an activating phosphorylation on threonine 160 (16). However, 500 nM rapamycin, 500 nM FKBP12, or 500 nM FKBP12-rapamycin did not inhibit a partially purified kinase activity that phos- phorylates ~ 3 4 ' ~ ~ ' " on threonine 160.' Inadequate levels of ~36""'" ''I in rapamycin-treated cells may account for the lack of association between p36'YcIi" I" and p9"knh"'. When rapamycin was added either 4 or 6 h after serum stimulation, there was little effect on the cytoplasmic levels of p36cyC1'n present at 8 h after serum stimulation (Fig. 3C). However, in the rapamy- cin-treated cells the amount of p36cY'1'" ''I associated with pSChhnl at 8 h was decreased relative to the amount of p36'Yc1'" DX associated with ~ 9 " ~ " ~ " ' at 8 h in untreated cells (Fig. 3C). Overall, these results suggest that p36'yC1'" can associate

M. W. Albers, R. T. Williams, F. L. Hall, and S. L. Schreiber. unpub- lished observations.

22828 Rapamycin Blocks a Cyclin-dependent Kinase Activity

A. pgCkshS1- associated B. lysate

"""""

1 2 3 4 5 6 7 0 9 1 0 PW*2

- "" """"-3,

C. Rapadded: - 4 - 6 - Harvested 4 8 6 8 0

cyclin D "c lysate 1 2 3 4 5

FIG. 3. Characterization of components of the rapamycin-sen- sitive histone H1 kinase activity. A, effects of rapamycin on the

prepared from quiescent MG-63 cells that were serum stimulated for 0, 2, 4, 6, and 8 h in the absence (-) and presence (+) of rapamycin. The lysates were normalized for protein concentration and precipitated with p9':k"h"'. The precipitates were washed and analyzed by SDS-PAGE and immunoblotting with anti-p36cYc1'"D1, anti-~34~'*, and anti-p3Fdkz an- tisera. B , levels of the components in the lysates. Five pg of detergent MG-63 lysates at the same time points described in A were analyzed by Western blotting with the same panel of antisera used in A. Since equivalent amounts of both ~34 '" '~ and ~ 3 3 ~ ~ were precipitated in the presence and absence of rapamycin, FKBP-rapamycin does not seem to impair the association of the cdks to p9':k"h"1. C, FKBP-rapamycin blocks p36~c11""1-p9'~knhn' association in lysates with control levels of p36cYc1i"1)1. Detergent lysates were prepared as described in B a t 4, 6, and 8 h following serum stimulation. Also, rapamycin ( 5 nM) was added a t 4 and 6 h after serum stimulation to synchronized MG-63 cells and then the cells were harvested a t 8 h. Each lysate was normalized for protein and cognate cdks were precipitated by p9Ck"h"'. These precipi- tates and 10 pg of each lysate was analyzed by SDS-PAGE and immu- noblotted with the polyclonal antisera recognizing p36'Y'l'" Although the level of cyclin Dl present at 8 h in cells treated a t six hours with rapamycin is comparable with the untreated control a t 8 h, the amount of cyclin D l associated with p9"k'h"1 in cells treated a t 6 h with rapa- mycin is significantly diminished.

either indirectly or directly with one or more members of the cdk family that interact with pSCknhn1 (28) and that FKBP- rapamycin blocks a step that facilitates this association.

Over the past 2 years, characterization of D-type cyclidcdk complexes has revealed a complex relationship between these two families of proteins. Cyclin D l and D2 have been reported to associate with a polypeptide antigenically related to ~34 '~ ' " and to bind a histone H1 kinase in vitro (19). Later, the same workers reported that cyclin D l in BAC1.2F5 murine macro- phages associates with cdk4 but not cdc2, cdk2, cdk3, PCTAIRE-1, or PCTAIRE-3 (20). More recently, they, working with others, found that either cyclin Dl , D2, or D3 coexpressed with cdk4 in Sf9 cells leads to the phosphorylation of the reti- noblastoma gene product but that only cyclins D2 and D3 co- expressed with cdk2 phosphorylated the retinoblastoma gene product in Sf9 cells (21). On the other hand, Xiong et al. (22) found that cyclin D l associates with cdk2, cdk4, and cdk5 in W138 human diploid fibroblasts. Moreover, cyclin D l and D3 have been reported to complex the retinoblastoma gene product in this fibroblast cell line (23). Thus, the function of cyclin D l in

association of p36cYCl8" 1)1 p34cdc2 and p33dk2 with pg"k*h*l In ' lysates

its interactions with cdks may be unique among the cyclin family, e.g. no kinase activity has been reported for cyclin D1- cdk2 complexes, and the association of cyclin D l with specific cdks and with the retinoblastoma gene product (14) appears to be highly regulated.

In light of the complexity of D-type cyclidcdk family member interactions, we wanted to confirm that p34*" and p33cd*2 are responsible for the histone H1 kinase activity present at 6 h following serum Stimulation and that cyclin D l associates with these cdks by immunoprecipitating MG-63 cell extracts pre- pared 6 h after serum stimulation with anti-p34'"'' and/or a n t i - ~ 3 3 ' ~ ~ ' antisera. As shown in Fig. 4A, histone H1 kinase activity associated with a n t i - ~ 3 4 ~ ~ ~ ' and a n t i - ~ 3 3 ' ~ ~ ' immuno- precipitates is detectable a t 6 h following serum addition, but is diminished in precipitates from extracts of rapamycin-treated cells prepared 6 h following serum addition. The inhibition of kinase activity by rapamycin was not due to lesser amounts of p34cdc2 precipitated from rapamycin-treated cells (Fig. 4B). but correlated (not necessarily cause and effect) with significantly lesser amounts of ~36"~"" associated with the immune com- plexes from rapamycin-treated cells (Fig. 4C). Anti-p36'yC"" ''I

antisera was able to precipitate some level of kinase activity, but significantly less than was precipitated by anti-~34~"" an- tisera. There are many possibilities for this, including differing efficiencies of the precipitating antibodies, modification of the

A 2h

1 2 3 4 5 0 7 8 Q 10 11 12

P C

" c- c-

"- "-e - . .

1 2 3 4 1 2 3 4 5 6 7 8

FIG. 4. Rapamycin inhibits histone H1 kinase activity precipi- tated by anti-p.74'dcP, anti-p33'd". and anti-p36"r""D1 antisera and prevents cyclin Dl coprecipitation with the antisera recog nizing cdks. A. quiescent MG-6.3 cells were serum stimulated for 2. 6. or 6 h in the presence of rapamycin before harvesting. The lysates were normalized for protein concentration and precipitated with protein A alone (control) or protein A and anti-~34'~''. anti-~33'~*'. and anti- p36'Yc1'""' antisera. The precipitates were washed and aRsayed for his- tone H1 kinase activity as described under 'Experimental Procedures." Rapamycin inhibited the histone H1 kinase activity associated with each antisera to near basal (2 h ) levels. R, lysates from quiescent MC-63 cells treated with serum for either 6 h or 6 h in the presence of rapa- mycin were precipitated with either protein A-agarose (control) or pro- tein A-agarose and anti-p34&" antisera as described under 'Experi- mental Procedures." These precipitates were analyzed by SDS-PAGE and immunoblotted with anti-p34'"' antisera. Similar levels of p34c'i'' were immunoprecipitated in the absence or presence of rapamycin. C. lysates were prepared as described in R and precipitated with with protein A alone (control) or protein A and anti-p34"'", anti-p33"'kZ. and anti-p36c~'"""1 antisera. The precipitates were analyzed by SDS-PAGE and immunoblotted with anti-p36i"'"""l antisera. Rapamycin blocked the association ofp36'.""'" with an"-p34'"''and antl-p39"'*' antisera.

Rapamycin Blocks a Cyclin-dependent Kinase Activity 22829

cyclin D l epitope (antigenic peptide has a putative cdk phos- phorylation site), relative amounts of cyclin D l and cdc2 in the lysates, and heterogeneous protein complexes involving cyclin D l and/or cdc2, each with different efficiencies for recognition by either antisera. Although these immunoprecipitation experi- ments indicate that rapamycin blocks a kinase activity that is present in a n t i - ~ 3 4 ~ ~ ' ~ immunoprecipitates and, to a lesser ex- tent, in a n t i - ~ 3 3 ' ~ ~ ~ immunoprecipitates and that p36"YC"" associates with p34cdc2 and p33cdk2, these experiments should not be taken to demonstrate that p36cYc1in interacts directly with or plays a role in activating either ~ 3 4 ~ ~ ' ~ or p33cdk2.4

In summary, these studies identify a temporal window for critical rapamycin-sensitive signaling events leading to G1 pro- gression in MG-63 cells. Within this window a rapamycin-sen- sitive cdk kinase activity has been identified as well as a rapa- mycin-sensitive cyclin Dl-cdk association. Morice et al. (24) recently reported that rapamycin arrests T lymphocytes in late G1 and inhibits a p34cdc2-associated kinase activity that nor- mally appears just prior to the onset of S phase in T lympho- cytes. T cells do not express cyclin D l (251, which may account for the fact that the rapamycin-induced arrest point in T cells is later in G1 than in MG-63 cells. In addition, rapamycin may be of considerable utility to probe cyclin Dl's association with cdk-containing protein complexes, as well as other proteins such as the retinoblastoma gene product (23), at a molecular level.

The molecular mechanism of action of rapamycin remains a mystery. Since the direct addition of FKBP-rapamycin com- plexes in vitro to pgCkshsl precipitates does not cause the dis- sociation of cyclin D l from these precipitates or inhibit their histone H1 kinase activity, cyclin-cdk complexes are not likely the direct targets of FKBP-rapamycin complexes. Moreover, we have not observed specific binding of cyclins or cdks to rapa- mycin-based affinity reagents. Genetic studies have identifed two genes, termed TOR1 and TOR2 (Target Of Rapamycin), that encode putative targets of the FKBP-rapamycin complex (7). The TOR2 gene from S. cereuisiae has recently been se- quenced and has homology with the catalytic domains of lipid kinases (26). Although it is possible that the TOR2 gene prod- uct is a direct target of the FKBP-rapamycin complex, a more likely explanation is that the TOR2 gene product lies down- stream of the direct target of rapamycin and that the TOR2 mutation caused the protein to be constitutively active. If the latter model is correct, then the TOR2 gene product joins p70S6k, cyclin-dependent kinases, and cyclin D l as proteins that lie downstream of the direct target of the FKBP-rapamy- cin complex and have been shown to play important roles in cell

Current studies are directed at addressing this issue (Williams, R. T., Albers, M. W., Wu, L., Liu, L., Carbonaro-Hall, D. A., Brown, E. J.,

ration). Tanaka, A., Schreiber, S. L., and Hall, F. L. Hall, manuscript in prepa-

cycle progression. The identification of the direct target of the FKBP-rapamycin complex will likely reveal an upstream com- ponent of the signal transduction pathway that leads to G1 progression and will help delineate the signal transduction pathways that link growth factor-mediated signaling events and cyclin-cdk activity required for cell cycle progression.

Acknowledgments-We thank J. M. Roberts for generously providing anti-cyclin E antibodies, D. T. Hung for providing the recombinant FKBP25, J. W. Harper for providing an expression system of p9Ck"hs', and R. Standaert for critically reading this manuscript.

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