Signal transduction from cell suthce development and disease MICHAEL

KARI1I

Department

of ?harmacolog

Univexaity of California

School #{225}f Medicine,

ABSTRACT Recent studies indicate that extracellular signals affect cell proliferation and differentiation by modulating transcription factor activity via protein phosphorylation cascades. This review discusses the basic outline of the eukaryotic signal transduction systems used to transmit information from the cell surface to the transcriptional machinery in the nucleus. Several examples that illustrate how these pathways control cell proliferation, differentiation and development are discussed Karin, M. Signal transduction from cell surface to nucleus in development and disease. FASEB J. 6: 2581-2590; 1992. Key

signal

Words:

transduction

transcription

factor

protein

phosphorylation

ABILITY TO RESPOND TO extracellular signals is essential for the survival and proper development of all living organisms. A common response to extracellular signals involves changes in the program and rates of gene expression. In

THE

prokaryotes induce

and

unicellular

differentiation

eukaryotes,

extracellular

into a form (spore)

expression

zymes and

and

adaptive

of unnecessary metabolic and inducing expression of others. responses

have probably served to the more complex differentiation

.50. © FASEB

San Diego, La Jolla, California

92093, USA

of protein-coding genes in eukaryotes (6). The bacterial activators controlled by phosphorylation behave very much like eukaryotic enhancer-binding proteins and are able to affect the activity of the RNA polymerase from a long distance. This suggests that these regulatory systems are the progenitors of the more complex signal transduction systems of eukaryotes. However, due to the increased physical distance and separation between cell-surface receptors and the nuclear transcriptional machinery, eukaryotic cells had to develop far more intricate pathways for signal transduction. Yet the basic operating principle remains the same: an extracellular signal affects the activity of a protein kinase cascade that modulates transcription factor activity by protein phosphorylation. This review is focused on three topics: 1) the control of transcription by signal transduction pathways in higher eukaryotes; 2) recent examples illustrating how these mechanisms control development, and 3) how aberrations in these pathways perturb normal differentiation and morphogenesis. These aberrations also provide clues about unknown components in the signaling systems that control these important processes.

to

unicellular orby repressing biosynthetic enSuch protective as evolutionary programs

precursors of multicellular organisms, most of which are also controlled by extracellular signals. Although multicellular organisms produce their own signals in the forms of hormones, growth factors, and morphogens, the basic mechanisms used in signal-regulated gene expression are identical to those operating in unicellular organisms. One evidence for these evolutionary relationships can be found in the high degree of sequence conservation among signal transducers (1, 2) and transcriptional regulators (3, 4). These types of molecules are the basic components of the signal transduction systems that connect the cell surface to the nucleus. The most common way to regulate gene expression in response to extracellular signals is to modulate the activity of sequence-specific transcription factors. In bacteria, the transduction of certain extracellular signals to the transcriptional machinery occurs via a two-component system (Fig. 1). One component serves as a sensor for the extracellular signal and transmits this information by a protein phosphorylation reaction to a second component that functions as a transcriptional activator (5). Phosphorylation in this case increases the ability of the regulator to activate transcription by affecting its ability to interact with RNA polymerase. The bacterial operons that are controlled in this manner are the ones whose regulation is most similar to that

0892-6638/92/0006-2581/$01

in

signals

that is resistant

environmental damage. Other signals allow ganisms to adapt to new nutritional conditions

to micleus

SIGNAL Receptors

TRANSDUCTION and

IN EUKARYOTES

ligands

The biochemical machinery involved in the control of eugene expression by extracellular signals is considerably more complex than the two-component bacterial signaling system. Due to space limitations, only the basic features of eukaryotic signal transduction will be described, and most attention and emphasis will be placed on the mechanisms involved in controlling transcription. For further detail the reader is referred to a number of excellent review articles (7-14). As in bacteria, signal transduction in eukaryotes is initiated by ligand-receptor interaction. Although most ligands identified in mammals are soluble proteins, peptides, or small organic molecules, some ligands are anchored in the karyotic

Abbreviations: PLC/3, phospholipase C/3; IP,, inositol triphosphate; DAG, diacylglycerol; Ras-GAP, Ras GTPaseactivating protein; PKC, protein kinase C; GH, growth hormone; PRL, prolactin; PKA, protein kinase A; CAM kinase, Car-activated mukifunctional kinase; CREB, cAMP response element binding protein; AP-1, activator protein-I; NFkB, nuclear factor kB; CRE, cAMP response element; TRE, TPA response element; SRF, serum response factor; SRE, serum response element; HIV-1, human immunodeficiency virus; IRF-1, interferon gene transcription factor; VPCs, vulval precursor cells; a-p, anterior-posterior; d-v, dora!ventral; isi, torso-like; 1(1) ph, 1(1)polehole;GRF, growth hormone releasing factor.

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ACTIL

&\L

TUCTOJ

516NA1

NITROGEN. PHOSPHATE. OSIIOLARITY

51N.c154’ ntr 6. pRo P. en, 1

A

1-c------ADP

hibit adenylate cyclase. Regardless of their specificity, the a subunits are active when complexed with GTP and inactive when in their GDP-bound state. Therefore, GTP hydrolysis is used to control the activity state of the G protein and its target. In addition to adenylate cyclase, other targets for G proteins include guanylate cyclase and phospholipase C/3 (PLC$). The latter enzyme generates the second messengers diacylglycerol (DAG) and inositol triphosphate (1P3). Other G proteins couple cell-surface receptors to ion channels.

Receptor-type Another

ntrc C. pRoD.

omp P

Figure 1. Gene regulation by extracellular signals in bacteria. An extracellular signal (osmolarity, availability of nutrients, etc.) activates a sensor protein embedded in the cell surface. The activated sensor catalyzes the transfer of a phosphate moiety from ATP to specific residues in the NH2-terminal domain of a sequence-specific regulator. This phosphorylat ion reaction modifies the ability of the regulator to interact with RNA polymerase, resulting in activation of transcription.

cell surface. The latter type of ligands, most of which were identified by genetic analysis of Caenorhabditis elegans and Drosophila melanogaster, act by direct cell-cell interactions. Whereas the structure and the chemical nature of ligands vary a great deal, their receptors can be grouped into a few structural families. One of these families is the seven-pass transmembrane protein family (9). Its members are glycoproteins composed of an extracellular NH2-terminal domain, a central core composed of seven membrane-spanning helices and connecting loops, and an intracellular COOH terminal domain. Ligands include neurotransmitters, neuropeptides, and various polypeptide hormones and growth factors. The ligand binding pocket is composed of the transmembrane helices; for those receptors whose ligands are large polypeptide hormones, additional interactions are provided by the NH2terminal domain. The intracellular domain contains several phosphorylation sites involved in receptor desensitization (15). Some of the intracellular loops connecting the transmembrane regions constitute the site of interaction with the next component of this signaling chain: the G protein. G proteins The G proteins are trimeric GTP-binding proteins composed of a, /3, and y subunits (7, 8). In response to receptor stimulation the a subunit, which has GTPase activity, dissociates from the membrane-attached /3 ‘y complex to affect the next elements of this signaling cascade: enzymes that generate second messengers. So far more than 40 distinct G proteins have been identified and most of their variability is due to the presence of different a subunits, which dictate target and response specificity (8). Whereas some a subunits (Ga,) stimulate adenylate cyclase, others (Ga1) in-

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May 1992

large

tyrosine family

kinases

of cell-surface

receptors

is composed

of

membrane-spanning tyrosine kinases (11). Although these proteins vary in structure, they all contain a conserved catalytic domain within their COOH-terminal cytoplasmic region responsible for phosphorylation of tyrosine residues. The NH2-terminal domains of these proteins are extracellular and some are covalently linked to a second polypeptide chain. The extracellular domain contains the site for binding of ligands that include a variety of polypeptide growth factors such as EGF, PDGF, CSF-1, and insulin. Although some tyrosine kinase receptors are monomeric in their resting state, others are loose dimers. A common mechanism for the activation of these receptors is ligand-induced dimerization or a ligand-induced increase in the association between a!ready dimerized subunits. The close apposition of two subunits results in their cross-phosphorylation on tyrosine residues, a reaction that is the hallmark of receptor activation (16). Although tyrosine kinase activity undoubtedly is required for signal transduction, it is not clear what are the immediate and critical substrates for these receptors (14, 16). Currently, one of the best candidates for a signal transducing substrate is PLC’y, which forms a physical complex with the cytoplasmic domains of several ligand-activated receptors (14). Furthermore, PLC-y activity is stimulated by tyrosine phosphorylation (17). Like the G protein-activated PLCI3, PLCy generates the second messengers DAG and 1P3 by hydrolyzing membrane phospholipids. Downstream

events

Another potential substrate of major importance is the Ras GTPase-activating protein (Ras-GAP). As indicated by its name, this protein stimulates the GTPase activity of the Ras proteins, which like the Ga proteins to which they show some homology, are active in their GTP.bound form and inactive when complexed with GDP (18). Ras-GAP is phosphorylated on tyrosine in response to EGF, PDGF, or CSF-1 and forms a complex with the activated PDGF receptor (14). Activation of tyrosine kinases affects the intracellular distribution of Ras-GAP and decreases its activity (19). The decrease in Ras-GAP activity should lead to an increased Ras activity. Indeed, growth factor stimulation increases the amount of GTP bound Ha-Ras protein (20). Although the direct downstream target for the Ras proteins is not known, microinjection experiments indicate that activated Ras proteins stimulate cell proliferation. On the other hand, neutralizing anti-Ras antibodies and dominant-negative Ras mutants block growth factor-induced DNA synthesis (14). Therefore, the Ras proteins are important mediators in the signaling cascade by binding of growth factors to membranespanning tyrosine kinase receptors. Other promising candidates for signal transducing substrates for tyrosine kinase receptors are several serine/threonine-specific protein kinases that function as switch

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kinases. The ability of these enzymes to phosphorylate teins on serines and threonines is stimulated by their phorylation

on

tyrosines.

The

members

of this

group

prophosmost

characterized are the MAP2-kinases, also known as ERK (21, 22). Although the MAP2-kinases are phosphorylated on tyrosine, a modification critical for their activation, they do not appear to be direct substrates for tyrosine kinase receptors and are probably activated by a factor that stimulates their autophosphorylation on tyrosines and threonines (23). This activator, however, may be regulated by a receptormediated event. The switch kinases may also include Raf-1, a serine/threonine kinase that appears to be associated with the PDGF receptor but not with other growth factor receptors (14, 24). Although Raf-1 is phosphorylated on tyrosine in response to receptor stimulation, the stoichiometry of this modification is very low and its significance is not clear. However, both serine- phosphorylation of Raf-1 and its catalytic activity are stimulated after activation of a variety of tyrosine kinase receptors (24). In addition, Raf-l activity and serine phosphorylation are stimulated by protein kinase C (PKC) activation. Recent experiments demonstrated that Raf-1 acts downstream to H-Ras (25). Therefore, it is possible that activation of Raf-1 by tyrosine kinase receptors is mediated through Ras. Other

receptors

and

mediators

Another class of receptors includes those recognized by growth hormone (GH), prolactin (PRL), and a variety of lymphokines and cytokines (26). Although structurally similar, these receptors do not possess any known enzymatic activity nor are they coupled to G proteins. Although their signal transduction mechanism is currently obscure, stimulation of several of these receptors results in rapid phosphorylation of other proteins on tyrosines (27). Hence, it is possible that these receptors are coupled to subunits that possess tyrosine kinase activity. Indeed, coprecipitation experiments suggest that at least one member of this class, the interleukin-2 receptor, is loosely associated (after activation) with tyrosine kinases of the Src family (27). Therefore, the mechanism of signal transduction by these receptors could be similar to the one used by receptors with endogenous tyrosine kinase activity. Some of the downstream effects of cell-surface receptor activation are mediated by second messenger-activated protein kinases (12). This group of serine/threonine kinases includes the cAMP-activated protein kinase A (PKA), the DAGactivated PKC, and Ca2/calmodulin-activated multifunctional kinase (CAM kinase). Ca2 functioning as a second messenger is generated either by the activation of receptormodulated Ca2 channels or more commonly by release from intracellular stores in response to 1P3 (10). Although the second messenger-activated kinases have many different substrates, it appears that most of their long-term effects are mediated by a specific class of recently identified substrates, the transcription factors.

TRANSCRIPTIONAL TRANSDUCTION

CONTROL

TRANSDUCTION

CREB CREB is a 43-kDa DNA binding protein that activates transcription of many cAMP-inducible genes via cAMP response elements (CRE) in their control regions (28, 29). CREB is phosphorylated by the catalytic subunit of PKA, which in response to cAMP dissociates from the regulatory subunit and translocates to the nucleus (30). Phosphoryladon of CREB on serine 133 dramatically increases its ability to activate transcription (31). Serine 133 resides within the transactivation domain of the protein, and its replacement by alanine using targeted mutagenesis interferes with its ability to activate transcription in response to the cAMP signal. The exact mechanism by which phosphorylation of this site modulates CREB activity is not known, but may involve a phosphorylation-induced conformational change of the CREB activation domain. This could increase its ability to interact with a yet-to-be identified target that is part of the basic transcriptional machinery (Fig. 2). According to a recent report, CREB activity is induced not only by cAMP but also by elevated intracellular Ca2’, which leads to phosphorylation of CREB on a site identical to the one recognized by PKA (32). These findings are intriguing because Ca2 does not activate PKA but a variety of other protein kinases, such as the CaM kinase. Furthermore, in most cells CRE-containing genes are not induced

CREBIS ACTIVED BY PHOSPHORYLATIONOF Ser 133 IN ITS ACTIVATION DOMAIN

cAMP

BY SIGNAL

CASCADES

Receptor stimulation results in transient production of second messengers, but its effects on cell proliferation and differentiation are long-lasting and sometimes irreversible. These effects are mediated by changes in gene expression brought about by specific transcription factors whose activity is modulated by phosphorylation. Although phosphorylation

SIGNAL

is likely to modulate the activity of many transcription factors, its role has been established only for a few, including: CREB (cAMP response element binding protein), AP-l (activator protein-i), and NFkB (nuclear factor kB). Signal- induced posttranslational activation of these factors results in induction of downstream genes, some of which encode other transcription factors, which in turn affect the expression of many target genes. Such cascades can generate long-lasting nuclear signals.

FROM CELL SURFACE TO NUCLEUS

OFF

ON

2. Control of CREB activity by cAMP. By binding to the regulatory (R) subunit, cAMP leads to dissociation of the PKA holoenzyme and translocation of its catalytic subunit (C) to the nucleus. The free and active catalytic subunit phosphorylates CREB (which is already bound to DNA) on serine 133 and thereby increases its ability to stimulate transcription. Figure

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by Ca2. In vitro, the CAM kinase was shown to phosphorylate the same site on CREB as PKA. However, it is possible that this convergence of the cAMP and Ca2 pathways is unique to PC12 cells in which it was found (and maybe to other neuronal cell types), but is not of general occurrence. The Ca2 activation of CREB could be indirect and involve activation of a calmodulin-sensitive adenylate cyclase that leads to activation of PKA. AP-! AP-i is a collection of homodimeric and heterodomeric complexes composed of the jun and fos gene products. These products interact with a common binding site, the TPA response element (TRE), and activate gene transcription in response to activators of PKC, growth factors, and transforming oncogenes (33, 34). The jun and fos genes are immediate-early genes whose transcription is rapidly induced by the same signals that induce AP-l activity (34). Most inducers of c-fos affect the activity of the serum response factor (SRF) that binds to the serum response element (SRE) within its promoter. SRF activity is thought to be regulated by a posttranslational mechanism as it exists in the cell before the initial stimulus (35). Usually c-fos induction is rapid and highly transient, resulting in appearance of the cFos protein within 1 h of the initial stimulus (36, 37). Induction of c-jun, on the other hand, is longer lasting and varies from a few hours to several days in a cell type and stimulus-dependent manner (34). Induction of c-jun is mediated by a TRE within its promoter (38) and requires the presence of preexisting cJun protein (39). Although c-jun is positively autoregulated (38), c-fos is subject to negative autoregulation (40). The elements and factors that control expression of other jun and fos genes are under investigation. The requirement for preexisting cJun for induction of cjun and AP-1 activity and its partial independence of de novo protein synthesis (33, 38) suggest a posttranslational mechanism. Many cell types contain considerable basal levels of cJun, which is phosphorylated on three closely spaced sites next to its DNA binding domain (41). Phosphorylation

of these

sites inhibits

DNA

binding.

PKC

Vol. 6

May 1992

NFkB

Originally scription

NFkB was thought to be a B cell-specific tranfactor that binds to an enhancer element of the im-

P1CC

PHOSPHATASE

1 L OFF

ON

H-Res

activa-

tion results in rapid dephosphorylation of one or more of these sites, an event that contributes to increased AP-1 binding activity. The kinase responsible for phosphorylation of these sites in resting cells is not known, but in vitro they are phosphorylated by glycogen synthase kinase 3 (41). The TPA-induced dephosphorylation of cJun is mediated by a yet-to-be identified PKC-activated nuclear protein phosphatase. cJun is positively regulated by phosphorylation of two sites in its NH2-terminal activation domain (42). The pathway responsible for phosphorylation of these sites, which potentiate the ability of cJun to activate transcription, involves the Ha-Ras oncoprotein. The kinase that mediates this effect is yet to be identified, although a recent report suggests that the NH2-terminal sites of cJun are phosphorylated by a MAP2-related kinase (43). Ha-Ras expression also decreases the phosphorylation of the inhibitory sites next to the cJun DNA binding domain (42, 44; Fig. 3). This dual effect is consistent with the ability of Ha-Ras to activate at least two signal transduction pathways, only one of which involves PKC (14). As Ha-Ras mediates the action of tyrosine kinase receptors, it is expected that growth factors will affect cJun activity similarly. Because c-jun transcription is positively autoregulated, these posttranslational modifications result in a cycle leading to further production of cJun protein, as observed in Ha-Ras transformed cells (44).

2584

Activation of PKC also stimulates phosphorylation of cFos (45). Although the TPA-inducible phosphorylation sites have not been mapped, most are located at the COOH-terminal region of cFos (45, 46). This part of the cFos protein contains one of its activation domains (47) and is also involved in transrepression of the c-fos promoter (40). Substitution of several serine residues that are putative phosphorylation sites by alanines abolishes the transrepression activity of cFos without affecting its ability to activate transcription (46). Therefore, inducible phosphorylation of cFos is likely to contribute to its transient synthesis, whereas in the case of cJun it prolongs its production and thereby generates a longerlasting nuclear signal. This basic difference between cJun and cFos may account for changes in composition of the AP-1 complex after cell stimulation (34).

/\

P1CC

KINASE

OFF ON 3. Posttranslational control of cJun (AP-1) activity. In nonstimulated cells cJun is present in a form that is fully phosphorylated on inhibitory sites next to its DNA-binding domain and therefore cannot bind DNA and activate transcription. The protein is also underphosphorylated at the NH2-terminal sites. A) In response to PKC activation, the inhibitory sites are dephosphoi-ylated by a specific protein phosphatase. The Figure

dephosphorylated protein binds DNA, leading to moderate transcriptional activation. B) In response to expression of activated HRas, PKC is activated, leading to dephosphorylation of cJun as described above. At the same time, another pathway activated by HRas increases the activity of a specific nuclear serine/threonine kinase that phosphorylates the NH2-terminal sites within the activation domain of cJun. This dual modification results in a cJun pro-

tein that leads to a strong transcriptional

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munoglobulin kappa light chain gene. However, it was soon found that its binding activity is induced in most cell types in response to stimulation of several signal transduction pathways by phorbol esters, antigens, cytokines, UV irradiation, virus infection, and double- stranded RNA (48). NFkB is a heterodimeric nuclear protein composed of 50and 65-kDa subunits (49). Both polypeptides participate in DNA binding (50). In nonstimulated cells the majority of NFkB activity is present in the cytosolic fraction as a cryptic form unable to bind DNA. This form can be activated by treatment of cytosolic extracts with mild denaturants, which dissociate the pSO:p65 heterodimer from a 37-kDa inhibitory polypeptide, IkB (51). IkB appears to be a target for several protein kinases, including PKA and PKC, all of which are capable of phosphorylating uncomplexed 1kB in vitro and prevent its interaction with NFkB. In addition, these protein kinases are capable of disrupting and activating the NFkB IkB complex (52, 53). The IkB sequence contains a putative PKC phosphorylation site (54). However, it remains to be demonstrated that IkB is a substrate for any of these kinases in vitro. The present model for NFkB regulation indicates that extracellular signals induce its activity by affecting phosphorylation of IkB, leading to its dissociation from the p5O:p65 heterodimer. The free NFkB heterodimer translocates to the nucleus where it binds to specific response ele-

1KB IS INHIBITED

BY PHOSPHORYLATION

NUCLEAR TRANSLOCATION

RESULTING

IN

OF ACTIVE NFkB

ments in the regulatory regions of downstream genes, leading to their activation (Fig. 4). The mechanism by which NFkB is permanently activated in B cells remains to be elucidated, but it could be due to inactivation of 1kB by constitutive phosphorylation or lack of its expression. Until recently, NFkB was thought to be regulated exclusively by this posttranslational mechanism; however, new findings demonstrate the existence of additional regulatory mechanisms. Sequencing of the p50 cDNA indicated that it is a member of the rel gene family (55, 56), which also includes the proto-oncogene c-rel and the dorsal developmental control gene of Drosophila (57). The open reading frame of the p5O cDNA encodes a larger, 105-kDa protein. Although full-length pl05 does not bind to DNA, a truncated form similar in size to p50 is fully active (55, 56). This suggests that p105 is the precursor for p50 and that its maturation requires a specific proteolytic cleavage. A protease encoded by the human immunodeficiency virus, HIV-1, is capable of processing plOS to p50 and could therefore contribute to activation of NFkB in HIV-1-infected cells (58). This is one of the first examples for activation of a transcription factor by proteolysis. The Dorsal protein is also activated by proteolysis, which is required for its nuclear translocation. Mutations in genes whose products are involved in the processing and nuclear translocation of Dorsal produce the same phenotype as loss-of-function dorsal mutations (59, 60). Like c-ret, which is a serum-inducible immediate early gene (61), the plO5/p5O gene was found to be inducible by the very same stimuli that activate NFkB (55, 62). Therefore, the NFkB system is analogous to the AP-1 system: in both cases posttranslational events increase the ability of a preexisting transcription factor to bind DNA, resulting in transcriptional activation of the gene encoding the factor, followed by synthesis of more protein. Analysis of the promoter of the pIOS/pSO gene will indicate whether it is a direct target for activation by NFkB. A similar autoregulatory loop was recently suggested to be involved in activation of the interferon gene transcription factor, IRF-1 (63). This may be a common mechanism for activation of signal-responsive transcription factors.

SIGNAL

> Figure 4. Posttranslational control of NFkB activity. In the cytosol of nonstimulated resting cells, NFkB (a pSO:pfiS heterodimer) is present as a nonactive complex with the inhibitory protein, IkB(I). In response to cell stimulation and activation of carious second messenger-responsive protein kinases (PKA, PKC), IkB is phosphorylated and dissociates from NFkB. The active NFkB heterodimer translocates to the nucleus where it binds to specific response elements and activates transcription of nearby genes. SIGNAL TRANSDUCTION FROM CELL SURFACE TO NUCLEUS

TRANSDUCTION

AND

CANCER

Cancer can be viewed as a disease of the signaling system that controls cell proliferation and differentiation. Any defect leading to constitutive activation of one of the components described previously may result in uncontrolled cell growth and, consequently, a neoplasia. Such aberrations are manifested by retroviral and cellular oncogenes that are derived from their normal counterparts by gain-of-function mutations. Another group of genes that negatively regulate cell growth was identified by loss-of-function mutations that contribute to the neoplastic phenotype. These genes are commonly known as tumor suppressor genes (64, 65). Because the relationship between signal transduction and cancer has been extensively reviewed (14, 64), only a brief summary will be provided herein. As illustrated by the v-sis oncogene, which encodes a protein highly similar to the B chain of PDGF, uncontrolled production of growth factors can result in the autocrine activation of cell proliferation (64). Although the genes encoding the PDGF receptor were not found to give rise to oncogenes, other genes that encode cell-surface tyrosine kinase receptors do so. A classical example is the erb-B oncogen, which encodes a truncated and constitutively activated version of the EGF receptor (14, 64). Further down this pathway, one finds oncogenic versions of

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the Ras proteins. In this case, activating mutations decrease the GTPase activity of these proteins or their response to

GAP and thereby increase the fraction of GTP bound Ras, the active species (18). The product of the NFl tumor suppressor gene shows considerable homology to Ras-GAP and is capable of exhibiting GAP activity in vitro (64, 65). Thus the loss of a negative regulator can have the same effects on cell proliferation as constitutive activation of a positive regulator. Other downstream mediators reincarnated as oncogenes include Raf-1, whose truncated oncogenic counterpart, vRaf, acts as a constitutively activated serine/threonine kinase (24, 25). Oncogenic activation also affects the most distal mediators of these signaling pathways, the nuclear transcription factors. Oncogenic versions of both c-jun and c-fos have been identified. In both cases, the major mechanism of oncogenic activation involves the fusion of these genes to a strong, constitutive retroviral promoter and loss of their 3’ untranslated regions that contain determinants of mRNA instability (66, 67). These changes result in overexpression of the vFos and vJun proteins, leading to constitutive activation of their target genes. Due to a frameshift mutation, vFos also contains a different COOH terminus from cFos and lacks the domain involved in transrepression (40). vJun, on the other hand, is missing a short NH2-terminal sequence present in cJun (67). In addition, a single amino acid substitution (Ser243-’Phe) prevents phosphorylation of vJun on inhibitory sites next to its DNA binding domain (41). Despite these changes, the DNA binding specificity and transactivating potential of vJun and vFos are similar to those of their normal counterparts (40, 68). A single oncogene is not sufficient for transformation of normal diploid cells. In the case of H-ras, its activity can be complemented by expression of a second oncogene, such as v-myc, or phorbol ester tumor promoters, a phenomenon known as oncogenic cooperation (64). Mechanistically, oncogenic cooperation can be explained by a requirement for one oncogene to augment the activity of another, which by itself is incapable of eliciting uncontrolled cell proliferation. Another possibility is the activation of two complementing pathways by the cooperating oncogenes, each of which can elicit only a part of the total changes required for uncontrolled proliferation. Although the molecular basis for oncogene cooperation may vary from one case to another, it was recently shown that the Ha-Ras oncoprotein can augment the activity of cJun by stimulating its phosphorylation (42, 43). Whereas neither Ha-ras nor c-jun (which was rendered oncogenic by cloning into a retroviral vector) is capable of transforming rat embryo fibroblasts by themselves, the two complement each other (69). It is possible that by converting cJun to a more efficient transcriptional activator (Fig. 3), Ha-Ras induces increased expression of downstream genes involved in cellular proliferation. These target genes remain to be identified.

SIGNAL TRANSDUCTION DEVELOPMENT

CASCADES

IN

Although it is well established that the pathways described previously control cell proliferation and metabolism, recent evidence indicates that some also participate in cell fate determination. As many components of these signaling pathways are ubiquitous proteins, it is surprising that the same basic hardware is used to construct unique developmental switches that control highly specific developmental decisions.

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May 1992

specificity in such cases is likely to be provided at two distinct steps of the signaling cascade. The first one is the ligand-receptor interaction, which is inherently specific. Additional specificity can be provided by localized and transient production of a ligand, which results in activation of a widespread receptor only within a small number of cells. The second level of specificity operates at the end of these pathways, by the direct or indirect activation of cell-type or lineage-specific transcription factors. The exact basis for celltype specificity and the ability of a single cell to respond differently to several extracellular signals is a splendid problem that must be solved before biochemical understanding of differentiation and development can be attained. The experimental systems discussed below offer some opportunities for detailed understanding of such processes. Biological

Vulval

induction

in C. elegans

The vulva is the egg-laying organ of C. elegans. It is formed by 22 cells that descend from six vulval precursor cells (VPC5). Although only three VPCs contribute to the vulva, the remaining VPCs also have the potential to generate vulval cells. The main decision that determines the fate of the VPCs occurs in response to an inductive signal generated by the anchor cell. This highly localized signal stimulates the VPCs closest to the anchor cell to form vulva, whereas the remaining VPCs form nonspecialized hypodermis. Some genes involved in the perception and execution of this signal were identified by genetic analysis (70). Several of these genes encode proteins with high degrees of homology to known signal transducers. Loss-of-function mutations in one of these genes, let-23, prevent the assumption of the vulval cell fate by the VPCs, whereas gain-of-function mutations in let-23 result in multiple vulva (2). The let-23 gene encodes a protein that is structurally related to the mammalian EGF receptor. The similarity is especially high between the tyrosine kinase domains of the two proteins (44% identity). Lossof-function let-23 mutations are suppressed by gain- offunction mutations in another gene required for vulval determination, let-60. Loss-of-function let-60 mutations prevent the response to the inductive signal, suggesting that the let-60 product acts downstream to the let-23 product. Sequencing of the let-60 gene indicates that it encodes a protein with striking similarity to the human N-Ras protein (70-72). These results suggest that let-23 encodes a receptor for the inductive signal which turns on its tyrosine kinase activity. Like the mammalian Ras proteins, the let-60 product is involved in converting the tyrosine kinase activity of let-23 into signals that affect downstream targets, which in this case are involved in vulval cell fate determination (Fig. 5). It is likely that some of the other genes required for vulval induction that act downstream to let-60 encode serine/threonine kinases and various transcription factors that activate the expression of vulval-specific genes. The gain-of-function let-23 mutations are analogous to those that constitutively activate the EGF receptor to generate the erbB oncoprotein (11). Likewise, gain-of-function mutations in let-60 are similar to those responsible for the oncogenic activation of the mammalian Ras proteins (71-73). It will be interesting to determine if the vulval induction pathway converges to regulate the activity of a vulval-specific transcription factor in a manner analogous to the control of AP-1 activity by the mammalian growth factor signaling pathway. Embryonic

development

of Drosophila

Localized production of growth factors also has an important role in polarizing the early Drosophila embryo along its

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A.

B. 6ROWIH

FACTOR

ANCHOR

Res

lIt 60

CELL

\

are known. These genes, tailless and bukebein, function as gap genes required for formation of terminal structures (1, 75). The tailless gene product is a protein that belongs to the steroid/thyroid hormone receptor superfamily and is therefore a putative transcription factor (76). It will be of interest to determine whether activity or expression of the Tailless protein is regulated by phosphorylation and whether it is subjected to positive autoregulation similar to the signalactivated transcription factors described above. Anterior

HYPODERIIAL FATE RWTH INHIBITION

VULVAL FATE

GNIWTN

STIMULATION

Figure 5. Homologous signaling pathways control mammalian cell proliferation and vulva! induction in C. elegans. A) Soluble growth factors, such as EGF and PDGF, activate a tyrosine kinase receptor, which in turn activates a Ras protein. The activated Ras protein generates signals that lead to activation of transcription factors, such as AP-l, that induce expression of growth stimulatory genes and, at the same time, repress activity of putative transcription factors that control expression of growth inhibitory genes. B) An inductive signal generated by the anchor cell activates the let-23 gene product, a tyrosine kinase receptor, in the vulva! precursor cell. The activated let-23 kinase leads to activation of the let-60 gene product, which shows considerable homology to the mammalian Ras proteins. The activated let-60 protein is likely to generate signals that activate transcription factors responsible for assumption of the vulva! cell fate and repress transcription factors that control the hypodermal cell fate.

anterior-posterior (a-p) and dorsal-ventral (d-v) axes. Several genes, whose products are deposited into the egg by the mother, control the activity of zygotic genes involved in cell fate determination. One such maternal-effect gene is torso (for). Mutations in this gene affect the formation of terminal structures. Loss-of-function for mutations result in loss of the head and tail, whereas gain-of-function mutations disrupt formation of thoracic and abdominal segments. The for gene sequence predicts a product with extensive homology to cellsurface tyrosine kinase receptors (1, 74). Because the Torso protein is ubiquitously expressed in the early embryo, it is likely that a ligand localized to the terminal region of the embryo provides the specificity to this pathway. The product of another maternal effect gene involved in formation of the terminal regions is a good candidate for a Torso ligand. This gene is torso-like (1st), mutations of which produce a similar phenotype to loss-of-function for mutations. Mosaic analysis suggests that the tsl product functions in the maternal follicular cells during oogenesis (1). In addition, tsl mutations are suppressed by gain-of-function for mutations. On the other hand, gain-of-function for mutations, which probably result in constitutively activated Torso tyrosine kinase, should be suppressed by mutations in genes that encode downstream targets. One such gene is 1(1)pole/wle[1(1)ph]. 1(1)ph encodes a protein, D-Raf, the Drosophila counterpart of the mammalian Raf-1 protooncoprotein (75). These findings are extremely pleasing because they support the results of biochemical and reversed genetic analyses which indicate that the mammalian Raf-1 protein is a downstream mediator in the pathways activated by tyrosine kinase receptors (25).

Although identified,

direct downstream two other

targets for D-Raf remain to be

genes that act at the end of this pathway

SIGNAL TRANSDUCTION FROM CELL SURFACE TO NUCLEUS

pituitary

development

An example of how extracellular signals control cell fate determination by modulating the activity of a cell type-specific transcription factor is provided by studies of somatotrophs differentiation within the anterior pituitary. This endocrine organ develops from an invagmation of the oral ectoderm known as Rathke’s pouch. After the detachment of Rathke’s pouch from the roof of the mouth, the progenitors of the hormone-producing cells start a combined program of cell proliferation and differentiation, leading to the appearance of the endocrine cell-types of the mature pituitary (77). One of the progenitors, the acidophiic cell, gives rise to somatotrophs, which secrete growth hormone (GH), and lactotrophs, which secrete prolactin (PRL). Mutations that affect anterior pituitary development and function are relatively easy to identify because they produce a visible phenotype dwarfism. One such mutation, dw, also known as the Snell dwarf, was mapped to a locus on mouse chromosome 16, the location of the GHF1/Pitl gene (78, 79). The GHF1 gene encodes a sequence-specific transcription factor, GHF1, which activates expression of the GH and PRL genes (80, 81). GHF1 is exclusively expressed in cells that belong to the somatotrophic lineage, and is therefore a truly cell typespecific transcription factor (77). The dw mutation causes a simple amino acid substitution within the DNA binding domain of GHF1 that reduces its binding activity by at least 20-fold (79). Like several other transcription factor genes, expression of GHF1 is positively autoregulated (82). In addition, it is induced in response to elevated cAMP via the CREB protein, which binds to two sites in the GHF1 promoter (82). The somatotrophic cAMP levels are elevated in response to the hypothalamic polypeptide-growth hormone releasing factor (GRF). In addition to its effect on GH expression and secretion, GRF is a specific mitogen for somatotrophs (83). Overexpression of GRF in transgenic mice results in specific somatotrophic hyperplasia (84). A similar disease in humans, GH-secreting pituitary adenoma, is caused by mutations that lead to constitutive activation of the Ga0 protein coupling the GRF receptor to adenylate cyclase (85). Constitutive activation of the somatotrophic adenylate cyclase can be induced by expression of a GH-cholera toxin fusion gene. Transgenic mice expressing this gene display pituitary hyperplasia and gigantism due to excessive proliferation of somatotrophs (86). On the other hand, expression of a nonphosphorylated variant CREB protein driven by the GH promoter leads to pituitary hypoplasia, GH deficiency, and dwarfism (87). These findings indicate that the GRFactivated CAMP signaling system has an important role in controlling the expansion of the somatotrophic lineage. The CAMP signaling system is likely to operate by controlling the level of GHFI expression (79). Analysis of Snell dwarf pituitaries indicates that the total cell number in the pars distahs of the anterior pituitary (the location of the somatotrophs) is markedly reduced, compared with the

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number of cells in normal pituitaries. Another part of the antenor pituitary (pars intermedia), which does not contain somatotrophs, is not affected. Thus the dw mutation results in specific anterior pituitary hypoplasia and not only in the mere absence of GH and PRL expression. Inhibition of GHFI expression in pituitary cell lines by antisense oligonucleotides prevents cell proliferation, whereas removal of the inhibitory oligonucleotides restores GHF1 synthesis and cell proliferation (79). Collectively, these findings suggest the following pathway for controlling the development of anterior pituitary somatotrophs (Fig. 6). Activation of adenylate cyclase by GRF binding to its receptor results in increased cAMP levels and activation of PKA. This in turn leads to phosphorylation of CREB, which stimulates transcription of the GHFJ gene. Increased GHFI expression leads to activation of target genes involved in both proliferation and the assumption of somatotroph cell identity. Although two GHFI target genes involved in expression of the somatotrophic cell fate are known (GH and PRL), those involved in cell proliferation remain to be identified. It is evident, however, that a common transcription factor can control expression of both classes of target genes.

GRF

PKA

CONCLUSIONS The examples described above illustrate an emerging principle governing cell proliferation and fate determination. Growth factors and morphogens, most of them produced in a rather localized manner, interact with cell-surface receptors that belong to three major functional groups: seven-pass transmembrane receptors coupled to G proteins, cell surface- spanning tyrosine kinases, and heterodimeric receptors that associate with Src-type tyrosine kinases. Although each receptor class uses different mechanisms for signal transduction, their activation results in activation of downstream serine/threonine kinases. These signal-activated protein kinases are responsible for transmitting the signals generated by receptor occupancy to the transcriptional machinery in the nucleus. This step is achieved either by direct phosphorylation of sequence-specific transcription factors or by phosphorylation of proteins that interact with transcription factors to modulate their activity. Phosphorylation can either stimulate or inhibit the activity of a given transcription factor and thereby induce or suppress the transcription of subordinate genes. These changes in gene transcription affect cell proliferation and expression of the differentiated phenotype. Mutations that interfere with the activity of any one of these steps have dramatic and similar effects on cell proliferation and/or cell differentiation. Although these basic principles are fairly well established, several important problems remain to be answered. For example, it is not clear how the activation of a given signaling pathway can stimulate the proliferation of one cell type while inhibiting the growth of another. A similar problem is the ability of a single growth factor interacting with a single receptor to elicit a wide variety of biological responses that vary from one cell type to another. The biggest mystery is the ability of a single cell type to respond differently to two distinct growth factors whose receptors appear to be hard-wired to the same signaling system. It is our hope that the combination of molecular, genetic, and biochemical dissections of a variety of model systems will lead to reasonable solutions to these important problems. The author acknowledges support by the National Institutes of Health, Environmental Protection Agency, Department of Energy, and Council for Tobacco Research. Ms. K. Sexton provided excellent secretarial help and Dr. J. L. Castrillo has helped with preparation of the figures.

REFERENCES

PROLIFERATION FATE DETERMINATION

Figure 6. Signal transduction and somatotroph cell determination. GRF is a specific growth factor secreted by the hypothalamus, and activates a cell-surface receptor linked to a G3 protein on somatotrophic progenitors. This results in activation of adenylate cyclase followed by activation of PKA. The catalytic subunit of PKA translocates to the nucleus where it phosphorylates and activates CREB. One target for CREB action is the GHFJ promoter. Increased GHFJ transcription leads to production of more GHFI protein, which feeds back to secure permanent activation of the CHF1 gene by a positive autoregulatory loop. Other targets for GHF1 action include genes that stimulate the proliferation of somatotrophic progenitors and determined somatotrophs and genes involved in assumption of somatotrophic cell identity.

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