Transmembrane receptors and intracellular pathways that control cell proliferation.

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Annu. Rev. Physiol. 1992. 54:195-210 Copyright © 1992 by Annual Reviews Inc. All rights reserved

TRANSMEMBRANE RECEPTORS AND INTRACELLULAR PATHWAYS THAT CONTROL CELL PROLIFERATION Jacques Pouyssegur and Klaus Seuwen Centre de Biochimie-CNRS, Universite de Nice, Parc Valrose 06034 Nice, France KEY WORDS:

G protein-coupled receptor, receptor tyrosine kinsase, MAP-kinase, growth factors, growth control, a-thrombin, PDGF

INTRODUCTION

It has long been known that extracellular factors such as hormones, growth factors, cytokines, and the extracellular matrix play a decisive role in the cell engagement toward either proliferation or differentiation. Often growth and differentiation are mutually exclusive. This notion has emerged from the common observation that tumors often arise as a failure of the cancer cells to differentiate. Therefore, to understand how cell proliferation is positively controlled, it is necessary to identify the key pathways that convey ex tracellular signals to the nuclei, where repression of the differentiation pro gram and induction of essential genes needed for cell cycle progression and replication are executed. Biochemical analysis of tumors that represent natu rally occurring dominant mutants of the proliferative response has been invaluable for progress in this field. Oncoproteins characterized over the past decade have been shown to span the entire signaling network, from growth factors, growth factor-receptor tyrosine kinases, cytoplasmic protein kinases, GTP-binding proteins, to nuclear transcription factors (for recent reviews on oncogenes and proto-oncogenes see 10, 18, 27, 31, 47, 92, 104). It has become rapidly evident that multiple signaling pathways exist, as is illustrated 195

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by the cooperation between oncogenes (45, 78), or by the synergy observed b�tween growth promoting agents (84). Although cooperation between dis tinct classes of growth factors appears to be the rule, some mitogens are so effective that they can act alone. This is the case for a-thrombin and platelet derived growth factor (PDGF), two potent mitogens for cells of mesenchymal origin ( 1 1 , 13, 29, 70, 74, 8 1). The scope of this review is not to cover the entire field of growth factor signaling, but to highlight recent advances on the early mitogenic events that originate at the plasma membrane receptors. First, with a-thrombin and PDGF as growth factor prototypes, we review the current knowledge of how two major classes of transmembrane receptors, G protein-coupled receptors (a-thrombin) and receptor tyrosine kinases (PDGF), initiate growth. Second, we briefly discuss the current views on raf-kinase and MAP-kinases activation and emphasize their role as possible cellular integrators of multiple signaling pathways.

G PROTEIN-COUPLED RECEPTORS-THE CASE OF a-THROMBIN Overview This class of receptors encompasses a superfamily of molecules that appears to have dominated the world of membrane signaling. They transduce ex tracellular signals that control physiological processes as diverse as vision, taste, olfaction, chemotaxis, secretion, differentiation, and growth. The re ceptors are characterized by seven transmembrane segments, often highly homologous even across receptor subfamilies. Their main feature is to interact with one or a small subset of specific heterotrimeric G proteins by which they transmit signals (8, 19, 99). To date, effector proteins shown to be controlled by G proteins include adenylyl cyclase (Ae) (24), cGMP-phosphodiesterase via transducin (98), phospholipases (PIP2-PLC, PLA2) (9, 15, 26), and ion channels ( 1 14). Certainly more effector systems remain to be identified as suggested by the multiplicity and diversity of G a-subunits (97). With such mUltiple actions of second messengers systems, it is not surpris ing that G protein-coupled receptors participate in the control of cell prolifera tion. At least two compelling pieces of evidence favor this notion. First, it was demonstrated that certain hormones, neurotransmitters, and vasoactive peptides, known to tran�mit their signal via G protein-coupled receptors, are either stimulators or inhibitors of cell proliferation. Among the growth promoting agents described are bombesin, vasopressin ( 1 15), a-thrombin, serotonin (33, 57, 64, 90, 91), substance P, angiotensin (32, 58), endothelin (93), and lysophosphatidic acid (l05). All these agents share the ability to activate, at least, PIPrPLC via G protein-coupled receptors. Other hormones


GROWTH FACTOR SIGNALING PATHWAYS

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like TSH, FSH, J3-adrenergic agonists, and prostaglandins PGEJ, PGI2, which activate AC, are potent growth inhibitors for many fibroblasts, smooth muscle and mesangial cells (52, 67), and are growth stimulators for endocrine and epithelial cells from thyroid and pituitary glands (20). The recent discov ery of constitutively activating mutations in Gas, the G protein subunit stimulating AC and expressed in pituitary and thyroid tumors, underscores the importance of cAMP as a positive modulator of proliferation in endocrine cells (46, 49). The second body of evidence was gathered by exploiting the ability of certain bacterial toxins to specifically ADP-ribosylate G protein a-subunits. Notably, the toxin instrumental in this approach is pertussis toxin (PTX). PTX specifically ADP-ribosylates GaOl, a02, aii> ai2, and ai3 subunits at a car boxy-terminal cysteine, thereby uncoupling their interaction with hormone activated receptors (103). The best example of PTX action is the uncoupling of receptors that inhibit AC and that in some systems activate PIPz-PLC (15). When this toxin was used in mitogenesis assays, it suppressed up to 95% of a-thrombin- and all of serotonin (S-HT) -induced reinitiation of DNA synthe sis in CCL39 cells, a cell line derived from Chinese hamster lung fibroblasts (12, 90). The fact that PTX had no effect on the mitogenic action of EGF, FGF, or IGF-1 in the same cells (12) is a key finding with several im plications. It proves that the biological effects of PTX are specific, it points to the existence of at least two independent growth-signaling mechanisms, and it strongly suggests that PTX-sensitive G proteins are key mediators in the mitogenic action of a-thrombin and 5-HT. A similar conclusion was in dependently reached by Van Corven et al (105) in rat I cells, where PTX abolishes mitogenic action of lysophosphatidic acid, but not that of EGF. Finally, the implication of G protein-coupled effector systems in triggering mitogenesis is further demonstrated by the direct use of non-hydrolyzable GTP analogues in intact cells. Addition of GTPyS to transiently depolarized CCL39 fibroblasts stimulates PI turnover and inhibits AC. Although GTPyS alone is not mitogenic, it does potentiate EGF and FGF-induced DNA synthe sis (65).

G Protein-Coupled Effector Systems Signaling Growth a-Thrombin was chosen initially as a model mitogen because of its extreme potency in CCL39 cells (70). Its action, like that of PDGF, is pleiotypic, involving multiple effector systems. At maximal mitogenic concentration (10 nM), a-thrombin exerts a dual action; it strongly stimulates PIP2-PLC (64) and inhibits stimulated AC (51). Since the discovery of the pluripotency of the phosphoinositide-derived second messengers, PI turnover has been the most appealing signaling system to be involved in the control of cell proliferation. The activation of protein kinase C (PKC) by diacylglycerol and the release of


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Ca2+ from endoplasmic reticulum by inositol trisphosphate (IP3) seemed to 2 account for most of the early mitogenic events [transient rise in Ca +, + + activation of the Na JH antiporter, protein S6 phosphorylation, and induc tion of immediate early genes (5, 60, 72)]. This notion was reinforced by the fact that all the growth promoting agents referred to above (a-thrombin, bombesin, substance P and K, vasopressin, angiotensin, endothelin, etc.) proved to be potent activators of PIPrPLC. It became clear, however, that this signaling pathway by itself could not drive Go-arrested cells into the cell cycle. This conclusion emerged from the following set of observations: first, the mitogenic action of serotonin, revealed in association with FGF, could be pharmacologically dissociated from its action on PIPrPLC (90); second, PTX, which almost totally abolishes a-thrombin-induced mitogenicity in CCL39 cells, inhibits only 40% of PIP2-PLC activation (64); third, 5-HT2, 5-HTlc, muscarinic Ml, or bombesin receptors, stably expressed in CCL39 cells, were found to couple efficiently to PLC via a PTX-insensitive G protein, presumably Gq (96, 100). In these transfected cells, serotonin, carbachol, or bombesin was as efficient as thrombin in eliciting a strong and persistent PIPTPLC activation leading to activation of most of the early mitogenic

events

(88).

In

contrast

to

a-thrombin,

however,

both

neurotransmitters and bombesin failed to induce a proliferative response on their own (88, 108, D. Roux & J. Pouyssegur, unpublished results). On the other hand, studies with PTX showed that pathways coupled to Gi(s) or Go(s) (see below) are crucial for the mitogenic action of serotonin and thrombin in hamster cells (90) and for lysophosphatidic acid in rat 1 cells (105). In agreement with this hypothesis, ectopic expression in CCL39 cells of the a2-adrenergic receptor that is negatively coupled via Gi to AC yielded cells that responded mitogenically to epinephrine when combined with FGF or EGF (89). Similarily to PLC, it must be stressed that activation of this . signaling pathway alone does not trigger mitogenesis. The nature of the effector system that participates in the mitogenic action of these hormones is still unknown. Although inhibition of AC (which would tend to lower cAMP, a growth inhibitor for these cells) is an appealing possibility, other systems controlled by Gi(s) or Go(s) cannot be excluded. At least four PTX-sensitive G proteins identified in CCL39 cells could participate in the potent mitogenic action of a-thrombin. These are Gi3 and Gi2 (73) and two Go subtypes that can be easily separated from Gi2 on urea gels (McKenzie, unpublished results). Finally, the mUltiple and potent action of a-thrombin is revealed by the fact that we have not been able to fully reconstitute the mitogenic signaling of a-thrombin. Indeed, full activation of PLC and inhibition of AC (in cells expressing either 5-HT2 and 5-HTl b receptors or muscarinic Ml and 5-HTlb receptors) by serotonin or the association of carbachol + serotonin was not sufficient to induce significant cell cycle reentry. Because FGF is needed to



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reveal the mitogenic potential of these G protein-activated pathways, it is tempting to propose that a-thrombin stimulates an additional pathway that might result in tyrosine kinase activation. So far, evidence that a-thrombin induces a rapid phosphorylation of cellular substrates at tyrosine residues has been illustrated in platelets (23) and in CCL39 fibroblasts (J. Pouyssegur et aI, unpublished results). However, the link between thrombin action and phosphorylation at tyrosine residues is still lacking. a- Thrombin

Receptor

Most of the biological actions of ex-thrombin are associated with its proteoly tic activity. In particular its mitogenic action on fibroblasts and smooth muscle cells, as well as its aggregating activity on platelets, is completely abolished by blocking the proteolytic site with serine protease inhibitors (38, 107). The first indication in favor of a specific thrombin receptor inducing Ca2+ demobilization was obtained by expression in Xenopus oocytes of poly(A +) RNA from thrombin-responsive hamster fibroblasts (106) or from human endothelial cells (71). The second and most conclusive evidence emerged from the cDNA sequencing and expression cloning of a human thrombin receptor (110) and then a hamster receptor homologue (77), both coupled to PLC. Vu et al (110) provided evidence that the human thrombin receptor not only possesses the hallmark of the G protein-coupled receptors, but that proteolysis of its N-terminal receptor domain is required for activa tion. Apparently the released peptide has no biological activity, whereas a synthetic peptide mimicking the new amino terminus stimulates PLC (110). This cleavage site, which is specific for thrombin, is conserved in the hamster receptor, and its mutation to an uncleavable receptor prevented activation by thrombin, but not by the new amino-terminal peptide. Knowing the complex action of thrombin to elicit a full mitogenic response, a key question is, does the cloned ex-thrombin receptor mediate all the biological effects? The answer is yes for platelet activation. Indeed, it is not surprising that the human receptor peptide that stimulates PLC induces platelet aggregation (110). As expected, the answer is more complex regarding the mitogenic action on Go-arrested CCL39 cells. The hamster receptor heptameric peptide, SFFLRNP, which mimics the new amino terminus, stimulates PLC and inhibits AC in CCL39 cells to the same extent as does a maximal thrombin concentration. However, in contrast to a-thrombin, the peptide alone is not mitogenic. Here again combination with FGF is required to reveal its mitogenic potential exactly as previously reported for serotonin action on 5-HT2 R-expressing cells (l08; V. Vouret-Craviari, E. Van Obberghen Schilling, U. Rasmussen, J. -P. Lecocq, J. Pouyssegur, in preparation). These results independently (a) reinforce our previous conclusion that full activation of these two G protein-signaling pathways ( t PLC, t AC) is not sufficient

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to commit Go-arrested fibroblasts to enter the cell cycle, and (b) indicate that thrombin must elicit an additional mitogenic signal, presumably via a distinct receptor. Obviously, elucidation of this signal is crucial to the reconstitution of the complete mitogenic signaling network elicited by a-thrombin and required for Go exit. The cDNA cloning of the putative new thrombin receptor subtype could give a clue to identifying this additional mitogenic pathway.

RECEPTOR-TYROSINE KINASES-THE CASE OF PDGF Overview This class of transmembrane signaling molecules, recently reviewed (29, 104, 113), does not suffer any ambiguity as to whether it plays a master role in the initiation of cell division. They transduce signals for a variety of growth factors and differentiating agents that include EGF, TGFa, FGFs, PDGFs, eSF-i, NGF, insulin, and IGF-I. These receptor-tyrosine kinases share a molecular topology. They possess a large extracellular ligand-binding do main, a single hydrophobic transmembrane segment, and a cytoplasmic portion that contains the tyrosine kinase catalytic domain. They are generally constitutively turned off and are turned on in response to ligand binding. Furthermore, they are subject to complex regulation by kinases and phospha tases. Their potent role in cell growth triggering is illustrated by the steadily increasing list of oncogenes that belong to the family of receptor- and non-receptor-tyrosine kinases (31). Although various degrees of potency exist among these different tyrosine kinases, it is clear that mutations that release the negative control of the kinase activity severely disrupt the control of cell growth. The signaling machinery that responds to the kinase-initiated signals is rather well conserved because these transmembrane molecules trigger growth even when they are ectopically expressed in cells of different origins. Good examples are provided by NGF-R (trk oncogene), CSFI-R (fms oncogene), and EGF-R (erbB oncogene), which are respectively expressed in neuronal cells, macrophages, and erythrocyte precursors, yet can transform cells from other lineages (39, 62, 83). It is now well demonstrated that the first and crucial event in the kinase activation is oligomerization of the receptor catalyzed by ligand binding. This concept, first demonstrated in cells for the EGF-R (14), was subsequently established for the receptor of the dimeric ligand, PDGF (28, 113). The concept of a dimer-mediated receptor activation mechanism for receptor tyrosine kinases is apparently universal as demonstrated by the functionality of receptor chimerae containing domains from different members of the family (80). Not only is the formation of the dimer essential, but two functional monomeric kinases are required to induce the activation process.



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This notion was confirmed by showing that expression of a negative PDGF-R mutant blunts the activation of a co-expressed wild-type PDGF-R molecule ( 1 02). It is obvious that the kinase domain, the most highly conserved portion of all receptor tyrosine kinase molecules, is central in the transmission of the signal. Replacement of the consensus lysine residue of the A TP-binding site in the EGF, insulin, and PDGF receptors completely abolishes their kinase activity (104 and references therein). Expression and membrane targeting of the modified receptor to the cell surface are not impaired, but induction of both early and late cellular responses, including mitogenesis and onco genicity, are completely lost (30, 55). This suggests that all receptor ty rosine kinase signaling activities depend on a functional tyrosine kinase and that these processes are mediated by tyrosine phosphorylation of cellular substrates. The key intracellular substrates phosphorylated directly by these tyrosine kinases have remained elusive, but recently a number of proteins with func tions potentially regulated by tyrosine phosphorylation have become appar ent. The first substrate in the signal transmission is the receptor itself. The current hypothesis is that following autophosphorylation the cytoplasmic domain of these receptors undergoes conformational changes exposing phosphotyrosine-containing stretches to src homology 2 (SH2) domains that have been identified in a variety of signaling molecules such as pp60c-src, PLC-l', GTPase-activating protein (GAP), and PI 3-kinase (5 4, 68). The SH2 domains are conserved sequences of approximately 100 amino acids, which represent a key element in the recognition of receptors with tyrosine kinase activity. This specific interaction leads to physical association of these mole cules to the plasma membrane via the activated receptors. In addition, the activated PDGF-R, as well as to some extent the EGF-R, can tyrosine phosphorylate the PLC-l', GAP, and the p85 subunit of PI 3-kinase. The next step in understanding this signal transmission is to elucidate the function of these associated proteins and to determine whether tyrosine phosphorylation modulates their activity. Second Messenger Systems PI 3-KINASE The PI 3-kinase was the first of this group of proteins found to be associated with tyrosine kinases. Discovered as tightly associated with middle T antigen/pp60c-src, the PI 3-kinase was then demonstrated to be separable from this complex (36, 1 1 2). It was also the first cytosolic enzyme found to be recruited in a ligand-dependent manner by the PDGF-R, with rapid recruitment to the plasma membrane detected in less than 1 min following stimulation with PDGF (35, 1 12). This association was shown to require the kinase insert domain, a unique sequence that splits the kinase


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catalytic domain of the PDGF-R (17). Interestingly, a tyrosine residue at position 751 of the human PDGF-R {3, which undergoes autophosphorylation, appears critical for PI 3-kinase recruitment (37). This is demonstrated by the fact that this association is lost in a kinase negative mutant; that it is competed for with a peptide mimicking this region only if the peptide is phosphorylated on tyrosine; and that this kinase insert region possesses some similarity with the sequence of polyoma middle T antigen that associates with PI 3-kinase (10). Besides middle T/pp60c-src complex and PDGF-R, PI 3-kinase has also been found associated with the insulin-R (85), the CSF-I-R (109), and EGF-R (6). The subunit composition of the active PI 3-kinase has remained elusive. It was known as a 85-kd phosphoprotein that co-imrnunoprecipitated with a 11O-kd companion protein. Now from purification and recent sequencing of p85 cDNA clones, it is clear that the 85 kd is not the kinase, but is rather a subunit that directs the association with the PDGF-R. It contains two SH2 and one SH3 regions (22, 61, 94). Specific antibodies directed against the 85 kd co-imrnunoprecipitate the PI-kinase activity together with the 110-kd protein that is expected to possess the kinase catalytic domain. How the association of the 85-kd subunit with the PDGF-R and its subsequent phosphorylation on tyrosine regulates the PI-kinase activity is still not understood. The role that this activated enzyme plays in the initiation of cell growth is unknown and remains a matter of speculation. Until recently the phosphorylation of phos phatidylinositol was thought to occur exclusively at positions 4 and 5 of the inositol moiety. The surprising discovery that the PI-kinase phosphorylates the 3 position (3) uncovered a new family of minor phosphoinositides that are not hydrolyzed by the various PLC isozymes described so far (79). The PI 3-phosphate as well as the relatives PI-3,4 P2 and PI-3,4,5 P3 are well conserved throughout evolution, thus pointing to a specific function. One possibility, suggested by the interaction of PIP2 with profilin, is that they could participate in the dynamics of the membrane-bound cytoskeletal ele ments that control cell shape and cell movement. These membrane-associated events, often disrupted after malignant transformation, are rapidly activated by growth factors. PHOSPHOLIPASE C-'Y Since the discovery that inositol lipid metabolism leads to the generation of important second messengers (diacylglycerol and Ca2i) (5, 60), almost all growth-promoting agents have been shown to activate this signaling pathway. However, its activation via G protein-coupled receptors is far more efficient than via receptor tyrosine kinases (42, 50, 64). In addition, activation of some receptors like insulin-R, IGFI-R, and CSFI-R have no apparent effect on PLC activation. EGF activates PLC mainly in cells that overexpress the receptors (44, 111), or like insulin and FGF, in cells where the PLC has been preactivated with an agonist (63). PDGF is unique in



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its capacity to induce by itself a significant fonnation of inositol-l,4,5 P3. The fact that a receptor tyrosine kinase could activate an effector system common to G protein-coupled receptors is intriguing. The paradox of this activation was rapidly resolved with the realization that each class of receptors activates different PLC isozymes (79). G protein-coupled receptors activate PLC f3 via Gq or members of the GIl subfamily (96, 100). In contrast, PDGF and EGF activate PLC-y, a soluble PLC isozyme that possesses SH2 domains required for proper interaction with tyrosine kinases. PDGF or EGF stimulate the phosphorylation of PLC-y on tyrosine, the association of PLC-y to their membrane receptors, and the production of IP3 and diacylglycerol (56, Il l ). Although most studies have shown that the properties of the PLC-y are not changed by tyrosine phosphorylation, one report suggests that tyrosine phosphorylation participates in its activation (59). However, the role of this pathway in PDGF- or EGF-induced mitogenic action is not clearly demon strated. For example, overexpression of the PLC-y in NIH-3T3 cells was associated with a higher rate of PI-turnover in response to PDGF, whereas the PDGF-induced mitogenic response remained unchanged (53). An interesting regulation implicating the small soluble actin-binding protein profilin has been reported. Profilin binds to PIP2 and inhibits the cleavage by PLC-y. Tyrosine phosphorylation of PLC-y releases this inhibition, which points to a possible key regulatory role of tyrosine phosphorylation (25). Although reinitiation of DNA synthesis can operate without the need to tum on PI breakdown, activation of PLC-y by PDGF and to some extent by EGF could contribute to reorganization of the cytoskeleton and promote membrane ruffl ing and cell movement. The ras proto-oncogene product is a 21-kd GTP-binding protein that plays a key role in the control of cell growth (4). It is hypothesized that growth factors switch the p21ras to the active GTP-bound state by at least two mechanisms: one by activation of a guanine nucleotide exchange factor, the second by inhibiting the rate of GTP hydrolysis. ras GAP (GTPase-activating protein) is a regulatory protein that stimulates the intrinsic GTPase activity of p21ras (101). Growth factors rapidly stimulate the ras�GTP/ras�GD P ratio, thus suggesting that either they activate the exchange or inhibit GAP or do both (86). In this context it is remarkable that in response to PDGF, ras GAP rapidly associates with the PDGF-R presumably via its two SH2 domains and becomes tyrosine phosphorylated (34). ras GAP is also phosphorylated in response to stimulation of cells with EGF or CSF-l and in cells transfonned by several oncogenes (21). Unfortunately, the physiological significance of this phosphorylation on the activity of GAP is still unknown. The tyrosine phosphorylated region of ras GAP could perhaps be involved in fonning secondary associations with proteins containing SH2 domains.

ras-GAP

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SWITCH KINASES, RELAYS FOR SIGNAL INTEGRATION-THE CASE OF MAP-KINASES

Reviewing the action of two potents growth factors, a-thrombin and PDGF, we have documented several major points: (a) reinitiation of DNA synthe sis can be operated through mechanistically separate transducing systems, and (b) Go to S-phase transition requires cooperation of mUltiple signaling pathways. Other growth factors, apparently less potent, need to be com bined to induce a strong mitogenic response, a situation that prevails in vivo. It is striking that the multiple cellular responses, referred to as the pleiotyp ic programs, are common to almost all growth factors. We believe that most of the cellular responses,including ionic changes (Na-H antiporter and Na/K/ CI cotransporter activation), increased protein synthesis (ribosomal protein S6 phosphorylation), and early gene transcription are initiated and maintained by phosphorylation changes occurring at serine and threonine residues. To account for the common activation of this pleiotypic program, we favor (75) the operation of switch kinases as relays for integration of extracellular signals. These kinases will pass the signals through a complex network of kinase-kinase cascades leading to activation of the multiple cellular targets. Such a picture for the mitogenic action of EGF is starting to emerge ( 1). Good candidates for switch kinases, defined as a serine-threonine kinase activated by tyrosine kinases and other types of transmembrane signals, are raf-I (76) and MAP-(mitogen-activated protein) kinases (82). Raf-I, the cellular homo logue of the oncogene v-raf, plays a determinant role in cell growth and differentiation. The kinase is ubiquitously expressed, activated by all growth factors, and when expressed in a constitutive form, it promotes proliferation (95). Conversely, its inhibition with raf-l antisense,or by expressing domi nant negative mutants, suppresses serum-induced proliferation of NIH-3T3 cells (41). These findings highlight its central role in the control of cell growth. However, its mechanism of activation, which was believed to be subsequent to its tyrosine phosphorylation,is still controversial (48),and the nature of the in vivo substrates remains to be determined. MAP-kinases p42mapk and p44mapk, also known as ERK2 and ERKI respec tively (7), are serine-threonine kinases ubiquitously expressed and homologous to yeast kinases KSSI and PUS3, which control Go events. These kinases,described earlier as pp41 and pp43 phosphorylated proteins in src-transformed and in mitogen-stimulated cells, are stimulated by a great variety of extracellular signals. These include activators of receptor tyrosine kinases (e.g. insulin, EGF, PDGF, FGF, NGF), protein kinase C (phorbol esters), and G protein-coupled receptors (a-thrombin, carbachol, serotonin) (7, 16, 4 0, 43, 82; S. Meloche, K. Seuwen, M. Cobb, J. Pouyssegur, in preparation). An important feature of the mitogenic response is the synergy



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between pathways. Often the synergy for reinitiation of DNA synthesis is mirrored early on by the activation of the initial events. We found that distinct pathways that synergize for DNA synthesis (FGF/thrombin and FGFI serotonin) (66, 90) also synergize in the activation of MAP-kinases,p42mapk and p44mapk (43; S. Meloche, K. Seuwen, M. Cobb, J. Pouyssegur, in preparation). These findings suggest an early point of convergence. The activation of these MAP-kinases was particularly appealing because it was demonstrated that a dual phosphorylation at tyrosine and threonine residues was required for full activation (2,69),which suggests that the MAP-kinases themselves are the site of converging signaling pathways. With the recent demonstration that phosphorylation at tyrosine and threonine residues is the result of an autophosphorylation (87; M. Weber, personal communication), the mechanism of activation appears more complex than orginally thought. It is tempting to propose that the pathway integrator is the MAP-kinases activa tor(s) defined recently by Krebs and co-workers (1), or is another element located upstream in the activating cascade. Work is in progress in several laboratories to elucidate the different steps of the activation as well as the role played by this new family of kinases in the control of cell growth and differentiation. ACKNOWLEDGMENTS

We thank Prof. Paul Pilch and Dr. Ellen Van Obberghen-Schilling for fruitful discussion and critical reading of the manuscript, and Martine Valetti for manuscript preparation. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the Association pour la Recherche contre Ie Cancer, and the Fondation pour la Recherche Medicale. Literature Cited 1. Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., Krebs, E. G. 1991. Multiple components in an epidermal growth factor-stimulated pro tein kinase cascade. J. Bioi. Chern. 266:4220:-27 2. Anderson, N. G., Maller, J. L., Tonks, N. K. , Sturgill, T. W. 1990. Require ment for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343:651-53 3. Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., Cantley, L. C. 1989. PDGF-dependent tyrosine phosphoryla tion stimulates production of novel polyphosphoinositides in intact cells. Cell 57:167-75 4. Barbacid, M. 1987. ras genes. A nnu. Rev. Biochem. 56:779--827

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