Deciphering the MAP kinase pathway

Pr,,~,cs., m Gn,wth

Pergamon

DECIPHERING

THE MAP

Factor Reseunh, Vol. 5, pp. 29 l-334. IYYJ Copyright 63 1994 Elm&r Science Ltd Printed in Great Britain All rights reserved OY55- x35/94 %2h 00

KINASE

PATHWAY

Gilles L’Allemain* Centre de Biochimie, University of Nice. 06108 Nice, France

MAP kinuses (MAPK) are serine/threonine kinuses which are activated by u dual phosphorylation on threonine and tyrosine residues. Their specific upstream activators, called MAP kinuse kinases (MAPKK). constitute u new family of duul-spec@ threonine/tyrosine kinases, which in turn are activated by upstream MAP kinuse kinase kinuses (MAPKKK). These three kinuse families are successively stimulated in a cascade of activation described in various species such as mammals. ,frog, fly, worm or yeast. In mammals. the MAP kinase module lies on the signaling puthway triggered b? numerous ugonists such us growth factors, hormones, lymphokines, tumor promoters, .stress factors. etc. Targets of MAP kinuse have been characterized in all subcellulur compartments. In yeast, genetic epistusis helped to characterize the presence of several MAP kinuse modules in the same system. By complementution tests. the relationships existing between phylogenetically distant members of each kinuse family huve been described. The roles of the MAP kinase cascade huve been analyzed by engineering various mutations in the kinuses of the module. The MAP kinase cascade has thus been implicuted in higher eukuryotes in cell growth, cell fate and differentiution. and in low eukuryotes. in conjugation, osmotic stress, cell wall construct und mitosis.

Keywords: Cell signaling, transcription

MAP kinase cascade, oncogenes, factors, complementation test.

nuclear

localization,

INTRODUCTION Historically, Ray and Sturgill first purified two proteins of 42 kDa and 44 kDa which were phosphorylated on tyrosine and threonine residues when 3T3-Ll adipocytes were challenged by insulin [l, 21. Then, Weber and Sturgill groups showed [3] that those proteins corresponded in fact to the growth factor-induced tyrosine phosphorylated proteins visualized several years before by bi-dimensional electrophoreses [4-61. *Address for correspondence: Centre de Biochimie. Part Valrose. 06108 Nice Cedex 02. France Acknowledgemmrs-Dr J Pouysstgur is sincerely thanked for critical reading of the manuscript. The author gratefully acknowledges all contributors from this laboratory and Drs M. Baccarini. 0. Bensaude. P. Cohen. M. Dunn, S. Grinstein, G. Johnson. C. Marshall, S. Meloche, S. Noselli, R. Seger and J. TavarC for communicating results before publication. The skillful secretarial assistance of Mrs Martine Valetti was highly appreciated.

291

The protein was named MAP kinase [I ,7] after its rather specific phosphorylating activity tovvards MAP-?. the brain 2/licrotubulc-Associated Protein 2. Thus, because various laboratories were workin,? at the sametime on the same protein in different cell systems, the same protein entity was given the names of: MBP kinase [7. 81, after another exogenous substrate. the myelin basic protein or MBP; MPK after a sea star meiosis-activated protein kinase [9]; RSK kinase [lo], after the endogenous substrate RSK, the 90 kDa ribosomal S6 kinase. Becauseit rapidly turned out that numerous agonists were able to stimulate MAP kinase activity. Sturgill and Wcbcr proposed that the same name of MAP kinase could stand for Mitogcn-Activated Protein kinasc. When the protein was first idcntiticd by cDNA cloning by M. Cobb ct ul. [I I], the gene was given the name of ERK for Extra-cellularly Regulated Kinase and numbered to take into account the different isoforms subsequently cloned (ERKI for the p44m3rk,ERK2 for the p4ZmZ’““,ERK3 for the p63”“‘Ph,etc.). The same story started again with the upstream activators of MAP kinase since the direct MAP kinase activator was first named MAPKK for MAP kinase kinase when purified from ,Ymopus oocytes ]I?], then, when cloned, it was given the name of MEK for Mitogcnically Extraregulated Kinase in an attempt to reconcile the two acronyms MAP and ERK [13]. Recently, one activator of this MEK (alias MAPKK) was cloned [14] and named MEKK for MEK kinase or MAPKKK for MAP kinase kinase kinase. The two other MEK activators described so far wcrc alrcady known as being the serineithreonine kinasesc-Raf and c-Mos (seesection on MAP kinase kinase kinasc family). Today. the generic names of MAP kinase, MAPKK and MAPKKK are used to designate any of the members in each i‘amily. The acronyms ERK, MEK and MEKK are numbered to help to identify a particular isozyme. THE MAP KINASE FAMILY Based on protein sequcncc hom~~logy. closely related members of the MAP kinase family have been described from yeast to man. In addition to the kinase subdomains present in all the serine/threonine protein kinases (see [15] for a review), most MAP kinase family members exhibit the two same regulatory phosphorylation sites: the amino acids threonine and tyrosine found in the -Thr-X-Tyrmotif [16] localized between the kinasic sub-domains VI1 and VIII (see Fig. 1).

*I&1------DSIAN-OLI---.--DSIAN-- l-l

MAPKK (hamster) MEKl (mouse) MEK2 ixenopbs) Byr 1 (S. pot-de) STE 7 (S. cerevisiae) Hemipterous (Drosophil MKKl (S. cerevisiae) PBS2 (S. cerevisiae) Wis 1 ( S. pornbe) MEKK (mouse) Byr 2 (S. pombe) MAPK (hamster)

IA-------DPEHTGP

SubdomaIn

FiGL‘RE

I.

Sequences

alignments

VII

of YblAPK

and

M/il’Kk.

Deciphering

the MAP

,793

Kinase Pathway

The rapidly expanding family of MAP kinases in animal species as different as mammal, yeast, fly, frog or worm indicates how ubiquitous are these proteins, the sequence homology at the amino acid level indicating a high degree of conservation during evolution. Mammalian

MAP

Kinases

Today no less than 5 mammalian isoforms of MAP kinases have been isolated: p4@“pk/Erkl, p42”“pk/Erk2, p63”“pk/Erk3, p46”“pk/Erk4, and p54”“pk. Among them, cell system p42mapk and ~44 mapk have been characterized in every mammalian checked so far; this is certainly the reason why these two proteins have been the most studied MAP kinase subtypes. Their expression pattern in tissues is very broad with the brain containing the larger amounts. MAP kinases are activated by different families of ligands (see Fig. 2), providing the presence of the corresponding receptors on the cell surface including: (9 (ii)

(iii)

serum growth factors, EGF, PDGF, FGF, IGF-I, HGF (see 1171for a review) or CSFl [18]; hormones like NGF, insulin, bradykinin, progesterone, methyl-adenine (see [17] for a review) but also thrombin [ 191, carbachol ([20] and G. L’Allemain. unpublished data), angiotensin [21], growth hormone GH [22,23], endothelin 1 [24], thyrotropin-releasing hormone (TRH) [25], neuropeptide PACAP [26] and the alpha,-adrenergic receptor agonists [27]: lymphokine family members such as: the interleukin-3 [28,29], interleukin-5 [28], the Steel factor (SLF) [28, 291, the granulocyte/macrophage colony stimulating factor (GM-CSF) [28,29], the interleukins -6 and -11 [30] and the two related cytokines, leukemia inhibitory factor (LIF) and oncostatin M [30, 3 I]: the inflammatory cytokine interleukin-1 [32]; the novel cytokine interleukin12 [33], which is secreted by macrophages and B lymphocytes, and acts on natural killer cells (NK) and T lymphocytes.

The MAP kinases have also been found activated by other kinds of agonists (see Fig. 2) such as: (9 (ii) (iii) (iv)

(v) (vi) (vii)

secretagogues such as nicotine or PGF, alpha { 171, thromboxane A2/prostaglandin H2 [34] and prostaglandin E2 (Y.-Z. Wang, personal communication); chemotactic factors like Net-Leu-Phe on neutrophils [35] or C5a. interleukin-8 and leukotrien B4 on leukocytes [36]; lipids like lysophosphatidic acid LPA [37] or platelet activating factor PAF [38]; tumor promoters such as phorbol esters [17], the protein kinase C (PKC) activators or thapsigargin [39], a calcium mobilizer which acts via the inhibition of the Ca’+/ATP-ase; surface immunoglobulins [40, 411 like anti-IgM antibodies [42]; phosphatase inhibitors like okadaic acid and sodium vanadate [17]; stress conditions were also described as inducing MAP kinase activity, such as mechanical stretch [43], UV radiation [44], heat shock [45], or even under electroconvulsive treatment [26].

G. L ‘Allemain

294

EGF.. .......................................(3, 8, 17, 115, 130, 133) FGF ................................. (17,19,127) PDGF ....................................(3, 17,115,135) IGF-1 .......................................(17, 114) HGF .........................................(17) CSF-1 ......................................(18) NGF .........................................(17,47,125,128,181,188 ) Insulin .....................................(1, 2, 11, 17, 47, 163) Anglotensin

II .........................(21)

Bradyklnin

..............................(17 )

Thrombin .................................(19, 123, 124) Endothelin-1

.............................(24)

TRH ..........................................(25 ) Growth hormone.. ................... (23 ) PACAP 38 ................................(28) Carbachol .................................(20, 99 ) IL-1 ...........................................(32 ) IL-3 , IL-5 ................................ (28, 29 ) GMCBF, SLF .......................... (28, 29 ) 11-8, IL-11, LIF, OSM ............... (30 , 31) (38 ) IL-8 ............................................ IL-12 .........................................(33) UV radlation ............. ................. (44) Heat shock ............................... (45) Mechanical stretch .................. (43) Eiectroconvulsive treatment .. (26)

secretagogues ....................... (17,34) Chemotacttc factors ............... (35) Lipids ........................................(37, 38, 98, 100 ) Tumor promoters ..................... (3, 17, 39, 94, 227 ) lmmunoglobullns ......................(40, 41, 42 )

FIGURE

2. Agonists

of MAP

kitme.

From sequence alignment methods, it has been established that the sequence homology between the two p42 mapkand p44mapk isoforms is very high but anyway lower than the one existing for one isoform between different mammalian species [46]. A very particular member of the MAP kinase family is ERK3. The amino acid sequence deduced from cloning a predicted protein of 63 kDa with a C-terminal

Deciphering

the MAP

Kinuse Pathway

295

extension of approximately 180 amino acids compared to p42mapk and p44mapk [47]. Truly, among all mammalian homologs, only Erk3 has: a highest expression level early in development and a very restricted expression pattern (mainly detected in neuronal cells and in muscle); a different substrate specificity (no phosphorylating activity towards MAP2 protein or MBP, the myelin basic protein); a limited sequence homology (only 4&45%, identical to Erkl); finally, a completely different regulatory site sequence with -SEG- instead of -TEY- between the kinasic consensus D-F-G of the subdomain VII and AS-P-E of the subdomain VIII [47]. The p54”“P”. first characterized in 1991 from cycloheximide-treated rat liver cells. was finally sequenced in 1994 1481. PCR cloning from a rat brain cDNA library allowed the isolation of independent clones encoding very homologous proteins [48]. From this remarkable study it appears that the ~54 MAP kinases: only exhibit an overall 40--45x homology with the p42/p44 MAP kinases (less than the identity between the mammalian p42/p44 with the yeast MAP kinases); possess the two same regulatory sites of the MAP kinase family. but separated by a different amino acid (the consensus TEY of the p42/p44 family is replaced by TPY in the p54m”r’a sequence); and are weakly (or not) activated by phorbol esters and growth factors. By contrast, this new MAP kinase subfamily is strongly stimulated by TNF alpha and stress factors (heat shock, etc.). For all these reasons, the ~54 family constitutes a particular MAP kinasc subfamily named SAPK for stress-activated protein kinases [48]. Today, another isoform, the ~45 Erk4 protein [47], remains poorly characterized. Non-mummuliun MAP Kinaw

Homologs of the MAP kinase family are also found in animal species as different as frog, clam, sea star, fly. worm or yeast (see Figs 3 and 4). The frog (Xenopus luevis), clam and sea star oocytes appear to express only one isoform: a p42”“pk in Xenopus [49] or in clams [50] and a p44mapk in sea star [51]. These three subtypes are activated during hormone-induced oocyte maturation [49 511 at the G2/M border of the oocyte meiotic cell cycle. A study comparing biochemical characteristics of the mammalian MAP kinases with invertebrate isoforms showed that they are indeed closely related [52]. In yeast, two major cell systems are studied: the budding yeast Succhuromyces cxm+siue (S. cerrv.) from which several MAP kinase-like proteins have been characterized like KSSl, FUS3, MPKl or HOG1 (see Fig. 3 and [53] for a review), and the fission yeast Schizosuccharomycespombe (S. pombe) in which only one isotype has been cloned so far, the spkl gene product which is highly similar (~50% identity) to KSSl and FUS3, but also to ERKl [54]. It is interesting to note that the MAP kinase homologs in budding yeast lie on parallel kinase cascade pathways (see [53] and Fig. 3): (9

(ii)

KSSl and FUS3 are involved in the mating pathway induced by pheromones [55,56]. The KSSl gene, when overexpressed, suppresses the Gl-specific cell cycle arrest induced by the mating pheromone alpha factor [55]. The overexpression of FUS3 increases the pheromone sensitivity whereas its absence causes sterility [56]; MPKl is implicated in a PKC mediated signaling pathway [57];

296

G. L ‘Allemain

Saccharomyces

cerevisiae

i. pombe

Mammals

am2,mam3 c QPQ 4 ‘?

t Osmotic stress

FIGURE

(iii)

3. Phylogenic

cell

t

t wall

Motlng

Mating

Mltorls

Melorls

Prollferatlon

conservation

from

yeast to man. Homologous

proteins

in the modules

are aligned.

HOG1 (for High Osmolarity Glycerol) is activated under production of internal glycerol [%I, which is an adaptive response of the yeast to high external osmolarity conditions.

A striking feature of all these yeast sequences is that they exhibit the two same regulatory sites as in mammalian sequences [59], suggesting the same mechanism for enzymatic activation. A Drosophila melanogaster homolog of Erk was cloned and named DmERK-A [60]. Because its tyrosine phosphorylation is enhanced in the torso pathway, it could play a role in the differentiation process of the anterior/posterior axis. Very recently, a new gain-of-function mutation in drosophila allowed to ubiquitously implicate MAP kinase in 3 different signaling pathways; the sevenless, torso and EGF pathways, respectively implicated in cell differentiation, cell fate and cell growth [61]. A MAP kinase homolog called Surl [62] or mpkl [63] has recently been cloned in

297

Deciphering the MAP Kinase Pathway

the worm species Caenorhabdiris elegans from loss-of-function mutations which provoked defects in Ras-mediated vulva1 ceil fate [62, 63]. Further, a MAP kinase homolog has also been isolated in the plant kingdom [64]. Today, more than 15 members of the MAP kinase family have already been discovered in eukaryotes and there are certainly more to come. The very remarkable conservation of regulatory sites through most, if not all, MAP kinase sequences suggests strongly that all MAP kinase members derived from an identical ancestral gene. THE MAP KINASE

KINASE

FAMILY

In 1992, a MAP kinase activator was purified from various cell systems (see [65] for a review) and the cDNA sequence that appeared the same year [13, 661 revealed a close homology to a family of yeast genes identified after genetic studies and directly acting upstream of the yeast MAP kinases. Mammalian

MAP Kinase Kinases

Purification of MAPKK was performed the same year by different laboratories working with various mammalian cell systems (see [65] for a review). The first mammalian MAPKK genes to be cloned were either from mouse 3T3 cells and called MEKl for MAP and ZiRK kinase [13] or from human T-cells [66]. The expression pattern showed a broad tissue distribution with higher amounts in brain and muscle [ 13, 661. In 1993, another MEK gene, MEK2, was cloned and shown to exhibit 80% identity with the MEKl gene [67,68]. A comparison of these two MEK sequences indicates that they are very close family members (>80% identical) as were ERKl and ERK2 in the MAP kinase family, with two significant differences in a proline-rich region located between the kinasic subdomains IX and X and in the N-terminus portion of the protein [67,68]. It is not known if these variable regions are responsible for MEK2 exhibiting a higher activity than MEKl, as recently shown [69]. A third mammalian gene called MEK3 has been isolated [66,69] and shown to be inactive towards ERKl and ERK2 [69]; it could, in fact, simply be an alternative splicing of MEKl. eon-mammaiian

MAP

Kinase Kinases

Following the first purification of a vertebrate MAPKK [12] and its cloning [70] in frog oocytes. two additional members of the MAP kinase kinase family in Xenopus laevis have been cloned by a polymerase chain reaction-based analysis of embryonic cDNAs and called XMEK2 and XMEK3 [71]. Although XMEK2 and XMEK3 exhibit a high degree (4565%) of sequence similarity with XMEKl, the first MAPKK cloned in any system [70], each isoform displays a unique pattern of expression [70,7 11. Such different properties suggest that parallel signaling pathways exist in frog, as in yeast. In yeast, several protein kinases (byrl in S. pombe, STE7, PBS2 and WISl in budding yeast) were already defined by genetic epistasis as acting upstream of yeast MAP kinases (see Fig. 3 and [53] for a review), when the first mammalian sequence

298

G. L’Allemain

was published [13]. Whereas byrl and STE7 are in the mating pathway, and PBS2 (Polymixin B Sensitivity) in the osmo-regulation signaling (see Fig. 3), the fission yeast kinase WISl has no upstream or downstream partners as yet characterized in the MAP kinase cascade. Since then, two others yeast MAPKKs (MKKl and MKK2 for MAP Kinase Kinase) have been isolated in the PKCl (for Protein Kinase C) pathway of budding yeast [72] which is essential to normal cell growth and division. These two redundant MAPKK homolog functions are downstream from the BCKl (for By-pass of C Kinase) gene product but upstream from the MAP kinase homolog. MPKl (see Fig. 3). The kinase domain of MKKl is 80% identical to MKK2, but only 40% homolog to those of STE7, byrl or MEKl [72]. Because of their location in the growth signaling, MKKl and MKK2 could well represent the yeast equivalent of mammalian MEKl and MEK2. By genetic screening of dominant mutants able to suppress the eye phenotype induced by D-Raf during Drosophila development, a MAPKK Drosophila homolog, Dsorl, was cloned and sequenced [73]. As expected, the Dsorl mutants were also dominant suppressor for genes acting upstream of D-Raf. The kinase domain of Dsorl is 75% identical to all mammalian MAPKK sequences identified so far and only 50% identical to those of yeast MAPKK (see [74] for a review). Recently, a new Drosophila gene called hemipterous has been found to encode a MEK-like gene and to be implicated in the cell fate (S. Noselli, personal communication). Truly, mutations in this gene result in dorsal closure defects of embryos. Altogether, the number of MAP kinase kinases is today almost as large as the MAP kinase family. The phylogenetic tree which can be established from cDNA sequences suggests that all members have diverged from only one ancestral gene. THE MAP KINASE

KINASE

KINASE

FAMILY

If the MAP kinase kinase protein seems to be the converging point of numerous signals, the upstream MAPKKK is represented by different families of protein kinases. Mammalian

MAP Kinase Kinase Kinases

The first mammalian MAPKKK was characterized in 1992. Both in viva and in vitro studies converged to demonstrate unambiguously that the proto-oncogene c-raf, which displays serine/threonine kinase activity, was responsible in activating directly the MAPKK (i)

(ii)

proteins [74-811. They include use of:

the yeast double hybrid system which allows, via the activation of its transcription system, the unraveling of new protein-protein interactions. Van Aelst et al. [75] established that a tight complex existed between MEK and Raf and, more precisely, by using various deletion mutants, that such an interaction is done via the catalytic (C-terminus) domain of Raf; mammalian cell systems which showed, when oncogenic forms of Raf are expressed, either a constitutive activation of MAPKK [74,76], or a modulated activation of MAPKK when Raf-1 is placed under the control of a conditional promoter [8 11;

299

Deciphering the MAP Kinase Pathway

(iii)

a whole set of in vitro kinase assays with endogenous, baculovirus expressed proteins [74,76-801.

recombinant

or

With such a parallelism in MAP kinase cascade pathways, both in yeast and in vertebrates, investigators started to look for mammalian equivalents of another yeast family of protein kinases located, by genetic epistasis analysis, upstream of MAPKK (see [53] and Fig. 3). Truly, the cloning of the very first mammalian MEKK (for MEK Kinase) was done in 1993 [14] by polymerase chain reaction using degenerated oligonucleotides derived from byr2, the fission yeast kinase upstream of byrl (see Fig. 3). Today, the sequencing of other MEKK isoforms exhibiting a different expression pattern is under completion (G. Johnson, personal communication). eon-rn~rnrn~~iun

MAP Kinase Kinase Kinases

Originally

discovered in germinal frog cells, the product of the proto-oncogene a particular case: it encodes a serine/threonine protein kinase whose activity is stimulated when oocytes are induced for maturation by progesterone. Mos synthesis is required in frog oocytes to render the Maturing Promoting Factor (MPF) complex active and to initiate egg maturation (see [82] for a review). Recent evidence shows that in Xenopus oocytes Mos is able to activate MAPK but also MAPKK in vitro [83-861, then playing the role of a MAPKKK in sexual cells. One can also envisage a divergent route for MAPKK activation as suggested in another study on CSF (cytostatic factor) activity of c-mos in Xenopus embryos [87]. CSF acts normally to arrest oocytes at metaphase of meiosis II until sperm arrival. Microinjection of Mos induces metaphase arrest, indicating a CSF activity for Mos. Truly, c-mos is a component of CSF which induces metaphase arrest via the activation of endogeneous MAP kinase. But in meiosis I, MAP kinase is active and no CSF-induced arrest occurs. So Mos is not the only component of CSF; another companion, as proposed by the authors [87], could be the cdk2 protein kinase which is required for arrest at meiosis II. Today, the idea that the presence of Mos or Raf is mutually exclusive (i.e. Mos playing in germinal cells the role of Raf in somatic cells) has been definitively excluded with the following results: Mos has also been implicated in the MAP kinase activating pathway of somatic cells [85,86]; a Xenopus form of Raf has also been cloned from oocytes [88]; Mos could act upstream of Raf because the blocking effect of antisense Mos oligonucleotides was overcome by co-injection with a human v-raf[89]. Therefore, it appears that not only the MAP kinase pathway in frogs is as complete as in mammalians (with Mos playing the role of MEKK), but the MAP kinase cascade could well diverge upstream of the MAPKK (see Fig. 4). Genetic epistasis in yeast indicated that at least three protein kinases belong to the MAPKKK sub-family (see Fig. 3); STEll and BCKl in budding yeast, and byr2 in fission yeast: c-mos constitutes

(i) (ii)

STEll is a mating-type specific gene which causes arrest of the mitotic cell cycle [90]; the BCKl (By-pass of C Kinase) gene product has been characterized as able to suppress the loss of PKCI function [72] and shown to exhibit almost 50% identity with STEl 1 and byr2 [53];

300

t y r 0 s ‘1 n a kinose r’ecepkolrs

c

4 CRB

Drk

?

i SOS

+ SOS1 (SOS2

i RPSl

4 p2lras

I

c D-raf

XeRaf,Mos + XeMEKlA3

4 Dsorl

4 HEKK,Raf,Mos w WEKl,WEKZ

c

I

DmErkR,rolled

Surl, mpkl

p42mapk

ERKl,

I

(iii)

4.

Ubiquity

ERK2

J

Cell Fate FlGURE

2

Differentiation

of the signaling

molecules

Meiosis

Cell

growth

in Metazoans.

byd was originally characterized as a protein kinase involved in the mating pathway of S. pombe (see Fig. 2) and able to suppress partially the Rasl mutation (91). Its sequence shows 60% homology with STEll [53].

In invertebrates, Raf analogs (see Fig. 4) are crucial for development: in the fly D. melangaster, a Raf-like gene called Drafparticipates in torso function [92]; in the worm Caenorhabditis elegans, the lin-45 gene product is a Raf-like protein implicated in vulva1 differentiation [93]. Finally, it appears that the whole MAP kinase cascade could be present in all living cells since a member of the Raf family of protein kinases, called CTRl, has recently been discovered in plants [94]. Another example is a member of the second family of MAPKKK, the NPKl gene, which has recently been cloned from tobacco [95]. With so many MAPKs and MAPKKs present in the MAP kinase module of various species, one can expect that the family of MAP kinase kinase kinases is going to expand rapidly.

Deciphering the MAP Kinuse Pathwa)

GOING The plasma during brates

UPSTREAM

IN THE MAP KINASE

CASCADE

gap existing in numerous signaling pathways between the cell receptor at the membrane level and the MAP kinase cascade has been completely filled the past year with a series of exciting results both in vertebrates, in inverteand in yeast (see Figs 3 and 4, and [96,97] for reviews). Coupling to the Receptor

Briefly, the signal starting from two types of surface receptors-seven transmembrane domain receptor and tyrosine kinase receptor-will activate the protein Ras [96] through different effector molecules: (i)

(ii)

the first receptor family is coupled to hetero-trimeric G proteins. If various studies [37, 9881001 indicated that activation of the Ras protein could be performed through G protein-coupled receptors, just a few originally identified the alpha subunit of the G protein as being the possible interacting molecule: G,,,,$ [loll, or Galphas and Galphaq [102]. In a different cell context, the beta/gamma subunits of the G proteins were specifically involved in the Rasmediated MAP kinase activation [102,103]. Transmission of the signal from beta/gamma to Ras could then be realized through binding of the beta/gamma dimers to the pleckstrin homology domain existing in several Ras-interacting molecules (see [104] for a review) as in GRF, the GDP releasing factor, in SOS. the GDP to GTP exchange factor, or in GAP, the GTP-ase activating protein (see Fig. 5a); the tyrosine kinase receptors will complex an adaptor protein called GRB2 in mammals, sem5 in C. eleguns or Drk in Drosophilu (see Fig. 4) via the interaction between the tyrosine phosphorylation motif of the receptor, a consequence of its intrinsic kinase activity, and the SH2 domains present on the adaptor protein [96,97]. This protein will use its SH3 domains to associate with the nucleotide exchange factor SOS which, in turn, will stimulate the Ras protein directly by acceleration of the exchange GDP+GTP onto Ras (see Figure 5a). Ras-Ruf

Interaction

In 1992, several results reported that Ras was able to activate Raf-I [IO51 but also MAPK when the oncogenic form of Ras was introduced in vertebrate cells [ 105,106]. But it is only recently that a remarkable array of independent studies have converged to clearly establish the direct RasRaf association: the double hybrid system showed that such an interaction takes place between the N-terminal (regulatory) portions of Raf or byr2 with the Ras effector domain [75,107]; in vitro reconstitution assays using recombinant Raf constructs [IOS] or Ras-affinity columns [77]; co-immunoprecipitation of Rafl with Ras antibodies [109]. The MAPKK protein which is found in Ras-Raf complexes is still present after Raf deprivation [77], suggesting the presence of other components in these complexes. This result is consistent with the presence of larger complexes containing Ras [1 lo] or Raf [l 1 l], Significantly, the Ras-Raf interaction has been demonstrated in intact mammalian cells, even if only up to 3% are found associated [112].

302

G. L ‘Allemain

As a consequence of RasRaf being upstream of the MAPK cascade, several laboratories demonstrated that in some examples MAPK activation is inhibited by expression of dominant-negative forms of Ras [37,113] or Raf [98]. Nevertheless, this effect cannot be generalized for all growth factor signaling pathways since first, a dominant-negative Raf mutant does not inhibit the EGF- or TPA-induced MAPK activation although the IGFl-induced stimulation is totally abolished [I 141, second, a dominant-negative form of Ras does not block the EGF-induced ERK2 phosphorylation [115], and third, in the baculovirus insect cell system, a dominant-negative mutant of Raf-1 was described as being unable to block the activation of MAPK by v-rus, suggesting that v-r-us could activate the MAPK in a Rafl-dependent and in a Rafl-independent pathway [80]. Altogether, these results, along with the presence of a different MAPKKK family [14], converged to demonstrate that the linear cascade of kinasic activation is interrupted at the MAPKK level (see Fig. 5). A Parallel with the Yeast System

In budding yeast, complementation experiments allowed description of at least two other genes upstream of the MAPK cascade components, namely STE5 and STE20, which act between the products of GPAl, STE4 and STEl8 (yeast equivalents of the alpha, beta, gamma subunits of the mammalian heterotrimeric G proteins) and the STEll gene product (see [53] and Fig 3). STE 20 encodes a serine/threonine protein kinase [53] and has a mammalian equivalent activated surprisingly by two Ras-related proteins which participate in cytoskeletal organization, Racl and CDC42 [116]. STE5 has no apparent activity but exhibits some significant homology [I 171 with FARl, a direct target of the yeast MAP kinase FUS3 (see section on MAP kinase targets). In this yeast system, Ras has only been involved in the regulation of adenylate cyclase [53]. By contrast, both Rasl and GPAl genes have been implicated in the mating/meiosis pathway of fission yeast S. pombe (see [53] and Fig. 3). Role

qf Src and Src-related Family Members

Two studies on the role of the cytoplasmic tyrosine kinase Src in the Ras-+Raf+ MAPK activation module used baculovirus-expressed proteins to indicate that: both Ras and Src are necessary to fully activate Raf [118]; 111) v-src which activates Rafl as efficiently as v-rug, cannot strongly induce ERKl activity as v-rus does [go], indicating that Ras activates Rafl in a way different from Src. This last result suggests that tyrosine phosphorylation of Rafl is not the activation mechanism responsible for the Rafl-mediated ERKl stimulation; (iii) in Rat1 cells, Src induced constitutive activation of MAP kinase, but not Ras or Raf [99]. A study from Johnson’s lab [119] indicates that MEK is activated by v-src only in cell lines where Src activates MAPK, as expected. Sensitivity to v-us or v-ruf followed the same rule of cell specificity [119]. Altogether, these data establish that signaling routes leading to MAP kinase activation depend on the cell context and diverge upstream of MEK.

303

Deciphering the MAP Kinuse Pathway

,,\I\

Serpentine receptors

&&

The Ras activating

RAS 58-;.

111)

pathways.

RAF

-b

MEK

-b

MAPK

Putative targets of Src and Src family members in the MAPK pathway.

RAF

RAS II)

?iI “I’ X -b

Pl$

--

? -+

MEK-

MAPK

MEKK

m FIGURE 5. dashed arrows

Central posltfonlng of PKC in the MAP kinase pathway. Interconnecting are for putative

networks upstream of MAP kioase. Solid interaction. (X) is for possible intermediary

arrows are for direct molecules.

activation,

Less is known on the exact signaling leading to MAP kinase activation in cells of hematopoietic origin which express various cytokine receptors. The tyrosine kinase activity of this receptor family is generally brought about by the Src family proteins Blk. Fyn. Lck or Lyn. Truly, those tyrosine kinases, which play in lymphocyte cells the role of early mediators of the signal issued from the antigen receptor, have been implicated as regulators of the MAP kinase cascade. These studies include:

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(ii)

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the in vivo regulation of MAP kinases by the CD4 surface antigen which requires functional Lck in a T lymphoma cell line [120]. In addition, murine baculovirus-expressed ~56’“~ was shown to directly phosphorylate and activate in vitro the sea star MAP kinase [120]; the in vitro association of Blk, Lyn and to a lesser extent Fyn with MAPK in B cells [121]. Similar quantitative differences were observed when fibroblastic lysates were analyzed for their capacity to exhibit MAPK-Src family kinases association, indicating that the kinase structure, not the cellular context, is responsible for the association.

The way Src family members regulate downstream molecules is certainly via their SH2 and SH3 domains which are involved in the recognition of. respectively, the phosphotyrosine motif and the proline-rich regions [121]. But altogether, if this series of results suggests the presence of a second branched pathway starting at the Raf level (see Fig. 5b), after the first one issued from the converging point constituted by the MAPKK protein (see section on MAP kinase kinase family and Fig, 4), they do not establish unambiguously the exact positioning of Src in the signaling pathway (see Fig. 5b). MAP KINASE

REGULATION

Not all MAPK isoforms are activated at the same phase of the cell cycle. Depending on the naturally occurring cell cycle arrest, the stimulation of MAP kinases will take place at the G, phase in germinal cells or at the G,+G, transition in somatic cells (see [122] for a review). Activation of MAP kinase in oocytes is a transient one-time event peaking after 15 min, which occurs at meiosis I only [50], and returning rapidly to basal levels. By contrast, the kinetics of MAP kinase activation in somatic cells appear to be bi-phasic: first, a very transient peak at 5-10 min and, second, an overshoot which slowly decreases to reach negligible values after several hours [123]. Recent data (S. Meloche, personal communication) indicate that releasing cells from various blocks in G, induces p42 and p44 MAP kinase activities which reach basal levels as cells enter into S phase. Interestingly, a negative regulatory role for serine phosphorylation on MAP kinase is also proposed (S. Meloche, unpublished results). An interesting feature concerning this kinetics of MAPK activation our laboratory has been strongly implicated in, was about the role of the second wave of activation in mitogenicity. Truly, although all agonists trigger the rapid first phase of activation (carbachol, serotonin, thrombin peptides, TPA), only those which are pure mitogens (FGF, thrombin, PDGF, EGF) are able to produce the sustained activation [20,124, and other unpublished data from our lab]. These results suggest that the maintaining of the MAP kinase activation appears to be required for cell growth. This sustained activation has also been found to be required in the differentiation process of the phaechromocytoma cell line PC12: (i)

data from Cohen’s lab demonstrate [125] that both kinetics of MAPK and MAPKK activation are sustained after 1 h when cells are challenged with the differentiating agent NGF whereas both activities are rapidly down-regulated after challenging with the growth promoting agonist EGF. Immunolo-

Deciphering the MAP Kinase Pathway

(ii)

305

calizations performed in parallel demonstrated that only NGF and not EGF is able to translocate MAP kinase into the nucleus, indicating that maintaining of the MAPK activation is required for a nuclear presence of MAPK where it will have access to its potential gene transcription targets (see section on MAP kinase targets); a different laboratory confirmed [126] these differential effects of NGF and EGF in PC12 cells and extended the study to the activation of p90rsk, a MAPK physiological substrate (again, see section on MAP kinase targets). Other pieces of data indicated that bFGF exhibits the same transient kinetics of MAPK activation than EGF in PC12 cells and that both NGF and FGF are able to reduce the number of high affinity EGF binding sites [127].

Aside from the physiological activation of MAP kinase by MAPK kinase (see section on MAP kinase kinase family), the MAP kinase activity was found to be modulated by other kinasic or phosphatasic activities. Role of Protein Kinase C

Studies involving the phorbol ester tetradecanoyl phorbol acetate (TPA) and derivatives started with the intriguing observations of Vila and Weber [S] who showed that an acute treatment with TPA, a direct activator of the serine/threonine protein kinase C (PKC), was sufficient to stimulate the tyrosine phosphorylation of a 42-kDa protein which is known today as p42 MAP kinase. This result indicated that the PKC effect on the MAP kinases could not be direct. It has been known for some time that depending on the agonist, the stimulation of MAP kinase can take place through various ratios of PKC-sensitive and PKCinsensitive pathways (see [59] for a review), depending on the cell system [ 1151. Leevers and Marshall [105] established that the constitutive activation of ERK2 in Swiss 3T3 cells transfected with oncogenic ras is almost totally PKC-dependent. a result which suggests that PKC is downstream of the Ras pathway (see Fig. 5~). Alternatively, the TPA-stimulated PKC was shown to directly activate the Raf-I protein kinase [ 1281. Two independent studies [80,114] demonstrated that a dominant-negative mutant of Rafl cannot block the activation of MAP kinase either by v-ras (801, or by EGF and a TPA analog [114], thus indicating the existence of both Rafl-dependent and Rafl-independent pathways for activating MAP kinase cascade. Whether PKC can also activate directly the MEK protein is not known yet. Alternatively, one can also envisage that PKC activates MEK through the activation of a Byr2-like mammalian MEKK protein (see Fig. 5c), as suggested by complementation experiments [225] showing that the BCKl yeast gene product can be replaced by mouse MEKK along the budding yeast MAP kinase module PKCI-+BCKl-+MKKI+ MPK I (see Fig. 3). Exciting results on cloning of an intracellular receptor for protein kinase C bring the final touch to the way PKC could be itself activated from the growth factor receptor, since this new receptor shows homology to the beta subunit of heterotrimeric G proteins [129]. Altogether, these results contributed to our proposed positioning of protein kinase C (PKC) in the mitogenic signaling pathway (see Fig. 5~).

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Also, the question of which PKC isotype (and with which specificity) is capable of relaying the MAP kinase activation remains uncompletely solved even if unpublished observations (J. Tavare, personal communication) indicate that TPA-induced MAP kinase activation is enhanced in cells stably transfected with the PKC isotypes alpha, beta or gamma but not with the epsilon isoform. Role oj’protein

Kinase A

Apart from the thyrocyte system on which addition on thyrotropin increases CAMP levels and initiates DNA synthesis without any MAP kinase stimulation [130], all the results presented so far indicate that CAMP variations will affect the MAP kinase response in one way or the other, depending on the type of signaling pathway analyzed. In the phaechromocytoma cell line PC12, addition of different CAMP increasing agents activates the MAP kinase enzyme and its upstream activator [131]. The synergistic effect of these agents on NGF in the MAP kinase activation can be correlated with the potentiating increase in NGF-induced neurite formation, a marker of differentiation in PC12 cells. In the transformed simian cell line COS7, an increase in CAMP also activates MAP kinase [loll. In cell systems where CAMP accumulation is inhibitory for DNA synthesis, the addition of various CAMP increase causing agents strongly inhibits the MAP kinase and/or MAPKK activities stimulated for 5 min by either EGF, LPA, TPA, insulin or PDGF [132-1371. In an attempt to determine on which step the CAMP acts to transmit its effect, two independent studies [133,134] showed that increase in CAMP also inhibits the activity of rafl, the MEK activator. Cook and McCormick [134] demonstrated that neither tyrosine kinase activity of EGF receptor, nor the association with the adaptor proteins She and Grb2, is inhibited by CAMP increase. Moolenaar’s group [137] excluded three early activated events as possible targets of CAMP pathway: and the Na’/H’ exchanger. general tyrosine phosphorylation, Ca2+ mobilization Bos et al. [132] indicated that the phosphorylation of another signaling molecule, the SOS protein-the nucleotide exchange factor responsible for increasing RasGTP levels-is inhibited by CAMP but with a slight delay compare to the MAP kinase inhibition. In fact, a recent study by Czech and co-workers on the phosphorylation of SOS by MAP kinase [138] suggests that the inhibition of SOS phosphorylation by a CAMP increase is the consequence and not the cause of the CAMP-induced MAPK blockade. Because the CAMP accumulation has no effect on Ras-GTP levels, several studies [133,134,137] localized the CAMP target downstream of p21’““.. Further, Sturgill, Weber and co-workers [ 1331 demonstrated that the PKA-mediated phosphorylation of Raf-1 both in vivo and in vitro is inhibitory for the Ras-Raf-1 interaction, which suggests that Raf is one direct CAMP target. Another one could be Rapt, the Ras-like protein analogous to the Krev gene product responsible for reversion of Ras transformation. Indeed, expression of the Rapl/Krev protein can also inhibit MAP kinase activation without interfering with Ras-GTP levels [37]. A previous study mentioned that Ras-induced transformation could be reversed after CAMP treatment [139]. Interestingly, Rap1 is phosphorylated in vivo after a CAMP increase and is an in vitro substrate for PKA [140]. Therefore, taken together, all these results suggest that the CAMP-induced Rap-l

Ikciphrring

the MAP

Kinase Pathwyv

.w7

phosphorylation could reverse the equilibrium of the genuine Rafl-Ras interaction towards an inactive Rafl-Rap1 interaction. Definite experiments on Raf-Rap binding are waiting to confirm or disprove this possibility. In particular, is RaplGTP, when phosphorylated by PKA, able to displace Ras-GTP from the interaction with Raf? In addition to the Ras-Raf interaction as an action site of CAMP, it could also be interesting to determine if another locus is targeted by the CAMP pathway. For example, an increase in CAMP could produce an acceleration of the MAPK-specific phosphatase activity (see below). All these studies used extreme conditions to reach CAMP levels never reached in normal conditions, except if we referred to the always possible but never demonstrated presence of subcellular macro-concentrations of CAMP. Under conditions where the growth inhibitory action of CAMP is observed, we did not detect in a previous study any MAP kinase inhibition in the absence of IBMX. the potent isobutyl-methylxanthine phosphodiesterase inhibitor. When reconsidering these assays with IBMX, the higher CAMP increase obtained is now inhibitory for MAP kinase but only shortly after its activation. By analysis of detailed kinetics, our laboratory (F. McKenzie, personal communication) has shown unambiguously that upon a high CAMP increase, the activation of MAP kinase in hamster fibroblasts is only delayed and still exhibits the same time-course profile as previously shown [ 123). Therefore, we do not believe that the antimitogenic effects of CAMP increase are mediated by the interference of the MAP kinase cascade. Role of’ Phosphatasrs

MAP kinase has been described as playing pivotal roles in many processes, and the way its activity was down-regulated interested many investigators. Early work showed in vifro that both the serine/threonine specific phosphatase PP2A and the tyrosine specific CD45 phosphatase were able to de-activate the MAP kinase [141]. But only one phosphate out of two could be removed by each enzymatic activity. During the past year, an array of closely related genes were cloned from differcnt species (see [142] for a review) and were called: (i)

CLlOO, a previously cloned protein identified from stressed human hbroblasts

(ii)

3CH134, the mouse homolog of CLlOO, was originally characterized as an immediate early gene [144]; the human HVHl, isolated from its homology to 3CH134, was so called after its identity to the active site of the serineftyrosine phosphatase VHI encoded by the vaccinia virus [145]; PACl, another VHl-related phosphatase whose activity is induced when T cells are challenged with mitogens [146]; Erp isolated from mouse is identical to 3CH134 and possesses an inhibitory effect on cell proliferation [147].

u431;

(iii)

(iv) (v)

Each of these gene products were found to encode proteins with motifs and activity of phosphatase. More interestingly, members of this phosphatase family arc growth factor-inducible early genes, exhibiting short half-lives and a rather specific in vitro and/or in vivo dephosphotylating activity towards the MAP kinase protein.

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Cycloheximide treatment maintains a high MAP kinase activity over hours, thus confirming that MAP kinase-specific phosphatases are truly early gene products [144] A striking homology has been detected [148] within two subdomains of CL100 and of a different dual-specificity phosphatase, the cdc25 gene whose activity is necessary for cdc2 stimulation. These regions of amino acid similarity could be involved in a consensus sequence specific for recognition by phosphatases of two adjacent amino acid residues to be dephosphorylated (which is the case in MAP kinase and cdc2 proteins). But the absence of cross-reactivity between CL100 and cdc25 [148] indicates that a higher specificity at the amino acid level is also present. Detailed kinetics of gene induction show that transcripts of this gene family are strongly induced 1 h after growth factor addition, with mRNA detectable as soon as 30 min following growth factor challenging. Therefore, these immediate early gene products can only be responsible for the down-modulation of the late phase activation of MAP kinase (see above). In particular, the same early gene products cannot explain the very rapid down-regulation of MAP kinase activity which follows immediately the initial peak of activation 15 min after growth factor addition (see above). To explain this very transient activation peak, one has to envisage the rapid activation of at least another phosphatase entity. Such a protein is still elusive but recent results [149] indicate that both MAP kinase and MAPK kinase activities are enhanced in cells infected with the SV40derived small tumor antigen (small t) and that these effects are mediated via the interaction of small t with the phosphatase PP2A. As SV40 small t was already known to bind to and inhibit the enzyme activity of PP2A [ 1501, one can expect that PP2A could, in physiological conditions, play a role in the de-activation of the MAP kinase pathway in vivo. These data can be reconciliated with previous results showing the activation of MAP kinase following a treatment of quiescent cells with okadaic acid, a potent inhibitor of PP2A. Truly, most recent yet unpublished data (P. Cohen, personal communication) suggest that the first peak of MAP kinase activation is down-regulated via the action of two different phosphatase entities; a new tyrosine phosphatase still under characterization would dephosphorylate MAP kinase on tyrosine, the first residue to be phosphorylated during the activation mechanism. From the above study with the small t antigen [149], one can then envisage that the phosphatase 2A could be responsible for the removal of the threonine residue during the first phase of MAP kinase deactivation. The fact that at least one member of the MAPK-specific gene family was found nuclear after growth factor addition ([146] and J.-M. Brondello, unpublished results) suggests strongly that MAP kinases, which start leaving the nucleus 3 h after addition of growth factors [151] are deactivated into the nucleus. A definite answer could be given from experiments with regulatory site-specific anti-MAP kinase antibodies. It is noteworthy that the ubiquity of the MAP kinase cascade is not restricted to kinase partners and can be extended to phosphatases since MAP kinase-specific dephosphorylating enzymes were characterized in Xenopus laevis in which the purified enzyme shows a strict specificity for tyrosine [ 1521 and in budding yeast where the MSGS gene product is active on the FUS3 protein kinase [153].

.w

Deciphering the MAP Kinase Pathway

MAP KINASE

TARGETS

Starting with the characterization of the site targeted by MAP kinase in myelin basic protein [154], investigators realized how specific the amino acid environment around the phosphorylated residue was. A MAP kinase consensus sequence was thus established as being P-X-S/T-P (see [155] for a review). Using this consensus phosphorylation site for MAP kinase targets, a series of putative in viva substrates were then characterized from receptor to nucleus (see Fig. 6), where the protein is translocated after growth factor treatment (see below). Cytosolic

Targets

Receptors Receptors

to EGF. In 1991, the EGF receptor was described for the first time as targeted by MAP kinase [156,157], at the threonine 669 site reported to regulate both receptor internalization and substrate phosphorylation on tyrosine residues. This phosphorylation had no effect on the tyrosine kinase activity linked to the receptor [158], nor is inhibited by the absence of this receptor-linked activity [159]. However, MAP kinase induces a decrease in receptor autophosphorylation via the activation of a tyrosine phosphatase [158]. Another study indicated [160] that the same phosphorylation occurs after a physical association between the EGF receptor itself and a new MAP kinase different from ERKl and ERK2 isoforms. This MAP kinase also associates in Wilm’s tumors [160] with the HER2 receptor, a related receptor encoded by the proto-oncogene c-neu. Receptors to NGF. The nerve growth factor has been described as promoting a rapid association between MAP kinase and ~140 trk, the high affinity NGF receptor [161]. By contrast, the association of MAPK and ~75, the low affinity NGF receptor, is independent of NGF presence; only the p75-linked MAP kinase activity is induced by NGF treatment [162]. The exact role of such an association is still unclear; ~75 does not appear to be a MAP kinase substrate [162] but other p75linked molecules could be ERIC substrates. Until the precise inter-connection between NGF receptors of high (~140”~) and low (~75) affinity can be depicted. it will remain uneasy to assess the physiological significance of such MAPK-NGF receptor associations. One original characteristic of such a complex is that the ERKl protein specifically favored the association with each NGF receptor type [161,162]. These results constitute one of the first examples allowing a discrimination between ERKl and ERK2. Kinases pYOr,skMKII.

Much interest in MAP kinase arose after an in vitro study in 1990 from Sturgill, Maller and collaborators in which they showed that the p90TSkenzyme (or S6 kinase II after its capacity to phosphorylate the ribosomal protein S6) could be phosphorylated and re-activated by p42mapk 11631. Since then, other studies have confirmed this result (see [164,165] for reviews). Another [166] identified the ~90’“~ protein in p44mapk immunoprecipitates. Although S6 phosphorylation is stimulated in cells re-activated after a G, or Gz arrest and could play a role in protein synthesis, no absolute physiological relevance

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in cell mitogenicity has been elucidated so far. Nevertheless, ~90’“~ goes into the nucleus, as well as MAPK, after mitogen stimulation where it could have access to other targets (167). Indeed, ~90’“~ enhances by phosphorylation the binding capacity of SRF (serum response factor) within the c-jios promoter [168], thus highlighting its role in the regulation of transcriptional activity. Finally, consistent with p42/p44 MAP kinases and ~90’“~ being in the same signaling pathway, there is no example to date of an agonist capable of activating one protein without the other. MAPKK. Mammalian sequences of MIX1 carry two MAP kinase consensus sites at the C-terminal end of the protein. Thus, if phosphorylated in a feedback loop by MAPK, those two sites could possess some regulatory role on MAPKK activity. Our laboratory engineered the double mutation of the two threonine 297 and 386 into alanine on the hamster MEKl sequence. We first found, by an in vitro kinase assay, that the double mutant of MAPKK retained its capacity to activate MAPK but was less phosphorylated by MAPK than the wild type MAPKK. This reduced phosphorylation resulted in an enhancement in the MEKl activation [169], even if the physiological significance is not yet clear. Expression of the double mutant did not deregulate cell growth or growth factor dependence. A parallel study indicated that MKKl (=MEKl) protein is negatively regulated by threonine phosphorylation [ 1701. Recently, works from Cohen’s and Marshall’s laboratories established that the two MAPK consensus sites which are phosphorylated in vitro by MAP kinase are also targeted in vivo with apparently no activation of MAPKK [171]. Another study led to the same conclusion by mutating one of the MAP kinase consensus sites [ 1721. Because phosphorylation of STE7 protein, one MAPKK family member in budding yeast (see section on MAP kinase kinase family and Fig. 2) is dependent on both FUS3/KSSl and STEll [173], it has also been suggested that STE7 is a target of yeast MAP kinases. Ruf A couple of years before having been characterized as one of the MEK activators, the protein kinase Raf was found to be a target of MAPK with no apparent effect on its activity [174-1761. Today, this phosphorylation can be located in a feed-back loop which could have some regulatory role on the MAP kinase cascade (see section on MAP kinase cascade). But the phosphorylation pattern of Raf is very complex [176]; Raf protein has been found phosphorylated on serine and threonine residues, as well as on tyrosine residues [177]. PKC and PKA affect differently its activity (see section on MAP kinase regulation). In addition, its phosphorylation state depends on type of agonist, cell line, experimental conditions, etc. Therefore, it will take some time before all the regulatory pathways acting through Raf can be deciphered with accuracy. MAPKAP kinase 2. This acronym was attributed to a MAP Kinase-Activated Protein Kinase, whose activation by phosphorylation was specifically due to MAP kinases [178]. In turn, the MAPKAP-2 inactivates by phosphorylation glycogen synthase, a crucial enzyme in the regulation of glucose levels. But MAPKAP-2 is also able to activate by phosphorylation tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis [ 1791.

Deciphering the M.4 P Kinase Pathway

311

Pro&mine kinuse. Purified preparations of MAP kinases have been shown to phosphorylate and activate protamine kinase, a cytosolic enzyme able in vitro to act on both the protein synthesis intiation factor 4E and the ribosomal protein S6 [180]. Other cytosolic turgets Phospholipase A,. The phospholipase

A,, the enzyme responsible for the hydrolysis of fatty acid chains in position 2 of the glycerol backbone, will generate mainly arachidonic acid, the precursor of two biosynthesis pathways involved in the inflammatory response: the prostaglandin/prostacylin and cyclooxygenase pathways. PLAz is found in two subcellular compartments: cytosol (cPLA,) and membrane (mPLA,). In vitro, MAP kinase induces cPLAz phosphorylation at a site required for in viva activation [18 1J. The same paper suggests that the complete activation of cPLA, is sequential: the calcium released after IP, (inositol trisphosphate) formation could induce the translocation of cPLA2 to the membrane where its enzymatic activation promoted by MAP kinase will give it access to its phospholipid substrate, arachidonic acid. It is noteworthy that the two products of the cPLA, enzymatic action. arachidonic acid and lysophospholipid are respectively intermediaries in the prostaglandinlprostacyclin (cyclooxygenase pathway) and leukotrienes (lipoxygenase pathway) synthesis or in the platelet activating factor formation. All these metabolites are potent mediators of the inflammatory response. MAP kinase signaling is certainly not the only way to activate cPLA2, but the blockade of MAP kinase could be useful to somewhat down-modulate the inflammation process. ‘~I~rosinr hyfroqluse. Located majoritarily in cell systems of neuronal or adrenal origin, tyrosine hydroxylase (TH) activity is enhanced by agonists of the catecholamine secretion pathway, thus allowing for the replenishment of catecholamine stores which were lost by secretion. TH has been found phosphorylated in a rat phacochromocytoma cell line by MAP kinases in vitro at a Ser31 site identical to the one targeted by NGF in intact cells [182]. Later, a comprehensive study [179] confirmed the MAP kinase site and identified the sites targeted by two downstream cffectors of MAP kinase: p90rsk/S6kII and MAPKAP-2. The same three phosphorylation sites arc targeted by agonists which stimulate TH activity and catccholamine synthesis [179]. Altogether, these results strongly suggest that only three members of the MAP kinase cascade are sufficient to activate the synthesis of catecholamine hormones, thus opening the way for more accurate pharmacological studies in this metabolic pathway. Stathmin. Multi-phosphorylation of stathmin, a purely cytosolic protein of 19 kDa which is located mainly in cells from neural origin, has been associated with the Ltctivation of an array of physiological processes (see [183] for a review). interestingly. two of the serine phosphorylation sites in stathmin which are targeted in vivo by NGF are also phosphorylated in vitro by MAPKK-activated recombinant MAP kinase [184]. The nucleotide exchange j&or SOS. SOS, the protein accelerating the exchange 01 GDP to GTP on Ras [185], has recently been described as phosphorylated in vitro by MAP kinase in a feedback regulatory loop [ 1381.

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Phosphatase 2C. The protein tyrosine phosphatase 2C (PTP2C), also called PTPl D, has been described to be phosphorylated and inhibited by p4.0apk ‘in vitro. Moreover, the EGF-induced kinetics of activation of the two proteins are inversely correlated [ 1861. STES. The budding yeast gene product STES has very recently been shown to associate with and to be phosphorylated by the yeast MAP kinase FUS3 [187]. As a strong suppressor of FUS3 mutations, a role of STE5 overexpression in suppression of Gl arrest has also been proposed [187]. Nuclear Targets The presence of nuclear targets for MAP kinases (see Fig. 6) can be easily explained by their nuclear translocation detected as soon as 15 min after growth factor addition [168,151]. Detailed kinetics of immunolocalization showed that the fluorescence signal reaches a maximum at 3 h, then MAP kinases leave the nuclei to become entirely cytosolic by 6 h [151]. This processus is independent of the activation state of MAP kinase since the mutation of one or two of the regulatory sites of MAP kinase do not alter the kinetics of nuclear localization [151]. Therefore, the phosphorylation state of the protein cannot explain why MAP kinase goes into the nucleus. As MAP kinase sequence does not exhibit a nuclear localization signal NLS-, the mechanism of its translocation remains obscure even if the presence of a chaperone-like protein can always be envisaged. The protein p90rsk, whose physical association with MAP kinase has been demonstrated [166], possesses such a NLS sequence and thus could play the role of a chaperone for MAP kinase. More investigation is required to determine, for example, if the translocation of MAP kinase is an ATP-dependent but cycloheximide-independent processus as in the case of estrogen receptors. Unpublished data from our laboratory (P. Lenormand, personal communication) indicate that MAP kinase is found cytosolic during at least 9 h after release from an hydroxyurea block. Thus, once returned into the cytoplasm, the MAP kinase protein does not appear to re-enter the nucleus later in the cell cycle. An immunofluorescence study from Davies’ lab indicates that an isoform of MAPK does not enter the nucleus after EGF treatment but is membrane-bound, thus appearing to co-localize with the growth factor receptors [188]. Apart from this MAPK isoform, the nuclear localization of all others suggests that several potential substrates of MAP kinase could belong to the transcriptional machinery. Transcription factors C-jun. In 1991, three MAP kinases isoforms were described [189] as positively regulating the transactivating activity of c-&n by in vitro phosphorylation in the N-terminal portion of the protein at the same two serine sites which were previously characterized as enhancing the c-&n activity when phosphorylated by growth factors. By contrast, an identical Jun phosphorylation induced by MAPK in vitro does not produce variations in transcriptional activity [190]. Further, rather intriguing results indicated [191] that MAP kinase could also inhibit the same transcriptional activity by phosphorylation of a negative regulatory site located in the C-terminus portion of c-&n where the DNA binding domain is.

Deciphering the MAP Kinase Pathwa)

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G. L ‘Allemain

Such a conflictual situation could be explained by the extraordinarily high number of complexes which can be formed between transcription factors of the c-&n and c-fis families to form APl homo- or heterodimers. but also between these two sub-families and the ATF-CREB family (see [192] for a review). This variable array of complexes depends on the expression level of such proteins in any given cell type, thus maybe explaining the discrepancies for MAP kinase effect on transcriptional activity of c+n. In addition, various protein kinases are able to target the same regulatory sites in vitro [190]. Another matter of controversy is if MAP kinase acts directly or not, but the presence of a Jun kinase [193] suggested that this MAP kinase effect was more indirect than expected. Other data (194) indicated that c-&n and APl interact through in vivo complexes with p42 mark and other MAPK-related proteins. The resulting numerous combinations between the APl proteins and the MAPK-related proteins could in turn target various genes [194]. Definite results came in April 1994 with the cloning of the Jun kinase activated after UV treatment, JNKl, which was identified as a member of the MAP kinase family [195]. JNKl is activated by dual phosphorylation on the same two sites as most of the other MAPK isoforms (see section on MAP kinase family). In turn, JNKl is capable of associating with the c-&n transactivation domain and to phosphorylate c-&n on the same two serine sites that MAP kinases were able to target in vitro, thus explaining previous results from various laboratories. Interestingly, another study definitively identified the c-jun kinases as being the p54 MAP kinases. also called SAPK [48]. ATFZ. The ATF2 protein, a member of the ATF (activating transcription factor)/CREB (cyclic AMP response element binding) family was found to increase its DNA binding activity after being phosphorylated in vitro by MAP kinase [196]. In vivo experiments are needed to establish unambiguously that ATF2 activation by MAP kinase can be physiologically significant. C-myc. The major regulatory site of c-myc, whose in vivo phosphorylation is associated with enhanced transactivation of gene expression has been found phosphorylated by MAP kinase in vitro [197]. Interestingly, transfection of MAP kinase provoked an enhancement in the phosphorylation state of the same site [197]. However, physiological significance is still unclear since other kinases are also targeting the same site [155]. Ets oncoprotein family. p62’cf/Elk 1: Originally, Elk1 was described as p62tcf, a ternary complex factor (TCF) protein of 62 kDa which associates with the p67srf (SRF, serum response factor) protein to bind the SRE (serum response element), a characteristic DNA sequence found in numerous promoters of mitogen-stimulated genes, like in the c-fos gene. First in vitro results indicated that phosphorylation of such a TCF by MAP kinase enhanced the transcriptional activity mediated by the SRE, independently [198] or not [199] from the ternary complex formation. Recently. Nordheim’s group described the in vivo phosphorylation of Elk1 by MAP kinase and the capacity for Elk1 to function without any SRF requirement [200]. In addition, the authors suggest that Sapl, another TCF of the Ets family, could also be a target for MAP kinase, but data from Baccarini’s group indicate that, although MAP kinase

Deciphering the MAP Kinase Pathway

215

can phosphorylate Sap1 in vitro, it does not target, in a DNA band shift assay, Sap1 bound to the SRE (personal communication). EtB: In a rat pituitary cell line, the transfection of an inhibitory form of MAP kinase prevents the activation of the EtQ-dependent prolactin promoter. But so far, no direct or indirect phosphorylation of Etsl or Ets2 by MAP kinase has been reported. Interestingly, normal c-jun activity is inhibitory on these highly differentiated neuroendocrine cells [201], thus demonstrating that the positive/negative role of transcriptional activity will depend on the cell context. NF-IM. Activation of MAP kinase increases the transcriptional activity and the phosphorylation of NF-IL6, a member of the basic-leucine zipper family of transcription factors [202]. TALI. The proto-oncogene product TALl, the most common locus genetically altered in T-cell acute leukemia, has been shown phosphorylated in vitro by ERKI on the same serine site targeted in vivo by EGF [203]. STEIN. By genetic epistasis, the yeast transcription factor STE12 has been found to be downstream of FUS3 and KSSl (see [53] for a review). Recently, the presence of significant amounts of STE12 in FUS3 immune complexes and its phosphorylation by FUS3 suggests that STE12 is targeted by FUS3 [204]. It remains to be established if the mechanism of this activation is direct or via one of the other proteins present in these complexes. Other nucleur targets Yeast inhibitor of cVvcfin-dependent kinase. In yeast, the FAR1 protein has been char-

acterized as a direct target of the MAP kinase FUS3 [205]. In turn, the phosphorylation of FAR1 induces its binding with the nuclear complex formed by one of the yeast Gl cyclins, Cln2, and the protein kinase CDC28, the yeast homolog of human cdc2. This association inactivates the kinase complex [205], thus provoking the yeast arrest in G, phase, a characteristic of pheromone pathway. Furthermore, biochemical and genetic data indicate that FAR1 could also be responsible for inhibition of Clnl-, Cln2- and Cln3-kinase complexes [206]. Anri-oncogene p-53. Recently, the tumor suppressor protein ~53, which is a target for several kinases, has been found phosphorylated by MAP kinases in vitro at two threonine sites which belong to one in vivo labeled tryptic phosphopeptide [207]. These data are consistent with previous results indicating that MAP kinase was induced after UV treatment [44]. Altogether with the ~53 protein accumulation after UV or gamma irradiation. it appears that MAP kinase could serve as a relay in the p53mediated stabilization of the cell machinery until DNA repair is performed. NHP6. The yeast NHP6 genes which encode High Mobility Groupl-like proteins associated to transcriptionally active chromatin, have been shown by genetic epistasis to step downstream of the PKCl+BCKl+MKK+MPKl (=SLT2) signaling pathway in budding yeast [208], but no direct interaction with the MAPK-like MPKl gene product has been detected so far.

316

G. L’Allemain Cytoskeletal

Targets

In the family of structural components, nuclear lamins have been reported as being phosphorylated in vitro by MAP kinase [209]. This result could be physiologically significant since lamins are phosphorylated on serine residues at interphase and mitosis (where nuclear envelope breaks down), and MAP kinase has been found activated during M phase (see section on MAP kinase regulation). Several microtubule-associated proteins (MAP) have also been reported to be targets for MAPK: MAP-4 [210] and MAP-2 proteins, the latter being the first exogenous substrate used for assessing MAP kinase activity [l]; the ~220 protein in Xenopus which appears to be also regulated by ~34~“~ protein kinase [211]; the tau protein on which MAP kinase induced a phosphorylation state close to the one characteristic of tau proteins found in paired helical filaments observed in brains with Alzheimer’s disease [2 121. As a general comment, the relevance of such phosphorylations is difficult to establish considering the number of phosphorylation sites present in this protein family (until 16 Pi per tau molecule), and the fact that maybe several protein kinases are targeting the microtubule-associated proteins. But one can conclude from these arrays of results that MAP kinase is playing an important role in microtubule reorganization, more particularly during the transition from interphase to mitosis where the cytoplasmic microtubule network disappears and the mitotic spindle responsible for chromosome segregation forms. Evidently, MAP kinase triggers microtubule dynamics in vitro, [2 131. Subcellular

Localization

sf the Upstream Activators

By contrast with the MAP kinases, the upstream activator MEKl is not translocated after the same growth factor treatment [151]. Very recent observations (Y.Z. Wang and M. Dunn, personal communication) indicated that in mesangial cells, the MAPKK can be found entirely nuclear after a prolonged treatment with TPA, an acute TPA treatment being inefficient. The physiological significance of this result is not clear but a role for a protease is proposed. In any case, this result cannot be extended to fibroblasts (P. Lenormand, unpublished observations). Recently, data from our laboratory indicate that the two MAPKKKs, Raf-1 and MEKK (either the auto-active N-deleted forms or the entire proteins) are not relocalized into the nucleus after serum addition (P. Lenormand, personal communication). Interestingly, parallel studies show that activated Raf is plasma membrane-associated [2 14, 2 151. Furthermore, once localized into the membrane, Raf no longer requires Ras to be activated [215]. This result suggests that the role of Ras-Raf interaction is to recruit Raf to the plasma membrane where Raf could have access to its targets. In any case, it appears that MAP kinase itself is the more upstream element of the whole MAP kinase cascade to translocate into the nucleus. This result will certainly lead more investigators to look for other nuclear targets of MAP kinase. Very recently, the p44mapk has been described as targeting the RNA polymerase 11, independently of transcription factors (0. Bensaude, personal communication).

317

Deciphering the MAP Kinase Pathway

MUTATIONS

IN THE MAP KINASE

MODULE

In order to generate kinase-dead molecules, a self-evident point mutation is the one located on any of the residues interfering with ATP binding and/or recognition, namely either on the invariant lysine of the kinasic sub-domain I or on the invariant aspartate of the -DFG- consensus in the sub-domain VII (see Fig. 1). This kind of mutant is crucial to demonstrate that the upstream activator(s) of any kinase is truly another kinase and not only a co-factor activating an autophosphorylation reaction. In addition, with the regulatory sites unchanged, it can be used as a substrate. To generate putative constitutive active or dominant negative forms of kinase, the regulatory sites (see Fig. 1) can be converted into negatively charged or hydrophobic amino acids. Mutations

in MAP Kinase

Mutations on the regulatory sites (see Fig. I) were generated and found to bc very fruitful to understand the mechanism of regulation of MAP kinase and its role in cell signaling. Several studies performed with mutants of the phosphorylation sites (T-+A, Y+F, T/A-Y/F, etc.) confirmed unambiguously that a dual phosphorylation was required to get full activation of MAP kinase [216, 2171. Surprisingly, no single or double mutant mimicking the phosphate charge (T+E, Y-+E or T/E-Y/E) generated onto p42”“pk or p44 mark exhibit auto-activity [216, 217 and our unpublished observations]. Interestingly, all these mutants translocate into the nucleus after mitogen treatment [151], thus demonstrating that the two mechanisms of translocation and activation are completely independent. The translocation appears to be crucial for mitogenesis since we have shown that only agonists which have mitogenie potentiality are able to trigger MAP kinase nuclearization [151]. An original study from our laboratory [218] demonstrated that the overexpression of the p44 mapkisoform carrying the point mutation Tl92-+A induced the inhibition of the endogeneous forms of MAP kinases (p42mapk and p44mapk), the growth factor-sensitive APl transcriptional activity, and the cell proliferation. To prove definitively that the dominant-negative effects of this MAP kinase mutant were not due to some artefact but to an absolute requirement of MAP kinases in the mitogenic signaling pathway, we used a different approach and showed that transfection of MAP kinase anti-sense mRNA, which works through a different mechanism, also induced a complete inhibition of mitogen-stimulated MAP kinase activity and, consequently, cell growth. As the mechanism of inhibition of anti-sense mRNA is supposed to be by titrating out the sense mRNA, this finding supports the notion that MAP kinases are crucial for cell mitogenesis. With more than 85% identity at the protein level between p42mapk and p44mapk, the inhibition of p42”“pk activity by anti-sense mRNA against total p41mapk sequence was in fact not so surprising. In C. elegatu, the loss-of-function mutations in the MAP kinase homolog Surl and mpkl were characterized: in the Surl gene, an alanine residue of the G-X-G-X-X--G kinase consensus of the sub-domain 1 was found mutated into valine [62], thus establishing how crucial are the X positions in the sequence -G- E-G-A-Y--G-, very well conserved among MAP kinase family members from

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yeast to man [59]; in the mpkl gene, the aliphatic Leu residue was changed into the aromatic Phe amino acid [63] in a sequence which is a putative protein kinase C site. From this naturally occurring mutant, a direct role for PKC on MAP kinase activation deserves to be more closely investigated. Another interesting mutation, Asp334 to Asn occurring in the Drosophila MAP kinase gene rolled, apparently induces a constitutive activation of the protein [61]. This recent finding opens an area of future investigations about the oncogenic potential of MAP kinase. Mutations

in MAP Kinase Kinase

Although many studies suggested that the MAP kinase activator could be a kinase, the unequivocal experiments came from experiments using the MAP kinasedead mutant (see [65] for a review). But it was only when the activator was cloned that the scientific community knew for sure that only one protein entity was responsible for triggering both phosphorylations of threonine and tyrosine onto the MAP kinase protein. It was not the first time that a protein kinase was suspected to be a dual specificity kinase [219], however, it was the first time that an in vivo substrate was clearly established. Until then, only serine/threonine kinases either with in vitro phosphorylating activity towards tyrosine residues or cloned from an anti-phosphotyrosine antibody screening were defined (see [219] for a review). No evident consensus sequence has been so far described for this new family of dual-specificity kinases. Consistent with our results described above demonstrating that the overexpression of the kinase-dead p44 MAP kinase (Thrl92--+Ala) totally blocks cell growth [218], the overexpression of a kinase-dead MAP kinase kinase (mutation of the conserved lysine of the ATP binding site in sub-domain I into an alanine residue) is inhibitory for both MAPKK and MAPK activities, for DNA synthesis and for clonal cell growth (R. Seger, personal communication). This result reinforces the role of the MAP kinase cascade in mitogenicity. If the regulatory sites were rapidly discovered for MAP kinase, the same story has not been reproduced for MEK and two years have passed since the first MEK gene was cloned [13]. The reason is the high complexity of the tryptic phosphopeptide pattern of MEK; not less than nine in vivo labeled peptides can be detected [169]. Indeed, several laboratories established that MEK activation was concomitant to a multi-phosphorylation on serine residues (see [65] for a review). Our laboratory was involved in the generation, on the hamster MEKl gene, of several point mutations affecting the regulatory phosphorylation sites. One of the most evident kinase region to mutate is located between the two kinase consensus sequences -DFG- and -A/S-P-E-, where the two regulatory sites of MAP kinase were disclosed [16]. Among four potentially crucial serine residues, the two located in the C-terminus end of this sequence (see Fig. 1) were mutated in alanine. One, the Ser222, corresponds to the position where MAP kinase, cdc2 and protein kinase A exhibit one of their key regulatory sites. When expressed in hamster fibroblasts, we established that the MEK protein carrying the mutation Ser222-+Ala exhibits no activity with or without growth factor addition and cannot be re-activated by the constitutive (i.e. N-deleted) form of MEKK. More importantly, when Ser222 is mutated into Asp, the very low basal activity of MEK is enhanced to about 50% of the full activation exhibited by growth

Deciphering

the MAP

Kinuse Pathway

319

factors. This mutant is now capable of activating MAP kinase in the absence of growth factor stimulation. From all these data, we concluded that Ser222 is a regulatory site involved in the regulation of the hamster MEKl protein [220]. At about the same time, a collaborative work between three laboratories [221] demonstrated, in complete agreement with our results, that two regulatory sites of rat MEKl, Ser217 and Ser221 (in the rabbit sequence) need to be phosphorylated by Raf protein to trigger full activity. Data from Raf-inducible activities of single/double mutants of MEKl (Ser217+Glu and/or Ser22l-+Glu) suggest that the phosphorylation of either one serine residue is sufficient to trigger activity and, consequently. both residues need to be dephosphorylated to de-activate the MEK enzyme. This result constitutes a remarkable feature on the mechanism of regulation of MEKl which is in sharp contrast to the MAP kinase one, for which dephosphorylation of either one residue is sufficient to de-activate the MAPK activity. More importantly, the double mutation (S217E/S22lE in rabbit MEKl or S218D/S222D in hamster MEKl) confers to MEKl a constitutive activation and potent transforming capacity (unpublished results from C. Marshall’s and J. Pouysdgur’s laboratories). Analysis of the mammalian MEKl sequence showed the presence of two threonine residues as MAP kinase consensus sites in the C-terminal end of the protein at positions Thr292 and Thr386 in the hamster sequence. Expression of the double mutant T/A-T/A in fibroblasts resulted in the disparition of two tryptic phosphopeptides along with an enhancement of the early MEKl activation [169]. This result suggests the existence at the MEKl level of a feed-back control in the MAP kinase cascade due to the MAPK-induced phosphorylation of these two threonine residues. Consistent with that conclusion, Rossomando et al. recently established that the Thr292 site is implicated in the inhibition of MEKl activation [170]. Other data indicated that the threonine sites T291 and T385 (equivalent to the T292 and T385 in the hamster sequence) were also phosphorylated in vivo by NGF but did not regulate, at least in vitro, the activity of MAPKK [171]. Mutations

in MAP Kinase Kinase Kinase

Constitutive activation of Rafl can be obtained either by truncation of the regulatory (N-terminus) domain of c-raf, by using v-rafwhich has a naturally occurring N-deletion (suggesting a regulatory role for the N-terminal portion of the protein). or by point mutation (see [222] for a review). In budding yeast, constitutive mutants of STEl 1 also exhibited a deletion in the N-terminus [223]. Thus, because the mammalian MAPKK kinase MEKKl was PCR-cloned from the fission yeast byr2 sequence, it was expected that deleting the 5’ coding region would create an auto-active form df MEKKl , as already described for the byr2-like gene STEll [223]. In fact, doing this not only auto-activates MEKKl but also precludes the isolation of any stable transfectants containing truncated MEKKl (unpublished observations from G. Johnson’s and J. Pouyss&ur’s laboratories). These results could be due to a lethal effect for the cell and argue for a negative regulatory role carried by the N-terminal portion ot MEKKl. Transient transfection of N-deleted MEKKl allowed us to perform an in vitro kinase assay reconstituting the MAP kinase cascade from MEKKl to MBP phosphorylation via MEKl and p44m”pk activation [220].

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Recent observations from our laboratory establish that the activity of the total MEKKl protein is already high in arrested cells and cannot be enhanced by growth factors (A. Brunet, personal communication). This result indicates that the MEKKl protein is no more regulated when overexpressed into mammalian cells, maybe because of a limiting amount of the endogenous regulatory protein. HETEROLOGOUS

COMPLEMENTATION KINASE MODULE

IN THE MAP

First described in the yeast system, the MAP kinase module has been characterized in all metazoans tested so far (see Figs 3 and 4). If complementation experiments in yeast allowed the definition of the order of kinases in the module, the vertebrate system established biochemically that no other molecular intermediate was lying in the sequence MAPKKK-+MAPKK+MAPK. With such a remarkable conservation of the complete module from yeast to man, every investigator in the field was then eager to know the results of heterologous complementation between various species. At the MAP Kinase Level Because the same sequence - TEY - of phosphorylation sites is also present in yeast (see Fig. l), it has been envisaged that all the MAP kinase family members could be regulated in the same way. Then several MAP kinases from vertebrates were tested for their capacity to complement strains defective in any of the yeast MAP kinases. Thus, Xenopus MAP kinase has been described as complementing in budding yeast, MPKl deletion mutants but not FUS3/KSSl double mutant [57]. FUS3 and KSSl are only partially redundant since KSSl is able to overcome the pheromoneinduced arrest of cell cycling [55] whereas FUS3 is required for the transition from mitosis into conjugation [56]. These opposite effects could explain why MAP kinase from Xenopus [57] or from mammalian cells (A. Brunet and 0. Nielsen, unpublished results) cannot complement the MAP kinase defects in the mating pathway of S. cerev. (see Fig. 3). We predict that the only budding yeast MAP kinase homolog that mammalian MAP kinases will efficiently complement is MPKl, which is located in the signaling pathway activated for normal growth and division of budding yeast (see Fig. 3). Cross-complementations of FUS3 or KSSl by spkl indicate that spkl shares a function with FUS3 that it does not share with KSSl 11731. In S. pombe, a mammalian MAP kinase was shown to partially restore sporulation in the sterile spkl yeast strain ([173] and A. Brunet, unpublished results). At the MAP Kinase Kinase Level Experiments of complementation between MAP kinase kinase family members appeared to be more restrictive than at the MAP kinase level. Homologous complementation between STE7 and byrl, the MAPKK genes of the evolutionary distant yeasts S. cerevisiae and S. pombe, suggests that these two MAPKK gene products share common activities [ 1731. Heterologous complementations with vertebrate MAPKK showed that:

Deciphering the MAP Kinase Pathway

(i)

(ii)

321

byrl strains can be complemented by mammalian MEK only if Raf, one of the MEK activating kinase (see section on MAP kinase kinase kinase family), is co-expressed with it [224]. The reason is certainly the poor interaction between MEK and byr2, the endogenous byrl activating kinase (see Fig. 2): the expression of the XMEK2 isoform in budding yeast suppressed the growth defect associated with loss of the MKKl and MKK2 genes [71], two redundant yeast MAPKK homologs implicated in the yeast PKC pathway (see Fig. 2). This effect was specific since XMEK2 expression did not suppress the downstream loss of MPKl, the corresponding yeast MAP kinase [71]. At the MAP Kinase Kinase Kinase Level

A comprehensive study on cross-complementation between MAPK cascade gene products in budding and fission yeast also envisaged the STEl l/byr2 complementation [ 1731. If overexpression of STE7 with byr2 restored some mating competence in a STEl l- strain of S. cerev., the data also established that byrl is necessary with byr2 to fully complement the STEl 1 defect [173]. Interestingly, heterologous complementation experiments between mammalian and yeast systems established [2253 that the mouse MEKK is capable in S. cerev. of specifically complementing the PKC-dependent MAP kinase pathway but not the MAP kinase pathways involved in mating or osmotic stress (see Fig. 3). Is the MAP Kinase Cascade Redundant?

Altogether, the complementation data along the MAP kindse module indicate how selective the interactions are between members of the various MAP kinase modules, thus leaving little space for redundancy. In addition, in S. cerev. it is now obvious that each kinase partner of the MAPK cascade is acting in a different signaling pathway: conjugation, cell division and wall construct, and osmolarity (see Fig. 3). Recently, complementation experiments with deletion or point mutants of the osmolarity/glycerol pathway suggest that the upstream regulators of the PBS2+HOGI MAP kinase cascade are functionally equivalent to the prokaryotic two-component signal transduction system, namely a transmembrane osmosensor histidine kinase (SLNl ) coupled to a SSKI response regulator [226]. Thus, it is clear that there is no redundancy but a reiteration of the kinase module in yeast to fulfill different functions. In metazoans, in contrast to yeast, no other complete MAPK module has been deciphered yet. The same MAP kinase module has been shown to play a crucial role in controlling cell cycle entry and cell proliferation as well as cell fate and cell differentiation (see Fig. 4). However, the MAP kinase cascade has been implicated in Raf stimulation through a large array of proto-oncogenes, thus showing little or no redundancy, as suggested by the authors [227]. In mammals, the fact that either MEKl or MEK2 is able to activate, at least in vitro, both p42”“pk and p44mapk [69] does not constitute an absolute proof for redundancy. The high expression levels obtained with baculovirus-expressed proteins could be responsible for such a cross-activation, which remains anyway to be established in vivo. In conclusion, we predict that parallel MAPK modules will be described soon in mammals, each one being involved in a specific pathway, but with the possibility,

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in cases of defect or overexpression, of a compensatory redundancy. Consistent with this idea is that the MEKl proteins, also called Jun kinases or SAPK [48,195]. FUTURE

effect different from a true cannot activate the p54”“pk

DIRECTIONS

Knocking Out the Genes With so many gene knock-outs in the air today, a self-evident long-term project would be to inactivate the MAP kinase gene by homologous recombination in mouse ES cells. Homozygous (-L) animals for each isoform of MAP kinase are expected to be viable because of possible compensation by closely related MAP kinase isoforms. However, discrete phenotypic alterations might be revealed by studying all lines derived from these animals. Finally, a crucial experiment will be to analyze the phenotype of animals made from crossing of homozygous animals (-/-) carrying either the ERKl gene deletion for one and the ERK2 gene deletion for the other. This double knock-out is presumably going to be lethal. Oncogenic Potential

of the MAPK

Module

The upstream components of the MAPK cascade are already known as oncogenes capable of deregulating cell growth (i.e. Ras or Raf). So, it is expected that components of the module could somewhat deregulate cell growth. Deregulation of the MAP kinase pathway could be obtained by: (i)

(ii)

Controlled modulation of the gene overexpression: to assess completely the role of the MAP kinase proteins in the general processus of cell division, it is necessary to reach different levels of expression because of the possible compensation existing between different members (see [218], in which overexpression of p44 mapkinhibits the close family member p42”“pk). Various levels can be obtained, either with a new kind of inducible promoter, like the tetracyclin dependent promoter for which induction factors of hundred-folds have already been obtained, or with a conditional system in which retroviruses, containing a fusion gene between the hormone-binding domain of the human estrogen receptor and the protein of interest, are used to transfect cells. As a result, expression or repression of the protein of interest will depend respectively on the presence or absence of estradiol in the growth medium. This elegant technique has already been applied with success for conditional expression of the protein kinase Raf-I [81]. Extended to the conditional expression of various molecules, such a technique will help in the elucidation of many signaling pathways, more interestingly those carrying proteins requiring a tightly controlled expression. Mutations on the MAP kinase-specific phosphatase(s): the first part of the challenge will be to determine how the first peak of MAP kinase activation is so rapidly down-regulated, before any expression of the MKPl phosphatase could take place. There is apparently another MAP kinase-specific phosphatase (see section on MAP kinase regulation).

Deciphering the MAP KinasePathwa-v

(iii)

.?31 -_

The second part will be to use phosphatase mutants to deregulate the activity. One can expect that a constitutive activation of the phosphatase would block the cell cycle in the G, phase, whereas a dominant-negative mutant would activate the MAP kinase pathway permanently, allowing the cells to become growth factor-independent for growth. Isolation of constitutive forms of MAPK or MAPKK: the presence of a parallel kinase cascade in yeast allowed the complementation of some yeast defects with various mammalian members of MAP kinase cascade (see section on heterologous complementation). So the next step would be to use this genetically very powerful cell system to isolate various mammalian mutants. Among them, mammalian mutants of MAP kinase exhibiting a constitutive enzymatic activity remain elusive so far, even if the characterization of a gain-of-function mutation in the MAP kinase-like Drosophila locus rolled [61] will lead investigators to overexpress it in mammalian systems in order to decipher the oncogenic potential of the protein, if any, and to analyze which step(s) is (are) deregulated. The recent crystallization of p42mapk [228] now renders possible the targeting of the amino acid environment around the regulatory sites, thus helping in engineering any deregulated mutant form of MAP kinase. Characterizing All the Targets

Look for the phosphorylation consensus sites of MAP kinase (-P-X- S/T-P-) through a search in protein data banks could not be as helpful in characterizing new targets of MAP kinase because other kinase families have a proline-directed serineithreonine kinase activity like the cdc2/cdk family, whose consensus (-S/T ~-P.X K/R-) overlaps the MAP kinase one. or the newly discovered sphingomyelin/ceramide-activated kinase family (see [229] for a review) whose consensus is exactly the same as the MAP kinase one. In fact, the sphingomyelin signaling pathway, triggered by the inflammatory cytokines TNF alpha and interleukin-1. is mediated by the p54 MAP kinase sub-family [48]. Thus, one has to be cautious on how significant these phosphorylations are since any member of the three families could be responsible in vivo for such phosphorylations. The technique of double hybrid system, which has already been very fruitful in demonstrating the direct interaction between Ras/Raf and Raf/MEK [75]. could help in isolating companions of MAP kinase. Filling the Gap Between the MAPK Module and the Cell Cycle Proteins

A whole set of universal inhibitors of cdk have recently been characterized in mammals (see [230] for a review). It would be very informative to know if some of these inhibitors represent physiological targets for mammalian MAP kinases. In yeast, the MAP kinase FUS3 directly phosphorylates the FAR1 protein which in turn will bind to and inactivate the CDC28-Cln2 complex (equivalent to a cdk Gl cyclin mammalian complex), thus provoking the G, arrest [205]. Following this line, it will be exciting to define if this regulatory interaction has been conserved in evolution. Namely, are mammalian MAP kinases able to regulate the cyclindependent kinases present in mammals via some FAR1 -related but yet undefined intermediary molecule?

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Already, the yeast FAR1 appears to belong to the family of mammalian inhibitors of cyclincdk complexes. The latter have been called universal inhibitors of cdk because of their in vitro capacity to block various combinations of cdk+yclins (see [230] for a review). Whether or not this new class of inhibitors exhibit a more restricted specificity in vivo is not yet known. Altogether, the next exciting challenge will certainly be to establish if MAP kinase is targeting directly or not one of these universal inhibitors of cdk. Finally, considering the numerous regulatory roles (some of them putative, others real) in such different physiological processes, and to reconcile every investigator in the field, we propose that MAP kinase could now more than ever stand for Mother of ,411 Protein kinases! NOTE ADDED

IN PROOF

While this manuscript was in the publication process, a series of important papers on MEKl-induced oncogenicity appeared in the literature from various laboratories: C. Marshall’s (Cowley et al. Cell 1994; 77: 841-852), N. Ahn and G. van de Woude’s (Mansour et al. Science 1994; 265: 966970) and J. Pouyssegur’s (Brunet et al. Oncogene 1994; in press). Concerning the role of Ras in Raf activation, two papers of great interest were published on the recruitment of Raf by Ras to the plasma membrane (Stokoe et al. Science 1994; 264: 1463-1466 and Leevers et al. Nature 1994; 369: 411414). The first publication describing a functional divergence between ERKl and ERK2 in the MAP kinase pathway (Chuang and Ng, FEBS Lett. 1994; 346: 229-234) reinforces our proposed hypothesis that in vivo the MAP kinase cascade is not redundant (see section on heterologous complementation). Finally, several pieces of data cited in the text as ‘personal communication’ have now been accepted for publication: work is in press from M. Baccarini’s group in Genes and Development, and from 0. Bensaude’s group in the EMBO Journal. REFERENCES I.

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