Downloaded from
`
`http://science.sciencemag.org/
`
`
`
`on June 13, 2018
`
`-
`
`15. C. Meyers and L. A. Laimins, unpublished material.
`16. A. E. G. Dunn and M. M. OgiMe, J. Ultrastruct.
`Res. 22, 282 (1968); C.R. Laverty, P. Russell, E.
`Hills, N. Booth, Acta Cytol. 22, 195 (1978); C.
`Morin and A. Meisels, ibid. 24, 82 (1980); J. Viac,
`D. Schmitt, J. Thivolet, J. Invest. Dermatol. 70, 263
`(1978); S. Pilotti, F. Rilke, G. De Palo, G. Della
`Torre, L. AJasio, J. C/in. Pathol. 34, 532 (1981).
`17. M. Favre, F. Breitburd, 0. Croissant, G. Orth, J.
`Viral. 15, 1239 (1975).
`18. D. H. Crawford and I. Ando, Immunology 59,405
`(1986); A. H. Davies, R. J. A. Grand, F. J. Evans,
`A. B. Rickinson, J. Viral. 65, 6838 (1991); a. X. Li
`et al., Nature 356, 347 (1992).
`19. S. J. Polyak, W. E. Rawls, D. G. Hamish, J. Virol.
`65, 3575 (1991).
`20. R. M. Lewis, J. C. Morrill, P. 8. Jahrting, T. M.
`Cosgriff, Rev. Infect. Dis. 11, 736 (1989).
`21. B. G. Weinshenker, S. Wilton, G. P. A. Rice, J.
`lmmunol. 140, 1626 (1988).
`22. B. R. Cullen and W. C. Greene, Cell 58, 423
`(1989); J. Laurence, H. Cooke, S. K. Sikder, Blood
`75, 696 (1990).
`23. C. Meyers and L. A. Laimins, in preparation.
`24. D. J. McCance, R. Kopan, E. Fuchs, L.A. Laimins,
`Proc. Natl. Acad. Sci. U.S.A. 85, 7169 (1988).
`25. We thank M. Turyk, G. Wilbanks, P. Larramendi, and
`S. Chou for assistance with electron microscopy, B.
`Roizman and S. Silverstein for critical reading of the
`manuscript, and the members of the L. Laimins
`laboratory for many helpful discussions. Supported
`by the Howard Hughes Medical lnstiMe and a grant
`from the American Cancer Society (L.A.L.).
`
`15 June 1992; accepted 15 July 1992
`
`R. J. Zaino, J. A. Weber, J. Viral. 61,590 (1987); J.
`W. Kreider, S. D. Patrick, N. M. Cladel, P. A.
`Welsh, Virology 1n, 415 (1990).
`4. J. Sterling, M. Stanley, G. Gatward, T. Minson, J.
`Viral. 64, 6305 (1990).
`5. L. B. Teichman and R. F. LaPorte, in (2), pp.
`109--140.
`6. C. M. Meyers and L.A. Laimins, Papil/ornavirus
`Rep. 3, 1 (1992).
`7. M.A. Bedell et al., J. Virol. 65, 2254 (1991).
`8. D. Asselineau, 8. A. Bernard, C. Bailly, D. Dar(cid:173)
`rnon, M. Prunieras, J. Invest. Dermatol. 86, 181
`(1986).
`9. R. Kopan, G. Traska, E. Fuchs, J. Cell Biol. 105,
`427 (1987).
`10. E. Fuchs and H. Green, Cell 19, 1033 (1980); R.
`Moll, W. W. Franke, D. L. Schiller, B. Geiger, R.
`Krepler, ibid. 31, 11 (1982); Y. J. Wu eta/., ibid., p.
`693; W. G. Nelson and T.-T. Sun, J. Cell Biol. 97,
`244 (1983).
`11. B. D. Ball, G. K. Walker, I. A. Bernstein, J. Biol.
`Chem. 253, 5861 (1978); B. A. Dale, K. A. Resing,
`J. D. Lonsdale-Eccles, Ann. N. Y. Acad. Sci. 455,
`330 (1985); 8. A. Dale, A. M. Gown, P. Fleckman,
`J. R. Kimball, K. A. Resing, J. Invest. Dermatol. 88,
`306 (1987).
`12. J. S. Rader et al., Oncogene 5, 571 (1990).
`13. Reproducible induction of differentiation occurred
`when raft cultures were incubated 16 to 24 hours
`every 4 days with cell culture medium that con(cid:173)
`tained 16 nM TPA. Raft cultures were grown for 16
`days then harvested, fixed in paraformaldehyde,
`embedded in paraffin, and sectioned for immuno(cid:173)
`histostaining (23).
`14. H. Pfister and P. G. Fuchs, in (1), pp. 1-18.
`
`Rapamycin-lnduced Inhibition of the
`70-Kilodalton 56 Protein Kinase
`Daniel J. Price,* J. Russell Grove,* Victor Calvo, Joseph Avruch,
`Barbara E. Bierert
`The immunosuppressant rapamycin inhibited proliferation of the H4IIEC hepatoma cell line.
`Aapamycin, but not its structural analog FK506, also inhibited the basal and insulin-stim(cid:173)
`ulated activity of the p70 ribosomal protein S6 kinase. By contrast, insulin stimulation of the
`p85 Ask S6 kinase and mitogen-activated protein (MAP) kinase activity were unaffected by
`drug. Aapamycin treatment of COS cells transfected with recombinant p70 S6 kinase
`completely inhibited the appearance of the hyperphosphorylated form of p70 S6 kinase
`concomitant with the inhibition of enzyme activity toward 40S subunits. Thus, rapamycin
`inhibits a signal transduction element that is necessary for the activation of p70 S6 kinase
`and mitogenesis but unnecessary for activation of p85 Ask S6 kinase or MAP kinase.
`
`Increased phosphorylation of multiple ser(cid:173)
`ine residues on the 40S ribosomal protein
`S6 numbers among the most rapid bio(cid:173)
`chemical responses exhibited by cells stim(cid:173)
`ulated with insulin or mitogens in vitro ( 1).
`Insulin or mitogen-stirnulated S6 phosphor-
`
`D. J. Price, J. R. Grove, J. Avruch, Diabetes Unit and
`Medical Services, Massachusetts General Hospital,
`Boston, MA, and Department of Medicine, Harvard
`Medical School, Boston, MA 02115.
`V. Calvo, Division of Pediatric Oncology, Dana-Farber
`Cancer Institute and Harvard Medical School, Boston,
`MA02115.
`B. E. Bierer, Division of Pediatric Oncology, Dana(cid:173)
`Farber Cancer Institute, Boston, MA; Hematology-On(cid:173)
`cology Division, Department of Medicine, Brigham and
`Women's Hospital, Boston, MA; and Department of
`Medicine, Harvard Medical School, Boston, MA 02115.
`*The first two authors contributed equally.
`tTo whom correspondence should be addressed.
`
`SCIENCE • VOL. 257
`
`• 14 AUGUST 1992
`
`ylation is catalyzed by one or both of two
`families of insulin or mitogen-activated S6
`Ser-Thr protein kinases--the Rsk or p85
`Rsk S6 kinases (2), and the p70 S6 kinases
`(3, 4). Both families of S6 kinases are
`themselves regulated by Ser-Thr phos(cid:173)
`phorylation, although the immediate up(cid:173)
`stream regulators of the two S6 kinase
`families differ, at least in part. The Xeno(cid:173)
`pus S6 kinase II, a p85 Rsk enzyme, is
`activated in vitro by phosphorylation with
`p42 MAP kinase (5). The MAP kinases
`are the dominant (perhaps only} immedi(cid:173)
`ate upstream activator of the p85 Rsk
`enzyme in situ in response to insulin or
`mitogens (6). Although the MAP kinases
`and cdc2 phosphorylate recombinant p70
`S6 kinase in vitro in a putative regulatory
`
`973
`
`termine if the structures we observed in nuclei
`of CIN-612 cells were indeed virions. Dot
`blot hybridization was performed on fractions
`from an isopycnic gradient purification ( 17) of
`HPV 3 lb virions produced in raft culture, and
`the presence of viral DNA was confirmed by
`Southern (DNA) blot hybridization (Fig.
`4A). Lanes 6, 7, and 8 of Fig. 4A demon(cid:173)
`strate the presence of HPV 3 lb DNA at low
`levels. From the copy number standards, we
`estimate the yield of viral particles to be at
`least 40 million per milliliter in lanes 7 and 8
`(Fig. 4A). Fractions positive for HPV 31b
`DNA contained viral particles (Fig. 4B). The
`density gradient in fractions where virions
`were found was between 1.3 and 1 .4 g/cm.3 as
`determined by weight. The presence of both
`HPV DNA and viral particles within the
`same fractions suggests that these are com(cid:173)
`plete HPV virions, not empty capsids.
`The ability to propagate papillomavirus in
`vitro is valuable not only for the understand(cid:173)
`ing of the virus, but also for the eventual
`development of anti-viral treatments and the
`prevention of papillomavirus-induced lesions.
`These studies also establish the tight link
`between epithelial differentiation and HPV
`virion production. The ability of TP A to
`induce an increased expression of differentia(cid:173)
`tion-specific markers suggests that a signal
`transduction pathway of epithelial differenti(cid:173)
`ation has been identified. Virion production
`induced by phorbol esters has been described
`in other systems such as Epstein-Barr virus
`(18), Pichinde virus (19), Rift Valley fever
`virus (20), cytomegalovirus (21), and human
`immunodeficiency virus (22). The induction
`of the complete vegetative life cycle of HPV s
`in vitro requires both stratification at an air(cid:173)
`liquid interface and protein kinase C (PKC)
`activation. In support of this hypothesis, the
`induction of viral particle biosynthesis by the
`addition of the synthetic diacylglycerol 1,2-
`dioctanoyl-sn-glycerol was observed (23). We
`believe that PKC activation of papillomavirus
`production is dependent on the induction of a
`more complex keratinocyte differentiation
`program and is not just a direct effect on
`capsid synthesis.
`In this report, we described a system
`whereby latent infection of keratinocytes is
`converted into a productive infection lead(cid:173)
`ing to the formation of papillomavirus vir(cid:173)
`ions in culture. This tissue culture system
`will be useful in studies of the mechanisms
`whereby latency is maintained and termi(cid:173)
`nated, and in the synthesis and assembly of
`papillomavirus virions.
`
`REFERENCES AND NOTES
`
`1. K. Syrjiinen, L. Gissman, L. G. Koss, Eds., Papil(cid:173)
`lomaviruses and Human Disease (Springer-Ver(cid:173)
`lag, Bertin, 1987).
`2. N. P. Salzman and P. M. Howley, Eds., The
`Papil/omaviruses, vol. 2 of The Papovaviridae
`(Plenum, New York, 1987).
`3. J. W. Kreider, M. K. Howett, A. E. Leure-Dupree,
`
`

`

`200
`
`120
`
`I 160
`0
`"b
`C
`!
`:§
`
`80
`
`0
`
`0.01 0.1
`1
`Rapamycln (nM)
`
`E t !c
`40 ~ 'e..
`10 100 ~ I ~
`I
`
`•
`
`•
`
`•
`
`D
`
`D
`
`C
`
`Fig. 1. Rapamycin-mediated inhibition of basal
`((cid:143)) and insulin-stimulated (e) proliferation of H4
`rat hepatoma cells. Cells were grown to near-
`confluence in Swims S77 medium (Sigma) with
`fetal calf serum (5%) and horse serum (15%) at
`37°C, 5% CO2 . Cells were cultured in serum-free
`medium 18 to 24 hours before assay. Serum-
`starved H4 cells were cultured in 96-well, flat-
`bottom plates (4 x 104 cells per well) in the
`absence ((cid:143)) or presence (e) of 10-5 M insulin.
`CsA (100 nM), FK506 (100 nM), and rapamycin
`(as indicated) were added at the start of the
`assay. Proliferation was assessed by the incor-
`poration of [3H]thymidine during 16 hours after a
`32-hour incubation. Mean ± SEM of triplicate
`determinations is shown, and this experiment is
`representative of four experiments.
`
`cells at the G 1 to S phase of the cell cycle ( 10,
`11). The molecular target of rapamycin action
`has not been defined. We now demonstrate
`that the signal transduction pathway leading
`to the activation of p 70 S6 kinase is selective(cid:173)
`ly inhibited by rapamycin.
`Rapamycin selectively inhibited the in(cid:173)
`corporation of [3H]-labeled thymidine into
`serum-starved H4 hepatoma cells in a con(cid:173)
`centration-dependent fashion both in the
`presence of ICso, half-maximal inhibitory
`concentration, of -0.1 nM and absence of
`-0.5 nM of the mitogen insulin (Fig. 1).
`Neither FK506 nor CsA, at 100-fold greater
`concentrations than those effective for
`rapamycin, inhibited basal or insulin-stim(cid:173)
`ulated H4 proliferation (Fig. 1).
`To analyze the rapamycin-mediated inhi(cid:173)
`bition of H4 proliferation, we examined early
`biochemical events in insulin signal transduc(cid:173)
`tion. Insulin treatment of serum-starved H4
`rat hepatoma cells results in the activation of
`cytosolic S6 protein kinases; assays of H4
`cytosolic extracts showed a progressive in(cid:173)
`crease in total S6 kinase to a plateau at 10 min
`that was sustained thereafter for at least 1 hour
`
`Downloaded from
`
`http://science.sciencemag.org/
`
`
`
`on June 13, 2018
`
`domain (7), this phosphorylation is not
`sufficient to activate the p70 S6 kinase,
`indicating the existence of other, as yet
`unidentified, insulin-mitogen-activated p70
`kinase-kinases (7).
`The macrolide immunosuppressant rapa(cid:173)
`mycin and its structural analog FK506 bind
`to the same family of intracellular receptors,
`termed FK506 binding proteins (FKBPs) (8).
`
`The complex of FK506 with FKBP binds
`to and inhibits the activity of calcineurin,
`a calcium-calmodulin-dependent Ser-Thr
`phosphatase (9). FK506, like the undecapep(cid:173)
`tide immunosuppressive agent cyclosporin A
`(CsA), inhibits T cell receptor-mediated
`events leading to lymphokine gene transcrip(cid:173)
`tion (JO). Rapamycin, but not FK506, inhib(cid:173)
`its
`lymphokine-dependent proliferation of
`
`A
`
`+
`
`p851 - - - _,. _______ _
`
`- • ••--~s6
`
`B
`
`·1r•l•
`
`1 D
`
`so
`
`I
`
`No substrate
`
`Rap•~:cl '" (•Ml 0
`
`'
`
`1
`
`5
`
`I 10
`
`50
`
`No substrate
`
`.... -.... I
`
`+ +
`
`+
`
`31 ·.
`
`_
`
`. . . , . . . 4 . .....
`
`Fig. 2. Effect of rapamycin
`on insulin-regulated protein
`kinases in H4 hepatoma
`(A) Serum-starved
`cells.
`(24 hours) H4 cells were
`incubated in the presence
`or the absence of 20 nM
`raparnycin for 30 min be(cid:173)
`fore the addition of insulin
`(10-6 M). At the indicated
`times, cytosolic extracts
`were prepared. Aliquots of
`total extract (20) (top pan(cid:173)
`el), extracts immunoprecip(cid:173)
`itated with affinity-purified
`polyclonal antibody to a
`peptide derived from p 70
`S6 kinase (14)
`(second
`panel), or p85 Ask S6 ki-
`nase (third panel), were as(cid:173)
`sayed for S6 kinase activity
`with 40S ribosomes as sub(cid:173)
`strate. Bottom panel, pro(cid:173)
`teins from H4 cell extracts
`Western-blotted with an an(cid:173)
`tibody to phosphotyrosine.
`The region containing IRS-1
`(P180) is shown. (B) Rapa(cid:173)
`rnycin-mediated inhibition of
`basal and insulin-stimulated
`S6 kinase actMty. Rapa-
`mycin (0 to 250 nm) was
`added to serum-starved H4 cells 1 hour before harvest. Thirty minutes before
`harvest, the cells were either treated with insulin (10-5 M) or left untreated.
`Cells were harvested (20), and the kinase actMty of cytosolic extracts
`detected as phosphorylation of 40S ribosomes (7, 12). (C) Effect of rapa(cid:173)
`mycin on incorporation of 32P into ribosomal protein S6 in intact H4 cells.
`Serum-starved (24 hours) H4 cells were incubated with serum-free medium
`containing 0.5 mCi of 32P; per 10-cm plate; after 1 hour, rapamycin (0 to 80
`nm) was added. One hour later, insulin (10-5 M) was added to half of the
`plates. The cells were harvested 30 min later in extraction buffer (20) without
`Triton X-100, with aprotinin (10 U/ml) (0.5 ml per plate), and homogenized
`
`C
`
`Rapamyd, (:Lr------=---=-__J
`
`D
`a
`
`£ =~ ~e 1.0
`i: ::: :st 0.5
`.,
`.,
`if :ll e 40
`(/) !-!~ {20
`(/) - 0
`
`0.0
`
`'ti
`Q.
`
`:: 60
`C
`
`No rapamycin
`
`Rapamycin
`
`C
`
`20
`
`40
`
`60
`20
`Fraction number
`
`40
`
`60
`
`and centrifuged (10 min, 1000g). After further centrifugation (1.5 hours, 2
`x 105g), sedimented material was subjected to SOS-PAGE. 32P-labeled
`S6 is indicated by the arrow. (D) MonoQ chromatography of extracts from
`insulin-treated H4 cells. Serum-starved (24 hours) H4 cells were incubat(cid:173)
`ed in the absence (a and c) or the presence (b and d) of rapamycin (20
`nM). After 30 min, insulin (10-5 M) was added to all plates. Cells were
`harvested 30 min thereafter into extraction buffer (20) without Triton
`X-100. Cytosolic extracts were chromatographed on a column (23).
`Fractions (fr) (1 ml) were assayed for kinase activities toward 40S
`ribosomes (a and b) and SKAIPS peptide (c and d) (4, 7).
`
`974
`
`SCIENCE • VOL. 257
`
`• 14 AUGUST 1992
`
`

`

`Downloaded from
`
`http://science.sciencemag.org/
`
`
`
`on June 13, 2018
`
`(Fig. 2A) (12). Incubation of H4 cells with
`rapamycin for 1 hour before addition of insu(cid:173)
`lin led to a dose-dependent inhibition of both
`basal and insulin-stimulated S6 kinase activity
`in the cytosolic extract that was essentially
`complete at 10 nM rapamycin (Fig. 2B). The
`rapamycin inhibition of cytosolic S6 kinase
`activity of H4 cells was accompanied by an
`inhibition of basal and insulin-stimulated
`phosphorylation of the ribosomal protein S6
`in situ (Fig. 2C).
`To determine whether the inhibition of
`total S6 kinase activity by rapamycin was a
`consequence of the inhibition of p70 or p85
`Rsk S6 kinase, or both, we resolved cyto(cid:173)
`solic extracts by anion-exchange chroma(cid:173)
`tography (Figs. 2D and 3A) and by immu(cid:173)
`noprecipitation (Fig. 2A). H4 cytosolic
`extracts contain a dominant peak of S6
`kinase activity that was eluted from a
`MonoQ anion-exchange column near 0.25
`M NaCl (Fig. 2D) . This peak corresponds
`to the p70 S6 kinase (13), and recombinant
`p70 S6 kinase expressed in COS cells ex(cid:173)
`hibits similar elution (14). This peak of S6
`kinase activity was completely inhibited by
`rapamycin pretreatment (Fig. 2D). Rapa(cid:173)
`mycin also caused loss of S6 kinase activity
`in immunoprecipitates prepared with anti(cid:173)
`bodies to a peptide from the p70 S6 kinase
`(Fig. 2A) . The p85 Rsk S6 kinase, which
`elutes from MonoQ between 0.05 and 0.1
`M NaCl (Fig. 3A), contributes less than
`5% of the total cytosolic S6 kinase activity
`in H4 cells (Fig. 2D). We examined the
`activity of p85 S6 kinase by immunoprecip(cid:173)
`itation with an antibody to a peptide from
`the p85 Rsk kinase protein; p85 Rsk S6
`kinase underwent activation in response to
`insulin, peaking in activity at 10 min (Fig.
`2A). Concentrations of rapamycin that
`caused maximal inhibition of mitogenesis
`(Fig. 1) and total inhibition of p70 S6
`kinase activity (Fig. 28) did not alter the
`time course or magnitude of the insulin
`activation of p85 Rsk S6 kinase (Fig. 2A).
`We 'further evaluated the differential sen(cid:173)
`sitivity of the p70 and p85 Rsk S6 kinases to
`inhibition by rapamycin by directly examin(cid:173)
`ing the activity of the recombinant S6 ki(cid:173)
`nase expressed transiently in COS cells.
`COS cells endogenously expressed both p85
`and p 70 S6 kinase activities, which were
`separable by MonoQ anion-exchange chro(cid:173)
`matography and independently regulated
`(Fig. 3A). Active phorbol esters (Fig. JA),
`serum, or epidermal growth factor (EGF),
`but not insulin, each stimulated the activity
`associated with the p85 Rsk kinase, but did
`not alter the activity of the p70 S6 kinase
`(15). The endogenous MAP kinase activity
`in COS cells was increased by the same
`stimuli that increased p85 Rsk S6 kinase
`activity (15) . Recombinant epitope-tagged
`(r-epi) (16) versions of p70 S6 kinase, p85
`Rsk S6 kinase, and rat p44 MAP kinase
`
`C
`
`t 1.0
`
`c 0.8
`111-
`0, E
`! in 0.6
`~:;;
`10 o 0.4
`Ill E
`~ S: 0.2
`a.
`
`4
`
`3
`
`2
`
`10
`
`0
`
`20
`
`8.0
`
`f I 6.0
`
`GIil)
`
`:l:;; 4.0
`C: 0
`S2 E
`11., S, 2.0
`m :.
`
`0.218
`0
`0.1 'a_
`
`8
`6
`4
`2
`Rapamycln (nM)
`
`10 FK506
`200nM
`
`~
`c e
`"' 5
`0.5 _§,
`0.4: ..
`
`0.3i1
`
`0
`
`4A
`
`~ 3
`
`-~-li .2 = i 2
`
`as ...
`C: -S2 :5
`
`10
`(/)
`
`B
`
`0.4
`
`0.3 ~
`0
`0.2 ~
`
`0.1
`
`5
`
`15
`10
`Fraction number
`
`a nl~~t!1
`
`vector p44
`p85
`p70
`DNA transfected
`
`:.
`
`D
`
`0
`
`1
`.1
`Rapamycin (nM)
`
`! ::,
`Recombinant p70
`m
`Vector
`(n~M_l_l_o_ ~1_1_-'--_0_~1_0_.0_3--'-,_o_._1-'-,_o_.~3~,_1'--~•-3=--~' kD
`
`10
`
`Fig. 3. Rapamycin inhibi(cid:173)
`tion of recombinant p70
`S6 kinase. (A) COS-M6
`cells were deprived of
`serum overnight, were
`treated with phorbol myri(cid:173)
`state acetate (PMA, 0.1
`µ,M) or carrier for 15 min,
`and then were harvested
`in extraction buffer (20) .
`After ultracentrifugation,
`the supernatants were
`matched for protein con(cid:173)
`tent and were chromato(cid:173)
`graphed on MonoQ as in
`Fig . 20 . S6 kinase activity
`ribosomes
`toward 40S
`was measured (7, 12). (0 )
`Untreated cells, (e ) PMA(cid:173)
`treated cells. U = pico(cid:173)
`moles of 32P transferred to
`S6 per minute. (B) PMA
`and EGF regulation of re(cid:173)
`combinant p70 and p85 S6
`kinases and p44 erk-1
`(24) . COS
`MAP kinase
`cells were transfected with
`~ - - - - - - - - - - - - -- - -~ - + 68
`vector only, with the epi(cid:173)
`tope-tagged S6 kinase expression constructs (r-epi p70 S6 kinase and r-epi p85 Rsk S6 kinase) or with
`the epitope-tagged p44 MAP kinase (r-epi erk-1) (24) . After 24 hours, cells were left untreated (solid
`bars) or treated with PMA (100 nM) for 15 min (open bars) or EGF (60 ng/ml) for 10 min (hatched bars),
`then were homogenized in extraction buffer (20) . Recombinant proteins were immunoprecipitated by
`incubation with the monoclonal antibody 12CA5 (22), and S6 kinase activity in the washed immunopre(cid:173)
`cipitates was determined (7, 12). The r-epi erk-1 activity was assayed similarly with myelin basic protein
`(MBP) as substrate. Error bars indicate standard deviation (n = 3). (C) Recombinant p70 S6 kinase but
`not p85 S6 kinase or erk-1 is inhibited by rapamycin but not FK506. COS cells were transfected with r-epi
`p70 and r-epi p85 Rsk S6 kinase cDNAs (top panel) , or r-epi p70 S6 kinase and r-epi erk-1 cDNAs
`(bottom panel) ; 48 hours later, the indicated concentrations of rapamycin were added to each plate 15
`min before harvest. Recombinant proteins were immunoprecipitated and assayed for kinase activities as
`in Fig . 3B. Error bars indicate standard deviation (upper panel, n = 5; lower panel, n = 6) . (D)
`Dose-response of rapamycin inhibition of r-epi p70 S6 kinase activity toward 40S subunits (top panel)
`(mean± SD, n = 6) compared with r-epi p70 S6 kinase autophosphorylation (middle) . COS cells were
`transfected with r-epi p70 S6 kinase cDNA After 48 hours, rapamycin was added at the indicated
`concentrations. PMA (0.1 µ,M) was added 45 min later, and 15 min thereafter cells were extracted and
`were subjected to immunoprecipitation as in Fig . 3A Autophosphorylation (middle panel) was measured
`by omitting the 40S subunits in the S6 kinase assay. The autoradiograph (middle) exhibits the 32P
`incorporation into r-epi p70 polypeptide during a 15-min incubation with ['y-32P)-labeled adenosine
`triphosphate. Portions of each immunoprecipitation were subjected to SOS-PAGE, were blotted onto
`PVDF membranes, and were probed with anli-p70 S6 kinase peptide antibody (14) (lower panel).
`
`•
`
`Vector
`
`0 I 1 I
`
`(nM)
`
`Recombinant p70
`I 0.031 0 . 1 I 0.3 I 1
`
`0
`
`• • • • •
`
`+-117
`
`+-66
`
`3
`
`I • I~"
`
`SCIENCE
`
`• VOL. 257
`
`• 14 AUGUST 1992
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`975
`
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`action in lymphoid cells is also likely to
`involve a ubiquitous signal transduction
`element shared with nonlymphoid lineages.
`The rapamycin target might be a proximate
`upstream activator of the p70 S6 kinase,
`such as an activating p 70 S6 kinase-kinase
`or a regulator of such an enzyme, and
`appears to be a crucial element linking
`growth factor receptors to subsequent intra(cid:173)
`cellular processes regulating proliferation.
`
`REFERENCES AND NOTES
`
`1. J. P. Gordon et al., Curr. Top. Cell. Regul. 21, 89
`(1982); R. L. Erikson, J. Biol. Chem. 266, 6007
`(1991).
`2. S. W. Jones et al., Proc. Natl. Acad. Sci. U.S.A. 85,
`3377 (1988); E. Erikson and J. L. Maller, J. Biol.
`Chem. 261, 350 (1986); D. A. Alcorta et al., Mo/.
`Cell. Biol. 9, 3850 (1989).
`3. P. Banerjee et al., Proc. Natl. Acad. Sci. U.S.A. 87,
`8550 (1990); S. C. Kozma et al., ibid., p. 7365; P.
`Jeno, L. M. Ballou, I. Novak-Hofer, G. Thomas,
`ibid. 85, 406 (1988).
`4. D. J. Price, R. A. Nemenoff, J. Avruch, J. Biol.
`Chem. 254, 13825 (1989).
`5. T. W. Sturgill, L. B. Ray, E. Erikson, J. L. Maller,
`Nature 334, 715 (1988).
`6. N. G. Ahn and E. Krebs, J. Biol. Chem. 265, 11495
`(1990); N. Gomez and P. Cohen, Nature 353,170
`(1991); J. Chung, S. L. Pelech, J. Blenis, Proc.
`Natl. Acad. Sci. U.S.A. 88, 4981 (1988); C. B.
`Barrett, E. Erikson, J. L. Maller, J. Biol. Chem. 267,
`4408 (1992); J. R. Grove, D. J. Price, P. Banerjee,
`M. Ahmad, A. Balasubrarnanyam, J. Avruch, un(cid:173)
`published observations.
`7. D. J. Price, N. K. Mukhopadhyay, J. Avruch, J.
`Biol. Chem. 266, 16821 (1991); N. K. Mukhopadh(cid:173)
`yay et al., ibid. 267, 3325 (1992).
`8. S. Schreiber, Science 251,283 (1991).
`9. J. Liu et al., Ce/166, 807 (1991); D. F. Fruman, C.
`Klee, B. E. Bierer, S. J. Burakoff, Proc. Natl. Acad.
`Sci. U.S.A. 89, 3686 (1992).
`10. F. J. Dumont, W. Staruch, S. L. Koprak, M. R.
`Merlino, N. H. Segal, J. lmmunol. 144, 251 (1990);
`B. E. Bierer et al., Proc. Natl. Acad. Sci. U.S.A. 87,
`9231 (1990).
`11. S. M. Metcalf and F. M. Richards, Transplantation
`49, 798 (1990); J. E. Kay et al., Immunology 72,
`544 (1991).
`12. R. A. Nernenoff, J. R. Gunsalus, J. Avruch, Arch.
`Biochem. Biophys. 245, 196 (1986).
`13. D. J. Price, J. R. Gunsalus, J. Avruch, Proc. Natl.
`Acad. Sci. U.S.A. 87, 7944 (1990).
`14. J. R. Grove eta/., Mo/. Cell. Biol.11, 2159 (1991).
`15. J. R. Grove, D. J. Price, B. E. Bierer, J. Avruch,
`unpublished data.
`16. M. F. White, R. Maron, C. R. Kahn, Nature 318,
`183 (1985); X. J. Sun et al., ibid. 352, 73 (1991).
`17. J. J. Siekierka, S. J. U. Ung, M. Poe, C. S. Lin, N.
`G. Sigal, ibid. 341, 755 (1989); M. H. Harding, A.
`Gala!, D. E. Uehling, S. L. Schreiber, ibid., p. 758;
`Y . .J. Jin, S. J. Burakoff, B. E. Bierer, J. Biol. Chem.
`26, 10942 (1992).
`18. Rapamycin failed to inhibit the total and Suc-1
`precipitable histone H1 kinase activity in H4 cells,
`further suggesting that cdc2 activity is unaffected
`by drug.
`19. V. Calvo, C. Crews, T. Vik, B. E. Bierer, Proc. Natl.
`Acad. Sci. U.S.A., in press.
`20. H4 hepatoma and COS cells were harvested by
`homogenization in an extraction buffer containing
`10 mM potassium phosphate (pH 6.5), 1 mM
`EDTA, 5 mM EGTA, 10 mM MgCl2 , 2 mM dithio(cid:173)
`threitol (DTT), 1 mM vanadate, 50 mM p-glycero(cid:173)
`phosphate, Triton X-100(0.1%),211,M leupeptin, 2
`11,M pepstatin, 0.2 mM phenylrnethylsulfonyl fluo(cid:173)
`ride (PMSF). Extracts were centrifuged for 1.5
`hours at 2 x 105g and matched for protein
`content before chromatography or immunopre(cid:173)
`cipitation. Immune complexes bound to protein A
`Sepharose were washed three times in extraction
`buffer containing 0.25 M NaCl, and once in 20 mM
`
`(erk-1), expressed transiently in COS cells,
`exhibited similar regulatory behavior. The
`r-epi p85 Rsk S6 kinase and r-epi p44 MAP
`kinase were activated by treatment of cells
`with phorbol
`12-myristate
`13-acetate
`(PMA) or EGF, whereas the recombinant
`p70 S6 kinase was constitutively active, and
`the activity was not altered significantly by
`PMA, EGF (Fig. JB), or withdrawal or
`readdition of serum ( 15). Treatment of COS
`cells with rapamycin abolished the activity
`of r-epi p70 S6 kinase, whereas the basal
`(15) or PMA-stimulated activities of the
`r-epi p85 S6 kinase and an r-epi p44 MAP
`kinase were not affected by 10- to 20-fold
`higher concentrations of rapamycin (Fig.
`JC). Rapamycin treatment did not inhibit
`the expression or recovery of the r-epi p 70
`S6 kinase polypeptide (Fig. JD). Moreover,
`the inhibition of p70 S6 kinase activiry was
`specific for rapamycin; treatment of cells
`with a 100-fold higher concentration of
`FK506 failed to inhibit r-epi p70 S6 kinase
`activity (Fig. JC).
`The p70 S6 kinase is activated by phos(cid:173)
`phorylation at multiple Ser and Thr resi(cid:173)
`dues (13). Both the endogenous and recom(cid:173)
`binant p70 S6 kinase polypeptide expressed
`in COS cells appear as a ladder of polypep(cid:173)
`tide bands after SDS-polyacrylamide gel
`electrophoresis (PAGE), of which only
`those with the slowest mobility coelute on
`MonoQ chromatography with the active
`enzyme (14). The slowed mobility on SDS(cid:173)
`PAGE is abolished by treatment with pro(cid:173)
`tein phosphatase and reflects a phosphoryl(cid:173)
`ation-induced conformational change asso(cid:173)
`ciated with the active state. Although vir(cid:173)
`tually all recombinant p70 S6 kinase
`polypeptides contain 32P when isolated
`from 32P-labeled COS cells, only a minority
`show the highly retarded migration on
`SDS-PAGE seen with purified active rat
`liver p70 S6 kinase, and coelute with S6
`kinase activity. After an in vitro autophos(cid:173)
`phorylation reaction, this electrophoreti(cid:173)
`cally retarded fraction of p70 S6 kinase
`polypeptides appears as a highly phospho(cid:173)
`rylated species migrating more slowly than
`the bulk of p70 S6 kinase; the latter shows
`much lower incorporation of 32P relative to
`its abundance (Fig. JD). The inhibition of
`the p70 S6 kinase activity seen at increas(cid:173)
`ing concentrations of rapamycin was corre(cid:173)
`lated with the disappearance of the slowly
`migrating 32P-labeled band observed after
`the autophosphorylation reaction in vitro.
`By contrast, the in vitro autophosphoryla(cid:173)
`tion associated with the faster migrating p70
`polypeptide (Fig. JD) and the overall 32P
`incorporation occurring in situ into the fast(cid:173)
`er migrating p70 polypeptides, which are
`catalytically inactive toward 40S subunits,
`were only diminished at much higher con(cid:173)
`centrations of rapamycin ( 15). Thus, rapa(cid:173)
`mycin inhibition of p70 S6 kinase activity
`
`976
`
`toward S6 in 40S subunits is paralleled by
`the selective loss of autophosphorylating ac(cid:173)
`tivity associated with the fully active (slow(cid:173)
`est migrating) p70 S6 kinase species.
`This result could be explained if rapa(cid:173)
`mycin, alone or as a complex with an FKBP,
`bound to and inhibited only the active
`conformation of p70 S6 kinase or prevented
`the accumulation of the active conformation
`of p70 S6 kinase by preventing its activation
`or accelerating its dephosphorylation. The
`effect of rapamycin, alone and as a complex
`with FKBP12 (17), an FKBP known to bind
`rapamycin with high affinity (8), on the
`activity of purified, fully active rat liver p 70
`S6 kinase (4, 14) was examined in vitro.
`Rapamycin ( 40 nM), added either alone or
`after prebinding to an equimolar concentra(cid:173)
`tion of recombinant FKBP12, did not alter
`p70 S6 kinase activity, or the rate at which
`rat liver p 70 S6 kinase was inactivated by
`phosphatase-2A (15). The lack of direct
`inhibition of active p70 S6 kinase by rapa(cid:173)
`mycin and FKBP12 suggests either that an(cid:173)
`other FKBP is required for rapamycin activ(cid:173)
`ity or that rapamycin inhibits p70 S6 kinase
`indirectly, by inhibiting an upstream activa(cid:173)
`tor that is also crucial for mitogenesis.
`The mechanism by which insulin medi(cid:173)
`ates activation of cytosolic p70 S6 kinase is
`incompletely understood. Intrinsic tyrosine
`kinase activity of the insulin receptor re(cid:173)
`sults in tyrosine phosphorylation of a 180-
`kD polypeptide substrate termed IRS-1
`(16). Insulin-stimulated tyrosine phospho(cid:173)
`rylation ofIRS-1 in H4 cells was not altered
`by concentrations of rapamycin that abol(cid:173)
`ished p70 S6 kinase activity (Fig. 28).
`Insulin activates an array of proline-direct(cid:173)
`ed protein kinases in H4 hepatoma cells,
`including MAP kinases and a form of cdc2
`that phosphorylate a putative regulatory
`domain on intact p 70 S6 kinase (3, 7). The
`activation of these enzymes can be moni(cid:173)
`tored with a synthetic polypeptide sub(cid:173)
`strate, S.KAIPS peptide, corresponding to
`these Ser Pro/Thr Pro-rich p 70 S6 kinase
`regulatory sequences (7). Rapamycin, at
`concentrations that completely inhibit ac(cid:173)
`tivation of p70 S6 kinase, did not alter the
`activation of these SKAIPS peptide kinases
`in response to insulin (Fig. 2D), indicating
`that MAP kinase and cdc2 activities are
`unaffected by drug ( 18). Thus, the target
`for rapamycin appears to be situated down(cid:173)
`stream of the insulin receptor kinase and
`tyrosine phosphorylation of IRS-1 on a
`signal transduction pathway distinct from
`that mediating activation of the erk-1/erk-2
`MAP kinases. In the interleukin-2-depen(cid:173)
`dent CTLL-20 cell line, interleukin-2 stim(cid:173)
`ulates, and rapamycin inhibits, S6 kinase
`activity, whereas the PMA-stimulated ac(cid:173)
`tivities of p85 Rsk S6 kinases and p42 MAP
`kinases are not inhibited by rapamycin
`(19). Thus, the mechanism of rapamycin
`
`SCIENCE • VOL. 257
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`• 14 AUGUST 1992
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`
`(Fig. IA), as would be expected if the
`agonist were opening inwardly rectifying
`K+ channels (9). Somatostatin (100 nM)
`increased this current by two- to tenfold in
`all mock-transfected cells and in cells trans(cid:173)
`fected with the WT «zAR or Asn 79 «zAR
`(Fig. 1, B through E). Maximally effective
`concentrations of UK 14 304 or clonidine
`produced a 1.5- to 8-fold increase in K+
`current in cells expressing the WT a 2AR
`(Fig. I, B through D). Concentrations of
`clonidine and UK 14304 that produced
`half-maximal activation {EC5o) of the K+
`current were 14 and 30 nM, respectively, in
`cells expressing the WT a 2AR {Fig. 2A);
`these are similar to the EC50 values for the
`inwardly rectifying K+ conductance acti(cid:173)
`vated by pharmacologically characterized
`a 2AARs in autonomic enteric and central
`locus coeruleus neurons ( 10). The actions
`of maximally effective concentrations of
`somatostatin and AR agonists were not
`additive {n = 22), which is evidence that
`the transfected WT a 2AR couples to the
`same set of K+ channels as does the endog(cid:173)
`enous somatostatin receptor. In contrast to
`the WT a 2AR, the mutant Asn 79 a 2AR did
`not activate K+ currents (Fig. 1, B, C, and
`E), even in the presence of 10,000-fold
`higher concentrations of clonidine or U