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(1990) Nature 343, 377-381 19 MORAN, M. F., POLAKIS, P., McCORMICK, F., PAWSON, T.

and ELLIS, C. (1991) Mol. Cell. Biol. 11,1804-1812 20 WENG, Z. et al. (1994) MoL Cell. Biol. 14, 4509-4521 21 DREYFUSS, G., MATUNIS, M. J., PIi~IOL-ROMA, S. and

8URD, C. G. (1993) Annu. Rev. Biochern. 62, 289-321 22 GIBSON, T. J., THOMPSON, I. D. and HERINGA, J. (1993) FEBS

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24 PAWSON, T. and SCHLESSINGER, J. (1993) Curt. Biol. 3, 434-442

7.S KORNBLUTH, S., DASSO, M. and NEWPORT, ]. (1994) I. Cell Biol. 125, 705-719

26 BISCHOFF, F. R. and PONST1NGL, H. (1991) Nature 354, 8O-82

27 BISCHOFF, F. R., KLEBE, K., KRETSCHMER, J., WlTrlNGHOFER, A. and PONSTINGL, H. (1994) Proc. Natl Acad. ScL USA 91, 2587-2591

Ras proteins are small membrane-associated GTPases that are essential for transmitting signals from recep- tors at the cell surface to internal signalling path- ways. Receptor activation converts Ras proteins from their inactive GDP-bound states to the active GTP- bound forms by promoting nucleotide exchange. The GTP-bound forms bind to proteins referred to as effectors, which transmit signals on downstream (Fig. 1). Signal transmission is terminated by GTPase-acti- rating proteins (GAPs) that catalyse conversion of Ras-bound GTP to GDPL Oncogenic mutants of Ras proteins occur in about 40% of all human cancers 2, and are impaired in their ability to hydrolyse GTP to GDP. As a result, they persist in their GTP-bound states, causing uncontrolled signalling.

One of the major signalling pathways controlled by Ras Is the MAP (mitogen.activated protein) kinase cascade, which Is Initiated by the serlne/threonlne kinase Raf :~s. Raf itself can exist in oncogenlc forms, although these have never been detected in human cancers. Oncogenic activation involves loss of the N- terminal region: this region is therefore presumed to repress klnase activity in the normal Raf counterpart. Raf activation during mltogenlc signalling in normal cells is also likely to involve release from the re- pressive effects of this domain. Until recently, the mechamsms by which Ras proteins activate Raf (and thus the MAP klnase cascade) had been completely unknown, but now the critical first step in this pro- cess is understood.

Ras and Raf Interact directly Many researchers speculated that Ras would acti-

vate Raf indirectly, via unidentified effectors. Few suspected that Ras transmits signals to Raf by a pro- cess involving direct physical interaction. An early indication that this might be the case came from the finding that Raf and MAP kinases bind to Ras pro- teins in vitro 6 with characteristics expected for the binding of a direct Ras effector; binding was depen- dent on GTP, and was eliminated by point mutations that render the Ras protein inactive (mutations in the 'effector-binding region'). These results suggested a signalling complex that contains Raf binds to Ras, but could not address the critical question of which component of the complex bound Ras directly.

The next major step came from an elegant demon- stration that Ras and Raf interact when tested in the

Raf: the Holy Grail of Ras biology?

Ras proteins regulate cell growth and differentiation, and their

mutation plays a major, direct role in causing human cancer.

For years, their precise function has been a mystery. One of the

pathways Ras controls has recently been identified. It consists of a

cascade of kinases (Ra~, MEK and MAP kinases) that transmits

signals fi'om tile plasma membrane to the nucleus. Tile role of Ras

is remarkably simple: it recnlits Raf from the cytoplasm to the

plasma membrane. Once in the membrane, Raf is activated and

tile kinase cascade is initiated. Is this tile whole story?

yeast two.hybrid systemL This system Is a powerful tool for detecting protein-protein interactions, and is based on the reconstitution of an active transcrip- tion factor when a protein fused to the factor's DNA- binding domain interacts with a hybrid protein containing the factor's transactivation domain s. In parallel with this investigation designed to test the hypothesis that Ras and Raf interact, the two-hybrid system was being used as a screen for proteins able to bind to Ras 9 or Rati °. The former screen identified a member of the Raf family, and the latter a member of the Ras family, Rapl. In complementary studies, Ras-Raf binding was detected by using biochemical approaches to assess the interaction of Ras and Raf in vitro ~ and in cell extracts 12. Collectively, these discoveries concluded that (1) Ras binding to Raf is dependent on GTP; (2) the effector-binding region of Ras is essential for Raf binding; (3) the Ras-related ................... protein Rapl, which shares the same effector-bind- l:rank McCormick ing sequence as Ras, also binds lo Raft and (4) the is at ONYX region of Raf that is necessary for binding is within PharmaceuUcals, the first conserved region of the regulatory domain Richmond, CA of the Raf protein. 94806, USA.

TRENDS IN CELL BIOLOGY VOL. 4 OCTOBER 1994 © 1994 Elsevier Science Ltd 0962.8924/94/$07.00 34 7

(. :()ll]lllC I l l

Ras-GDP

GAPs GEFs

Ras_~GTP

Y Raf-1

(MAP kinase kinase kinase)

MEK (MAP kinaso kinase)

ERKs (MAP kinases)

Nuclear regulatory proteins Other substrates

FIGURE 1

Regulation of the Raf-lVlAP-kinase pathway by Ras proteins. The level of Ras in the active GTP-bound state is determined by the relative activities of GAPs and GEFs. The hierarchy of

kinases shown here is conserved throughout evolution.

These data make a very strong case for Raf as a major effector of the Ras protein, particularly within the biological context discussed above, However, Raf is not the first mammalian protein to fulfil the cri- teria expected of a Ras effector. The GAPs p120.GAP and neuroflbromln bind to Ras (and Rap) with simi- lar properties, which led to speculation that these regu- lators may also have effector functions ~, However, genetic analysis of GAP function in yeast and flies argues strongly against an effector role for these proteins within the known Ras-regulated path- ways, Furthermore, Saccharomyces cerevislae adenylate cyclase has long been known to be a Ras effector protein ~3. Remarkably, the three classes of proteins - Rafs, GAPs and adenylate cyclase - now known to bind to Ras proteins at the effector-blnding region, in a GTP-dependent manner, share no detectable sequence similarity,

Ras-.Ref Interaction recruits Raf to the membrane Binding of Ras to Raf must represent the first step

in the process of Ras-dependent Raf activation. What is the consequence of binding?

One possibility is that binding of Ras to Raf directly activates the kinase activity of Raf, by causing a con- formational change in Raf. This would be analogous to the activation of mammalian adenylate cyclase by physical association with the Gs~ subunit 14. Support for this model comes from the demonstration that recombinant Ras proteins, in their GTP-bound states, activate MAP kinases when added to extracts from Xenop,s oocytes ]s,]6, and activate adenylate cyclase in membranes from S. cerevisiae ~7. Against this model,

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Raf purified in a stable, active state is not associated with Ras, and thus appears to be locked into the active state through a covalent modification, or through high-affinity interaction with an unknown regulatol ~.]8. Also against the direct-activation-by- binding model, many groups, including my own (S. Macdonald and F. McCormick, unpublished), have reconstituted Ras-Raf binding in vitro, but have failed to detect increased kinase activity as a result of binding.

A second possible outcome of Ras-Raf binding is a conformational ~*. :'~ange in Raf that allows subsequent activation by an unknown event. For example, bind- ing of Ras to Raf might unfold the protein to allow subsequent covalent modification by an activator: Raf would then be in a conformation that is stable, with persistent activity independent of Ras (Fig. 2a shows this model for a membrane-associated Ras- dependent activator).

A third possibility is that binding of Raf by Ras leads to activation of Raf simply as a result of its recruitment to the membrane (Fig. 2b), as suggested by Traverse and colleagues 19. This model differs from the others in that no activating conformational changes occur during binding: physical association of the two proteins is sufficient to account for the role of Ras in the process. The major difficulty with this third model is that it fails to explain how recombi- nant Ras proteins could activate MAP kinases when added to cell-free extracts in vitro, but this could be resolved if the in vitro systems in fact contained intact membrane material to allow Raf recruitment.

Indeed, recent results indicate that the third model is the correct interpretation. If the role of Ras in Raf activation Is merely to recruit Raf to the membrane, then localizing Raf to the membrane by engineering targeting signals onto it should replace the need for Ras In the process of Raf activation. Two groups have shown that this Is the case 2~z. Raf Is normally a cyto- plasmic protein, but Raf proteins fused to Ras Caax motifs become associated with the plasma mem- brane. Forced association with the membrane acti- vates Raf kinase and instigates the downstream kinase cascade shown in Fig. I. Most importantly, Raf activation is independent of Ras under these circumstances.

Ref activation In the plasma membrane What biochemical steps occur after Raf is recruited

to the plasma membrane to turn on Raf kinase ac- tivity? We do not yet have an answer to this question. However, once deposited in the plasma membrane, Raf becomes associated with insoluble structural el- ements, as it cannot be released by treatment with detergent 2z. Ras, however, is liberated from mem- branes under these conditions, showing that it has parted company with the Raf protein that it brought to the membrane. The nature of the insoluble mem- brane components that Raf associates with after deposition in the membrane are not known: their insolubiUty suggests cytoskeletal structures, but many possibilities exist. Identification of these com- ponents is essential, as they are expected to contain the missing activating components that lock Raf in its kinase-active state. One set of components that

348 TRENDS IN CELL BIOLOGY VOL. 4 OCTOBER 1994

FORUM '1111

(a)

may be necessary for Raf activation are proteins of the 14-3-3 family, which are associated with Raf in cells and move with Raf to the plasma membrane dur- ing the process of activation 23.24. Other components of the activation process are expected to be kinases, including pro- tein kinase C 2s,26, but the precise roles of these complex events during acti- vation have not been fully delineated.

The dissociation of Ras from Raf at the membrane allows for a catalytic aspect to Raf activation, in which a single Ras (b) protein in its GTP-bound state could, in [,rinciple, deliver any number of Raf proteins to the plasma membrane. In cells, the number of cycles of delivery, and thus the degree of signal amplifi- cation, is presumably regulated by inter- action of Ras with GAPs (which turn Ras-GTP to the GDP-bound state) and exchange factors, such as Sos (which recharge Ras with GTP).

Interestingly, Rap, which is 50% ident- ical to Ras and binds tightly to Raf 27, cannot replace Ras as a Raf activator. In fact, it inhibits Ras signalling, and FK;URE 2 reverts transformation of cells caused by ras oncogenes; indeed, it was identified as the product of the Krev-I gene, which is named after its ability to cause mor- pholc~gical reversion of cells trans- formed by Ki-ras zS. The simplest model to explain these data draws on the fact that Ral)l is located in the Golgi com- plex and not in the plasma membrane -~'~. We there- fore speculate that binding of Rap to Raf forms a non- productive complex because membrane components that are necessary for Raf activation are missing in Golgi membranes. Alternatively, Golgl membranes may contain enzymes that Inactivate Raf in some way. This model Is currently being tested.

Does Ras have other functions? Ras proteins produce a range of effects when over-

expressed or microinjected into cells, including in- itiation of DNA synthesis, and morphological changes :¢~. Early analysis suggested that the effects of Ras on cell morphology and DNA synthesis occur through distinct pathways :~]. More recently, Cowley and colleagues :~z have suggested that stimulation of DNA synthesis is mediated by the MAP kinase path- way, whereas morphological changes utilize a dis- tinct Ras-dependent pathway that may make an important contribution to malignant transformation by oncogenic Ras mutants. This pathway is likely to involve regulation of the Ras-related proteins Rac and Rho, since these proteins regulate cell shape ~.:~*~,:~4, and may be downstream of Ras or perhaps coordi- nately regulated with Ras.

In conclusion, the discovery that Ras binds directly to Raf has focused attention sharply on the bio- chemical steps involved in the activation process, and has led to a simplifying concept for Ras action as

Plasma membrane

I

Active Raf F MAP kinase

cascade Inactive Raf

Plasma membrane

I

Active Raf

MAP kinase cascade

Inactive Raf

Two models for the role of Ras in the process of Raf activation by membrane components. (a) Binding of Ras to Raf causes a conformational change that allows Raf activation by plasma membrane components. (b) Ras acts solely as a recruitment factor, and all activation steps occur subsequent to membrane localization. Raf proteins (A-Raf, B-Raf and c-Raf) contain three highly conserved domains, shown by the numbered boxes. Ras binds to conserved region 1. The kinase domain is conserved region 3. Oncogenic mutants of Raf proteins lack the first two conserved regions.

a i~lasma membrane recrl~itment factor. The future should reveal how gener;~ this phenomenon is (i.e. how many other protehv Ras recruits to the mem. brane) and the physical nature of the Ras-Raf binding event Itself. In addition, it will now be possible to dis- sect the biological functions of Ras into those that work through Raf activation and those that do not: no doubt Ras biology still has some surprises in store.

References 1 BOGUSKI, M. S. and McCORMICK, F. (1993) Nature 366, 643-653 2 BOS, J. L. (1989) Cancer Res. 49, 4682-4689 3 RAPP, U. R. (1991) Oncogene 6, 495-500 4 ROBERTS, T. (1992) Nature 360, 534-535 5 MARSHALL, C. J. (1994) Curt. Opin. Genet. Dev. 4, 82-89 6 MOODIE, S. A., WlLLUMSEN, B. M., WEBER, M. I. and

WOLFMAN, A. (1992) Science 260, 1658-1661 7 VAN AELST, L, BARR, M., MARCUS, S., POLVERINO, P. and

WIGLER, M. (1993)Prec. NatlAcad. ScL USA 90, 6213-6217 8 FIELDS, S. and SONG, O. (1989) Nature 340, 245-246 9 VOITEK, A. B., GOLLENBERG, S. M. and COOPER, ~. A. (1993)

Cell 74, 205-214 10 ZHANG, X-F. et al. (1993) Nature 364, 308-313 11 WARNE, P. H., VlClANA, P. R. and DOWNWARD, I. (1993)

Nature 364, 352-355 12 KOIDE, H., SATOH, T., NAK~.FUKU, M. and KAZIRO, Y. (1993)

Prec. Natl Acad. Sci. USA 90, 8683-8686 13 WlGLER, M. et aL (1988) Cold Spring Harbor Syrup. Quant. Biol.

53, 649-655

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i / i l ~ ~ i ! i i i =

..... i~iiiiiii ..... FORUM

i

14 BOURNE, H. R., SANDERS, D. A. and McCORMICK, F. (1990) Nature 348,125-132

15 ffOH, T. et al. (1993) Proc. Nail Acad. Sci. USA 90, 975-979 16 SHIBUYA, E., POLVERINO, A., CHANG, E., WIGLER, M. and

RUDERMAN, I. (1993) Proc. NatlAcad. Sci. USA 89, 9831-9835 17 FIELD, J. etal. (1988) MoL Cell. Biol. 8, 2159-2165 18 MACDONALD, S. G. etal. (1993) MoL Cell. Biol. 13, 6615-6620 19 TRAVERSE, S. etal. (1993) OncogeneS, 3175-3181 20 LEEVERS, S. I., PATERSON, H. F. and MARSHALL, C. J. (1994)

Nature 369, 411-4i4 21 MARSHALL, C. J. (1994) Curr. Opin. Genet. Dev. 4, 82-89 22 STOKOE, D., MACDONALD, S. G., CADWALLADER, K.,

SYMONS, M. and HANCOCK, I. F. (1994) Sdence 264,1465-1467 23 FREED, E., MACDONALD, S., SYMONS, M., McCORMICK, F.

and RUGGIERI, R. Science (in press) 24 IRIE, K. et al. Science (in press)

25 SOZERI, O. et al. 0992) Oncogene 7, 2259-2262 26 KOLCH, W. et aL (1993) Nature 364, 249-252 27 BOLLAG, G. and McCORMICK, F. (1991) Annu. Rev. Cell. Biol. 7,

601--632 28 KffAYAMA, H., SUGIMOTO, Y., MATSUZAKI, T., IKAWA, Y. and

NODA, M. (1989) Cell 56, 77-84 29 BERANGER, F., GOUD, B., TAVITIAN, A. and DE GUNZBERG, J.

(1991) Proc. Natl Acad. Sci. USA 88, 1606-1610 30 BAR-SAGI, D. and FERAMISCO, J. R. (1986) Science233,1061-1068 31 LLOYD, A. C., PATERSON, H. F., MORRIS, J. D., HALL, A. and

MARSHALL, -C. I. (!989) EMBO J. 8,1099-1104 32 COWLEY, S., PATERSON, H., KEMP, P. and MARSHALL, C. J.

(1994) Cell 77, 841--852 33 RIDLEY, A. I. and HALL, A. (1992) Cell 70, 389-399 34 RIDLEY, A. I., PATERSON, H. F., IOHNSTON, C. L., DIEKMANN,

D. and HALL, A. (1992) Cell 70, 401-410

Is plasma membrane lipid

composition defined in the exocytic or the

endocytic pathway?

/ laL, A ~ I'A1 m a m t i a I Istilt m t im. I [ ~ / a

Compared with intracellular membranes, the plasma membrane

is rich in cholesterol and sphingomyelin. How does this distinct

composition arise? Here David Allan and Karl-losef Kallen take

a critical view of the belief that these lipids arrive at the

plasma membrane via vesicular traffic from the Golgi complex and

propose instead that they may be accreted in the endocvtic

recvcling pathway.

There is a dramatic difference between the lipid com- position of the plasma membrane and that of most intracellular membranes, in particular the endoplas- mic reticulum (ER). The plasma membrane is rich in cholesterol, sphingomyelin (SM), glycolipids and phosphatidylserine and poor in phosphatidylinosi- tol, cholesterol ester and triacylglycerol, whereas the ER (the main site of lipid biosynthesis) is rich in

phosphatidylcholine and phosphatidylinositol but contains only small amounts of those lipids that are characteristic of plasma membranes 1.

A substantial bulk flow of ER lipids to the plasma membrane must be a consequence of the vesicular transport processes that move proteins to the cell sur- face from their site of synthesis in the ER, but how does the cell generate and maintain the peculiar lipid composition of the plasma membrane if large quan- tities of ER llpids are continually entering it from the secretory pathway?

Despite the recent interest in lipid trafficking 2, this question has still not been answered satisfactorily. There is no obvious lipid equivalent of the sorting signals that target proteins to particular organelles, although cytosollc lipid-transfer proteins have been identified that can exchange Ilplds between different membranes s. These may play a role in glycerollpld translocatlon ~,2 but it is difficult to imagine how they could maintain lipid gradients between the ER and plasma membrane or affect the distribution of SM or glycolipids. The sphingoliplds are generally locked into lumenal membrane surfaces and do not undergo significant transbllayer movement (flip-flop) so they can move between organelles only by vesicular trans- port processes. Although a small proportion of sphingolipids and cholesterol may be associated with caveoli in the plasma membrane 4 and there is con- siderable evidence for limited lipid microdomains, in general it is assumed that most lipids diffuse freely in membranes. Thus, most liplds are not sorted between compartments of a vesicular transport pathway - i.e. lipids generally follow bulk membrane flow.

The Golgl lipid gradient Nonetheless, i t has become c o m m o n l y accepted

that the l ip id compos i t ion of t ransport vesicles changes gradual ly across the Golgi complex, so that the vesicles u l t imate ly fusing w i t h the plasma membrane are r ich in cholesterol and SM s.6. This l ip id gradient model is wide ly believed because i t is

350 © 1994 Elsevier Science Ltd 0962-8924/94/$07.00 TRENDS IN CELL BIOLOGY VOL. 4 OCTOBER 1994