30. Buffer A consists of 10 mM tris-acetate (pH 7.5 at4TC), 40 mM 0-glycerophosphate, 1.5 mM EGTA,0.5 mM EDTA, 25 mM sodium fluoride, 1 mMsodium pyrophosphate, 0.5mM sodium orthovan-adate, 1 mM dithiothreitol, 1 mM benzamidine,0.5% (vN) Triton X-100, phenylmethylsulfonylfluo-ride (0.1 mg/mI), 1 p.M pepstatin A, leupeptin (2gg/ml), and aprotinin (2 pLg/ml).

31. B. Margolis et al., Mol. Cell. Biol. 10, 435 (1990).32. A. J. Rossomando, D. M. Payne, M. J. Weber, T.

W. Sturgill, Proc. Nati. Acad. Sci. U.S.A. 86, 6940(1989).

33. For the in vivo studies, cells were quickly chilled andwashed twice with ice-cold phosphate-buffered sa-line, sedimented, and resuspended in p21 buffer (3)(100 pI per 15-cm plate). In later experiments,additional phosphatase inhibitors (10 mM Na2VO4,10 mM p-nitropherrl phosphate, 40 mM p-glycerolphosphate, 25 mM NaF, 1 gM microcystin-LR, 0.01gM calyculin A, and 0.2 mM sodium pyrophos-phate) were included in all buffers and appear to beimportant for preserving the effect of forskolin onRaf-1. Resuspended cells were homogenized (27),and the lysates were centrifuged (100,000g at 40Cfor 30 min). Approximately 40 jig of supernatantprotein was incubated (200 p1 total volume at 4°C for30 min) with 50 pig of immobilized c-Ha-Ras, loadedwith GMP-PNP or GDP (3). Beads were sedimentedby brief centrifugation (1g5,000 for 1 min), andsupernatants were removed. Beads were resus-pended in 200 p1A of p21 buffer containing CHAPS

(0.1% wN) and layered onto 5x p21 buffer (1 ml ina 1.5-mI tube) and centrifuged. Recovered beadswere washed (4x with 1 ml of p21 buffer withCHAPS). Adsorbed proteins were released withSDS sample buffer and processed by SDS-PAGE(on 8% gels) for protein immunoblotting with anti-bodies to Raf-CR2 (conserved region 2) (anti-Raf-CR2); immunoblots were developed by enhancedchemiluminescence. Raf band intensities of immu-noblots were quantitated on the basis of the localbackground by a densitometer (Molecular Dynam-ics, Sunnyvale, CA).

34. Supported by grants from NIH to T.W.S.(DK41077) and to M.J.W. (CA39076) and from theAmerican Cancer Society to T.W.S. (BE69D) andby Young Investigator Awards to P.D. and J.W.from the Virginia affiliate of the American DiabetesAssociation. Fellowship support for J.W. camefrom the Juvenile Diabetes Foundation Interna-tional. We especially thank D. Morrison (ABL-Basic Research Program, Frederick, MD) for pro-viding recombinant Raf(K375M) baculovirus, theanti-Raf-CR2, synthetic Raf-1 peptides, andmany helpful discussions. We also thank L. A.Vincent for technical assistance; J. Schlessinger(New York University) and R. Schatzman (Syntex,Palo Alto, CA) for antibodies to the EGFR andRaf-COOH-terminus, respectively; and J. Corbin(Vanderbilt University) for PKA.

17 August 1993; accepted 5 October 1993

Inhibition by cAMP of Ras-DependentActivation of Raf

Simon J. Cook* and Frank McCormickActivation of the Raf and extracellular signal-regulated kinases (ERKs) (or mitogen-activated protein kinases) are key events in mitogenic signaling, but little is known aboutinteractions with other signaling pathways. Agents that raise levels of intracellular cyclicadenosine 3',5'-monophosphate (cAMP) blocked DNA synthesis and signal transductionin Rati cells exposed to epidermal growth factor (EGF) or lysophosphatidic acid. In thecase of EGF, receptor tyrosine kinase activity and association with the signaling moleculesGrb2 and Shc were unaffected by cAMP. Ukewise, EGF-dependent accumulation of theguanosine 5'-triphosphate-bound form of Ras was unaffected. In contrast, activation ofRaf-1 and ERK kinases was inhibited. Thus, cAMP appears to inhibit signal transmissionfrom Ras by preventing Ras-dependent activation of Raf-1.

Cyclic adenosine 31,5 '-monophosphate(cAMP) was the first second messenger tobe identified, and its role in regulatingphysiological processes is well established(1). Hormone receptors increase the intra-cellular concentration of cAMP by increas-ing the amount of the free a subunit of theguanosine triphosphate (GTP) binding pro-tein G, (Gas.GTP), which activates ade-nylyl cyclase (2, 3). Despite literature dat-ing back 25 years (3), the precise role ofcAMP in regulating cell growth and prolif-eration remains a matter of considerabledebate (3, 4). In some cells, such as Swiss3T3 cells and thyrocytes, cAMP is a mito-genic messenger and promotes the G, to Sphase transition in the cell cycle (4). In

contrast, cAMP inhibits the proliferation ofmany cell lines, including fibroblasts, Tcells, neuroblastoma and astrocytoma cells,and cells transformed by Ras, Src, andpolyoma middle T antigen (3).

Kinetic analysis indicates cAMP exerts a

cytostatic effect in the early Go to G, phaseas well as mid-G, phase (5) and, in a fewcases, G2 (3). Cyclic AMP inhibits prolif-eration by growth factors irrespective oftheir ability to activate the polyphospho-inositide (PI) pathway; therefore, the abil-ity ofcAMP to inhibit full activation of thePI pathway is unlikely to explain its effects(5). Because cAMP inhibits proliferationstimulated by receptor tyrosine kinases or

GTP binding protein-coupled or "serpen-tine" receptors, it is more likely to exert itseffect downstream of receptor activationand second messenger generation, wheresignal transduction pathways converge.

SCIENCE * VOL. 262 * 12 NOVEMBER 1993

A common point of convergence formany, if not all, growth factors is the activa-tion of the Ras proteins (6), which regulatesignals to the mitogen-activated protein ki-nases ERKI and ERK2 (7). These are serine-threonine kinases that require phosphoryla-tion at both tyrosine and threonine for acti-vation; both phosphorylation events are cat-alyzed by the same dual-specificity mitogen-activated protein (MAP) kinase kinase calledMAP or ERK kinase (MEK) (8). MEK is inturn regulated by at least two upstream ki-nases: the c-Raf-1 proto-oncogene product (9)or MEK kinase (10). The stimulation ofquiescent cells with various growth factors orphorbol esters or the introduction of onco-

genic Ras proteins leads to the activation ofERKs within minutes (7). Indeed, Ras isrequired for growth factors to fully activate theRaf-MEK-ERK pathway (7, 11), which sug-gests that Ras is a common target for growthfactors and that this kinase cascade is impor-tant for regulating the G1 to S phase transi-tion. In Ratl cells, Ras is a point of conver-

gence for two different classes of growth fac-tor, lysophosphatidic acid (LPA) or epidermalgrowth factor (EGF), to fully stimulate DNAsynthesis and ERK activation (1 1). Here, wehave examined the effect ofcAMP on growthfactor stimulation of ERK1 and ERK2.

Quiescent Ratl cells were incubatedwith 1 mM dibutyryl cAMP for 10 minbefore stimulation with LPA or EGF, andERK activation was assayed on protein im-munoblots by the appearance of activated,hyperphosphorylated forms with retardedmobility on SDS-polyacrylamide gel elec-trophoresis (SDS-PAGE). Dibutyryl cAMPalone had no effect on the activation ofERKl and ERK2 in Ratl cells, but a 10-mintreatment with dibutyryl cAMP completelyinhibited the LPA- or EGF-induced mobil-ity shift (Fig. 1A); 8-bromo-cAMP was alsoeffective (12). With a more sensitive im-mune complex kinase assay (13), we foundthat the ability of the phorbol ester PMA(phorbol 12-myristate 13-acetate) to acti-vate MAP kinases, albeit a submaximalactivation compared with that of EGF, wasalso inhibited by dibutyryl cAMP (Fig. 1B).These effects were not due to contamina-tion of dibutyryl cAMP with butyrate ormetabolic release of butyrate within the cellbecause butyrate itself (up to 1 mM) did notinhibit ERKI (Fig. 1B) or DNA synthesis(12). Furthermore, a 10-min exposure todibutyryl cAMP did not affect the amountof immunoreactive ERK1 and ERK2 (Fig.1A), so the loss of ERK activity (Fig. 1B)did not result from a decreased amount ofthe enzyme. The 50% inhibition concen-tration (IC50) of dibutyryl cAMP uponLPA- or EGF-stimulated ERKl was approx-imately 0.1 to 0.2 mM, which agreed with

that for inhibition of DNA synthesis (12).The adenosine diphosphate (ADP) ribo-

1069

Onyx Pharmaceuticals, 3031 Research Drive, Rich-mond, CA 94806.*To whom correspondence should be addressed.

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syl transferase activity of cholera toxin (CT)specifically adds an ADP ribose group toGa,, which results in inhibition of the in-trinsic guanosine triphosphatase (GTPase)activity of Gas GTP and constitutive acti-vation of adenylyl cyclase (14). Treatmentof Ratl cells with increasing concentrationsof CT resulted in a dose-dependent inhibi-tion of subsequent ERK1 activation by EGFmeasured by the ability of the immunopre-cipitated ERK1 to phosphorylate myelin ba-sic protein (MBP) (Fig. 2A). The IC50 forthe effect of CT (0.17 + 0.15 ng/ml) fellwithin the range of the IC50 for CT-mediat-ed inhibition of DNA synthesis by EGF(0.03 + 0.01 ng/ml) (Fig. 2B). The effect ofsubmaximal doses ofCT on EGF-stimulatedERK activation was greatly potentiated bythe cAMP phosphodiesterase inhibitor3-isobutyl-1-methylxanthine (IBMX) (50jxM), which resulted in a shift of the CTdose-response curve to the left by one toone and a half orders of magnitude (Fig.2A). This potentiation of low doses of CTby IBMX was also noted for its inhibition ofEGF- or LPA-stimulated DNA synthesis(12) and represents the ability of IBMX toblock metabolic conversion of cAMP,thereby resulting in more persistent andincreased concentrations of cAMP evenwith small amounts of CT (15). The factthat both ERK activation and DNA synthe-sis were inhibited by both cAMP analogsand CT, together with the observed synergybetween IBMX and small doses of CT,strongly suggests that CT exerts its effectsthrough elevation of cAMP in this system.

The major elements required for func-tional coupling of the EGF receptor(EGFR) to ERKs have been identified(16). The Src homology 2 (SH2)-SH3adaptor protein Grb2 recruits the Rasexchange factor Sos to the activatedEGFR at the plasma membrane where it

activates Ras (16). We studied the effectof cAMP on EGFR phosphorylation andsignaling complex assembly by assayingthe binding of the activated EGFR to theglutathione-S-transferase-Grb2 fusionprotein (GST-Grb2) in vitro (17). Celllysates from control or EGF-treated Rat 1cells previously incubated with or withoutCT were incubated with GST-Grb2.Bound proteins were detected by proteinimmunoblotting with monoclonal anti-bodies to phosphotyrosine (PY20 andPY69; ICN, Irvine, California).

Phosphotyrosine-containing proteinswere not detectable in GST precipitatesfrom either control or EGF-stimulated cells(12, 18). In GST-Grb2 precipitates, weobserved the association of several phos-photyrosine-containing proteins with GST-Grb2 in an EGF-dependent manner, in-cluding a broad, heavily phosphorylatedband at approximately 170 to 180 kD. Thisprotein appears to be the autophosphoryl-ated EGFR because it comigrated with thehuman EGFR in similar experiments donein HER14 cells (12). We also observedother tyrosine phosphoproteins (p52, p55,p66, and pl 16) that coprecipitated with theEGFR in an EGF-dependent manner. Pro-tein immunoblotting of the same samplesresolved on the same gel with a polyclonalantibody to Shc confirmed the identity oftwo of these proteins as p52s1c and p66s`IShc is an SH2 domain-containing proteinthat is implicated in the coupling of recep-tor and nonreceptor tyrosine kinases to Rasand that can transform cells when overex-pressed (19). Thus, EGF stimulation resultsin the assembly of multimeric signalingcomplexes at the EGFR that include Grb2,Shc, and other phosphoproteins. In cellspretreated with CT, we observed no inhi-bition of EGFR autophosphorylation or as-sociation of EGFR signaling complexes

Fig. 1. Effect of dibutyryl A Controll cAMP BcAMP on the activation of p44ERK1 _ 100,000ER1and ERK2 in Rati a4E K-i4mP a Control

cells. (A) Inhibition of LPA- CnL.EC L E m *EGFor EGF-stimulated phosphorylation of ERK1 and ERK2

PMA

by dibutyryl cAMP. Quiescent Rati cells were incubat-ed with Dulbecco's modified Eagle's medium (DME)(control) or DME containing dibutyryl cAMP (1 mM) for10 min before the addition of DME (C) or DME con- O 40000taining LPA (10 p.M) (L) or 10 nM EGF (10 nM) (E) and 3incubated for a further 5 min. Detergent lysates were 20,000 -

resolved by SDS-PAGE, transferred to nitrocellulose,and blotted with antibodies to ERK1 and ERK2 (MK1 2; 0Transduction Labs). (B) Inhibition of activation of Controt CAMP SoiumERK1 by dibutyryl cAMP but not sodium butyrate. butyrate

Quiescent Rati cells were incubated with DME (control) or DME containing dibutyryl cAMP (1 mM)or sodium butyrate (1 mM) for 10 min before addition of DME (black bars) or DME containing EGF(10 nM, hatched bars) or PMA (100 nM, stippled bars) for a further 5 min. Detergent lysates were

immunoprecipitated with E1.2 antiserum, which specifically recognizes ERK1 (11), and kinaseactivity was assayed against MBP in the immune complex. Results are expressed as the amount of32p incorporated into MBP (counts per minute) and are from a single experiment representative ofthree that gave similar results.

with GST-Grb2 (Fig. 3A).EGF activation of ERK1 and ERK2 pro-

ceeds through a Ras-dependent pathway (7,1 1), and EGF treatment of Ratl cells for 5min resulted in an increase in the percentageof Ras in the GTP-bound form from 12 to 30to 35%. Treatment of Ratl cells with 1 mMdibutyryl cAMP for 10 min had no effect onthe ability ofEGF to increase the amount ofRas-GTP in Ratl cells (Fig. 3B). Thus,under conditions in which EGFR phospho-rylation, EGFR signaling complexes, andEGFR signaling to Ras were all intact, theability of EGF to activate ERKI and ERK2was completely inhibited.

Raf-1 has been defined genetically andbiochemically as being a likely target of Ras(20), and Ras is critical for activation of

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Fig. 2. Effects of CT on EGF-stimulated ERK1activity and DNA synthesis in Rati cells. (A)Inhibition by CT of EGF-stimulated ERK1 acti-vation in Rati cells. Quiescent cells were incu-bated for 1 hour with various concentrations ofCT alone (a) or in the presence of IBMX (50aM) (D) before stimulation with 10 nM EGF for5 min. After lysis, ERK1 immunoprecipitateswere assayed for MBP kinase activity. Resultsare expressed as the amount of 32p incorporat-ed into MBP (counts per minute) and are from asingle experiment representative of three thatgave similar results. (B) Inhibition of EGF-stim-ulated DNA synthesis in Ratl cells by CT.Quiescent cells were stimulated with 10 nMEGF in the presence of increasing concentra-tions of CT. [3H]Thymidine (3 p.M unlabeledthymidine) was added for the last 2 hours, andthe reactions were terminated after 22 hours byaddition of 5% trichloroacetic acid (1 1). Resultsare expressed as mean + SD in counts perminute of [3H]thymidine incorporated into acid-precipitable material and are from a singleexperiment representative of three that gavesimilar results.

SCIENCE * VOL. 262 * 12 NOVEMBER 19931070

. .. ~~~~~~~~~~~~~~~~~~~~~.... ....1,0.

Raf-1 by growth factors (7). Fractions fromRaf-1-transformed cells have MEK kinaseactivity (9), and Raf-1 activates MEK invitro (21); thus, Raf is likely to be the linkbetween Ras and the ERK cascade. Stimu-lation of Rati cells with LPA or EGFresulted in the activation of Raf-1, as mea-sured by the phosphorylation of recombi-nant, catalytically inactive MEK B in im-munoprecipitates of antibodies to Raf-1(anti-Raf-1) (22); irrelevant pre-immuneantiserum precipitated no MEK kinase ac-tivity (Fig. 4). LPA was at best half aseffective at activating Raf-1 as was EGF(Fig. 4), and this correlates with the smallerincrease in RasGTP activated with LPAcompared to that activated with EGF (11).Treatment of Ratl cells with CT (100ng/ml) resulted in a complete inhibition ofEGF- or LPA-stimulated Raf activationmeasured in anti-Raf immune complexes(Fig. 4). Thus, cAMP appears to inhibitERK activation by uncoupling Ras.GTPfrom the activation of Raf-1.

The effects ofcAMP are similar to thoseof the Rapla or Krevl Ras-related GTPase,which can revert Ras transformation whenoverexpressed (23, 24): Rap inhibits acti-vation of ERKs by either LPA or EGF (1 1)by inhibiting the coupling of Ras-GTP to

Raf (25). Increased concentrations ofcAMP result in protein kinase A (PKA)-mediated phosphorylation of Rapla andRap GTPase activating protein (24); thus,Rap may itself be a mediator of the effects ofcAMP in this system. However, our prelim-inary results indicate that dibutyryl cAMPdoes not increase the percentage of Rap inthe GTP-bound form in Rati cells (12). Analternative and simpler model is that PKAphosphorylates Raf, thereby inhibiting itsactivation. Raf-1, but not A-Raf or B-Raf,contains a PKA phosphorylation consensussite in the NH2-terminal regulatory do-main, and preliminary experiments haveconfirmed that PKA is able to phospho-rylate recombinant Raf-1 in vitro (12).

Our results are consistent with a modelin which cAMP inhibits growth of Ratlcells by inhibiting Raf activation, althoughcAMP is also likely to affect cell regulationthrough other Raf-independent mecha-nisms (26, 27). Analogs of cAMP inhibitthe growth of Ras- and Src-transformedcells (3), which suggests that the targeteduse of tissue-specific hormones that increasethe intracellular concentration of cAMPmay represent a possible avenue of researchfor therapeutics directed against the Raspathway.

Fig. 3. Signal transduction from the EGER to Ras A Anti-pTyr Anti-Shc

is not impaired by cAMP. (A) EGFR autophos- Control CT Control CT

phorylation and in vitro binding of GST-Grb2 andShc in Rati cells. Quiescent Rati cultures wereincubated with CT (100 ng mi-1 for 1 hour)before stimulation with EGF (10 nM) for 5 min. 116

Duplicate cleared lysates were incubated with 8p66shcGST-Grb2, and bound proteins were resolved by p52SDS-PAGE, transferred to PVDF membranes, 49 GST-Grb2and immunoblotted with either antibodies tophosphotyrosine (anti-pTyr) (PY20 and PY69, E + +left) or antibodies to Shc (right). The positions ofprestained molecular size markers (in kilodal- B

tons), the GST-Grb2 fusion protein, and p52shc EGF+ cAand p66shc are indicated. (B) Lack of effect ofcAMP on the EGF-stimulated increases in the EGF

percentage of Ras in the GTP-bound state. Ele-vated cAMP does not impair EGF-stimulated Control

increases in Ras.GTP. Quiescent Rati cultureswere metabolically labeled with [32p]P04 for 3 10 15 20 25 30 35

hours and received DME or DME containing 1 Ras.GTP/Ras.GDP + Ras.GTP (%)

mM dibutyryl cAMP for the last 5 min. Cells were then stimulated with EGF, and the amount of Rasin the GTP-bound state was determined as described (1 1). Results (mean ± SD of duplicates) are

from a single experiment representative of three that gave identical results.

Fig. 4. Cyclic AMP inhibits activation of Raf. Quies- Pre-immune Anti-Raf-1cent Rati cells were incubated for 1 hour with DME Control Control I CTalone (control) or DME containing CT (100 ng/ml)

before stimulation with LPA (L) (10 &LM) or EGF (E)(10 nM) for 5 min. Proteins from cell lysates were

immunoprecipitated with irrelevant antiserum or an- 30

tiserum to Raf-1 and assayed for MEK kinase activ-ity in the immune complex. Samples were resolved C L E C L E C L E

by SDS-PAGE and detected by autoradiography. The positions of catalytically inactive MEK, the

immunoglobulin heavy chain (1g HC), and pre-stained molecular size markers (in kilodaltons) areindicated. Results are from a single experiment representative of three that gave similar results. C,control, no stimulation.

SCIENCE * VOL. 262 * 12 NOVEMBER 1993

REFERENCES AND NOTES

1. T. W. Rail etal., J. Biol. Chem. 224, 463 (1957); E.W. Sutherland, Science 177, 401 (1972).

2. L. Stryer and H. R. Bourne, Annu. Rev. Cell Biol. 2,391 (1986); E.-J. Choi, Z. Xia, E. C. Villacres, D. R.Storm, Curr. Opin. Cell Biol. 5, 269 (1993).

3. I. H. Pastan et al., Annu. Rev. Biochem. 44, 491(1975).

4. A. L. Boynton and J. F. Whitfield, Adv. CyclicNucleotide Res. 15, 193 (1983); E. Rozengurt,Science 234, 161 (1986); M. M. Gottesman and R.D. Fleischmann, Cancer Surv. 5, 291 (1986); J. F.Whitfield et al., Cancer Metastasis Rev. 5, 205(1987); J. E. Dumont et al., Trends Biochem. Sci.14, 67 (1989).

5. I. Magnaldo et al., FEBS Lett. 245, 65 (1989); C.0. Rock, J. L. Cleveland, S. Jacowski, Mol. Cell.Biol. 12, 2351 (1992).

6. G. Bollag and F. McCormick, Annu. Rev. Cell Biol.7, 601 (1991); A. Hall, Curr. Opin. Cell. Biol. 5,265(1993).

7. S. L. Leevers and C. J. Marshall, EMBO J. 11, 569(1992); S. M. Thomas et al., Cell 68, 1031 (1992);K. W. Wood etal., ibid., p. 1041; A. M. M. deVriesSmits et al., Nature 357, 602 (1992).

8. C. M. Crews, A. Alessandrini, R. L. Erikson, Sci-ence 258, 478 (1992).

9. P. Dent etal., ibid. 257,1404 (1992); J. M. Kyriakiset al., Nature 358, 417 (1992); L. R. Howe et al.,Cell 71, 335 (1992).

10. C. A. Lange-Carter, C. M. Pleiman, A. M. Gardner,K. J. Blumer, G. L. Johnson, Science 260, 315(1993).

11. S. J. Cook, B. Rubinfeld, I. Albert, F. McCormick,EMBO J. 12, 3475 (1993).

12. S. J. Cook and F. McCormick, unpublished re-sults.

13. Cells were deprived of serum for 36 hours beforestimulation with growth factors at 370C for 5 min.Incubations were terminated by aspiration of themedium and addition of ice-cold lysis buffer [20mM tris-HCI (pH 8), 1% Triton X-100, 10% glycer-ol, 137 mM NaCI, 1.5 mM MgCI2, 1 mM EGTA, 50mM NaF, 1 mM Na3V04, 1 mM phenylmethylsul-fonyl fluoride, 20 pLM leupeptin, and 10 ±g mlaprotinin]. Detergent-insoluble material was re-moved by centrifugation at 14,000g for 10 min at4'C; protein concentrations were assayed by theBio-Rad assay and varied by less than 10%. Celllysates were incubated for 2 hours with E1.2 andE2. 1 antisera (1 1), and protein A-Sepharosebeads were added for the last 30 min. Immunecomplexes were washed three times with lysisbuffer and once with kinase buffer [30 mM tris (pH8), 20 mM MgCI2, and 2 mM MnCI2]. ERK kinaseactivity was assayed by addition of kinase buffer(30 p1) containing adenosine triphosphate (ATP)(2 p.M), MBP (7 pg), and 5 ,Ci of [y-32P]ATP andincubation at 300C for 30 min. Incubations wereterminated by addition of hot 4x SDS-PAGE sam-ple buffer (10 pi), and reaction products wereresolved by SDS-PAGE (12% gels) followed byautoradiography. The MBP band was excised,and incorporated radioactivity was quantitated byliquid scintillation counting.

14. C. Van Dop et al., J. Biol. Chem. 259, 696 (1984);D. Cassel et al., Proc. Natl. Acad. Sci. U.S.A. 74,3307 (1977).

15. G. Kaur et al., Cancer Res. 52, 3340 (1992).16. L. Buday and J. Downward, Cell 73, 611 (1993);

M. Rozakis-Adcock, R. Fernley, J. Wade, T. Paw-son, D. Bowtell, Nature 363, 83 (1993); N. Li etal.,ibid., p. 85; N. W. Gale, S. Kaplan, E. J. Lowen-stein, J. Schlessinger, D. Bar-Sagi, ibid., p. 88; S.E. Egan et al., ibid., p. 45.

17. Induction and purification of GST-Grb2 fromEscherichia coli were as described previously(18). Equal quantities of Rati cell lysates wereincubated with GST agarose beads followed byincubation with 10 pg of immobilized GST orGST-Grb2 for 2 hours at 4°C. Samples were thenwashed five times with lysis buffer (inclusion ofradioimmunoprecipitation assay buffer washesyielded identical results), drained, and boiled inSDS-PAGE sample buffer. Samples were resolved

1071

by SDS-PAGE (10% gels by Novex) and trans-ferred to poly(vinylidene difluoride) (PVDF) mem-branes, which were then stained with Coomassieblue to confirm equal sample loading. Filters werethen incubated with either antibodies PY20 andPY69 to phosphotyrosine (1:1500 dilution; ICN,Irvine, CA) or polyclonal antibodies to Shc (1:250;Transduction Labs, Lexington, KY). Detection waswith horseradish peroxidase-conjugated second-ary antibodies and the enhanced chemilumines-cence system (ECL, Amersham).

18. E. J. Lowenstein et al., Cell 70, 431 (1992).19. G. Pelicci et al., ibid., p. 93; J. McGlade, A.

Cheng, G. Pelicci, P. G. Pelicci, T. Pawson, Proc.Nati. Acad. Sci. U.S.A. 89, 8869 (1992); M. Roza-kis-Adcock et al., Nature 360, 689 (1992).

20. W. Kolch, G. Heidecker, P. Uoyd, U. R. Rapp,Nature 349, 426 (1991); L. Van Aelst, M. Barr, S.Marcus, A. Polverino, M. Wigler, Proc. Nati. Acad.Sci. U.S.A. 90, 6213 (1993); A. B. Vojtek, S. M.Hollenberg, J. A. Cooper, Cell 74, 205 (1993);X.-F. Zhang et al., Nature 364, 308 (1993); P. H.Warne, P. R. Viciana, J. Downward, ibid., p. 352.

21. S. G. Macdonald et al., Mol. Cell. Biol., in press.22. Proteins from equal quantities of cell lysates from

control or stimulated Rati cells were immunopre-cipitated with 10 glI of Raf-1 polyclonal antiserum[R. Schatzmann, Syntex, Palo Alto, CA] for 2.5hours, and protein A-Sepharose beads were add-

ed for the last 45 min. Immune complexes werewashed three times in lysis buffer and once inkinase buffer [30 mM tris (pH 8), 20 mM MgC12,and 1 mM dithiothreitol] before being resuspendedin 30 pll of kinase assay cocktail containing kinasebuffer, 0.5 gg of recombinant, baculovirus-ex-pressed catalytically inactive MEK B, 2 AM ATP,and 5 pCi of [y-32P]ATP per sample. Incubationswere for 30 min at 300C and were terminated by theaddition of hot 4x SDS-PAGE sample buffer (10 pug)followed by boiling for 5 min at 950C. Samples wereresolved by SDS-PAGE (10% gels), and gels werestained with Coomassie brilliant blue, dried, andsubjected to autoradiography.

23. H. Kitayama et al., Cell 56, 77 (1989).24. M. Noda, Biochim. Biophys. Acta 1 155,97 (1993).25. T. Sakoda et al., Oncogene 6, 1705 (1992).26. J. F. Habener, Mot. Endocrinol. 4,1087 (1990).27. F. Lamy et al., J. Biot. Chem. 268, 8398 (1993).28. We thank T. Evans and G. Bollag (Onyx) for

critical reading of the manuscript, T. Palmer (DukeUniversity Medical Center) for advice, S. Macdon-ald and T. Evans (Onyx) for the recombinant MEKB, and R. Schatzman (Syntex) for antiserum top74raf-1. S.J.C. and F.M. are supported by a grantfrom the National Cancer Institute (U01 CA51992-03) to F.M.

3 August 1993; accepted 13 October 1993

Unidirectional Coupling of Gap JunctionsBetween Neuroglia

Stephen R. Robinson,* Edith C. G. M. Hampson, Mark N. Munro,David 1. Vaney

Gap junctions permit the passage of ions and small molecules between cells, therebyproviding a basis for direct intercellular communication. In the rabbit retina, the lowmolecular weight dyes Lucifer yellow and biocytin passed readily from astrocytes intoadjacent astrocytes, oligodendrocytes, and Muller cells. However, the dyes rarelypassed from either oligodendrocytes or Muller cells into astrocytes. Unidirectional pas-sage of dye suggests the presence of an asymmetric barrier to the movement ofmolecules through heterologous gap junctions and indicates the potential for a hierarchyof command between interconnected cells.

Many vertebrate and invertebrate cells are

connected by "gap junctions" that providea route for intercellular communication(1). Cellular coupling can be revealed bythe intracellular injection of a membrane-impermeant dye of low molecular weight,such as Lucifer yellow (457 daltons) (2) or

biocytin (373 daltons) (3, 4). The assump-tion that molecules can pass through gapjunctions equally in both directions was

challenged by Flagg-Newton and Loewen-stein's (5) experiments on cocultures ofBalb/c and B fibroblasts. They reported thatdye injected into B cells diffused readilyinto neighboring Balb/c cells but that little,if any, dye transferred from injected Balb/ccells into neighboring B cells. This remark-able finding has not been replicated, and its

Vision, Touch, and Hearing Research Centre, Depart-ment of Physiology and Pharmacology, University ofQueensland, Brisbane, Australia 4072.

*To whom correspondence should be addressed.

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functional significance is unclear becausethe two cell lines are unlikely to encountereach other outside a petri dish. We now

report that asymmetric diffusion of dye oc-

curs between different types of neuroglia(astrocytes, oligodendrocytes, and Mullercells) in the myelinated band of the intactrabbit retina (6).

Retinae from adult rabbits were stainedwith Hoechst 38317 (7), and labeled peri-axonal astrocytes (8) were injected withLucifer yellow (9). We photographed 91periaxonal astrocytes that had been inject-ed, and in every instance dye had diffusedinto the majority (>90%) of stained cellswithin their dendritic fields (mean numberof coupled cells, 70; range, 20 to 200; Fig.1, A and B). Many of these coupled cells(50 to 70%) were neighboring periaxonalastrocytes, whereas the remainder were ei-ther oligodendrocytes (Fig. 1, E and F) or

astrocytes belonging to other classes. Inaddition, 58 periaxonal astrocytes were in-

SCIENCE * VOL. 262 * 12 NOVEMBER 1993

jected with biocytin (10). They were cou-pled to 30 to 300 cells (mean number ofcoupled cells, 156; Fig. LG), of which 50 to70% were periaxonal astrocytes, and theremainder were either oligodendrocytes orastrocytes from other classes.

Some periaxonal astrocytes were alsocoupled to Muller cells (radial glia): 10 ofthe 91 injected with Lucifer yellow werecoupled to 1 to 12 Muller cells (meannumber of coupled cells, 5), whereas 40 ofthe 58 filled with biocytin were coupled to1 to 20 Muller cells (mean number ofcoupled cells, 3). These Muller cells couldbe found up to 250 gum from the soma of aninjected astrocyte. Because the lateral pro-cesses of rabbit Muller cells are only about25 pum long (1 1), such labeling must be dueto the diffusion of dye through gap junc-tions between astrocytes and Muller cellsrather than to inadvertent impalement ofMuller cell processes. We injected morethan 100 Muller cells with Lucifer yellow orbiocytin, but they were never coupled toother Muller cells or to astrocytes.

Oligodendrocytes were identified bytheir characteristic morphology (12) (Fig.1, C, D, F, and H) and by their absence ofimmunoreactivity to glial fibrillary acidicprotein. Approximately half of the stainednuclei at the margin of the myelinated bandwere oligodendrocytes. Of the 92 oligoden-drocytes injected with Lucifer yellow, 90exhibited no dye diffusion into adjacentcells (Fig. 1, C and D). The remaining 2oligodendrocytes were surrounded by 8 to10 lightly labeled oligodendrocytes andMuller cells. Similarly, 49 of the 70 oligo-dendrocytes injected with biocytin were notcoupled. The remaining 21 oligodendro-cytes were surrounded by lightly labeledoligodendrocytes and astrocytes (meannumber of coupled cells, 49; range, 1 to150; Fig. lH).

Our results show that, when periaxonalastrocytes are injected with Lucifer yellowor biocytin, most of the nearby oligoden-drocytes and some Muller cells fill with dye.By contrast, when oligodendrocytes are in-jected, few (if any) of the nearby astrocytesshow coupling. Furthermore, when Mullercells are injected, nearby astrocytes arenever labeled with dye (Fig. 2).

It is conceivable that the unidirectionalspread of dye is attributable simply to dam-age to the oligodendrocytes (and Mullercells) caused by the injection procedure andconsequent closing of their gap junctions.To investigate this possibility, oligodendro-cytes were injected with either Lucifer yel-low or biocytin, and nearby astrocytes wereimmediately injected with the other dye(13). Of the 23 successful fills, 21 oligoden-drocytes were double-labeled. In these 21cases, dye had passed from astrocytes intooligodendrocytes after the oligodendrocytes

.ll5--- :- Mi. 11.