Genetics of signal transduction in invertebrates

64

Introduction

Genetics of signal transduction in invertebrates

Barry Dickson and Ernst Hafen

Universittit Ziirich, Ztirich, Switzerland

Receptor tyrosine kinases regulate a number of different cell fate decisions during invertebrate development. Genetic analysis of the signal transduction pathways activated by these kinases suggests that they converge upon a common pathway involving Ras and a cascade of cytoplasmic kinases, diverging again in the nucleus with the regulation of specific transcription

factors.

Current Opinion in Genetics and Development 1994, 4:64-70

Rapid progress has been made in recent years in the attempt to trace the pathways of intracellular signal transduction from receptor tyrosine kinases to the nucleus. Biochemical studies with vertebrate systems and genetic studies with invertebrates have converged to reveal a highly conserved signal transduction cascade. Here, we shall review the contribution of invertebrate genetics to the elucidation of this pathway.

Receptor tyrosine kinases control cell fate determination

The genetic analysis of cell-cell interactions mediated by receptor tyrosine kinases (RTKs) has focussed mainly, but by no means exclusively, on three signalling events, two in Drosophila and one in the ne- matode Caenorhabditis elegans. During C. elegans development, an inductive signal from the anchor cell of the gonad induces the formation of the vulva from a group of nearby hypodermal cells (Fig. la). The anchor cell signal is the-product of the h-3 gene, which bears homology to the mammalian epidermal growth factor (EGF) [l’l. The receptor for this signal is most probably the product of the let-23 gene, a RTK closely related to the mammalian EGF receptor [21.

In Drosophila, the RTK encoded by the gene torso is required for the correct specification of cell fates at the unsegmented termini of the embryo (Fig. lb) [3,41. The torso protein is uniformly distributed in the egg cell membrane, but is locally activated at the termini in response to an activity present in the terminal regions of the perivitelline space 15,6.,71. Genetic experiments suggest that the gene torso-like encodes this

activity, which may be either the torso ligand itself, or a substance required for its production, release, or interaction with torso 181.

Another RTK, encoded by the sevenless (SW) gene, is required later in Drosophila development for the specification of the R7 photoreceptor cell fate in the com- pound eye of the adult (Fig. lc) @-121. The sev protein is expressed on the surface of a number of cells, most of which are also competent to respond to its activation 113-151. The sev tyrosine kinase activity is stimulated in the R7 precursor cell upon binding of the ligand, the product of the bride of sevenless (boss) gene, which is expressed on the surface of the neighbouring R8 photoreceptor cell M-181.

A feature common to all three signalling events is that the restricted distribution of the ligand results in a non-uniform activation of the RTK, and ultimately the selection of different cell fates amongst a group of cells with equivalent developmental potentials (an equivalence group). It should also be noted that in each case the response to RTK activation is not cell proliferation, as seen, for example, in the responses of mammalian cells to the activation of various growth factor receptors. Rather, R’IX activation affects the terminal differentiation of the cell.

Convergence

One of the immediate consequences of the stimulation of all three RTKs is probably the activation of the p21”-m homologues encoded by the genes Rasl (or Drasl) in Drosophila [191, and let-60 in C. elegans 1201. In Drosophila, Rasl mutations have been isolated both as enhancers of a partial loss-of-function sev allele 1211, and as suppressors of a gain-of-function

Abbreviations boss-bride of sevenless; nw-corkscrew; OrI-Downstream of receptor kinases; EGF-epidermal growth factor;

ERK-extracellular signal-related kinase; CNRF-guanine nucleotide release factor; hk6-hckebein; MAPK-mitogen-activated protein kinase; MEK-MAPK or ERK kinase; RTK-receptor tyrosine kinase;

sebsevenless; SH-Src homology; h-Son of sevenless; t/Ltai//ess.

0 Current Biology Ltd ISSN 0959-437X

Invertebrate signal transduction Dickson and Hafen 65

(a) Caenorhabditis elegans let-23 pathway

Anchor cell

(b) Drosophila (c) Drosophikl torso pathway sevenless pathway

Blastoderm Photoreceptors

Vulva1 fate Terminal fate Neural fate

0 1994 Current Opinion in Genetics and Development

Fig. 1. Signalling pathways controlled by receptor tyrosine kinases in invertebrates. (a) Vulva1 induction in C. elegans. An inductive signal from the anchor cell specifies 1 ‘, 2’ and 3’ fate in the vulva1 precursor cells. Cells that assume the 1. or the 2’ fate will form the vulva. (b) The terminal system in Drosophila. At the blastoderm stage, a signal in the perivitellinic fluid activates locally the torso receptor tyrosine kinase, and thereby triggers the differentiation of terminal structures. (c) Specification of the R7 photoreceptor cells. The boss protein expressed on the differentiating R8 photoreceptor cell activates the sev receptor on the neighbouring R7 precursor, and thereby triggers its differentiation as an R7 cell.

torso allele 1221. Thus, under certain conditions, Rasl is a rate-limiting component of both pathways. Fur- thermore, the expression of a dominant Rasl allele (RaslVj’lz) in all seu-expressing cells 123.1, and the injection of activated p2l”-rus protein into embryos 124’1, have shown that Rasl activation alone can induce the normal response to RTK activation. Similarly, in C. elegans, both recessive loss-of-function and dominant gain-of-function alleles of the let-60 Ras gene have been recovered, and produce phenotypes consistent with disruption and constitutive activation of the let-23 pathway, respectively 1251.

Activation of Ras, therefore, represents a point of convergence for all three signalling pathways, and there is evidence that other RTKs in Drosophila may also act through Rasl [22,26,271. One pathway by which Ras is activated in response to RTK stimulation has now been fairly well characterized in both vertebrates and invertebrates. This involves SH3-SH2SH3 adaptor proteins (Drk in Drosophila [28”,29”1, Sem-5 in C. elegans [30”1, and Grb2 in vertebrates [311>, which act as a link between activated RTKs and the Ras guanine nucleotide release factor (GNRF) SOS. Gain- of-function mutations in SOS (Son of sevenless) have been isolated as suppressors of a weak sev mutation 1321. Loss-of-function mutations both in SOS and Drk (Downstream of receptor kinases) were isolated in the same screen for components of the sev signalling pathway as were Rasl mutations 1211, and SOS alleles were also recovered along with Rasl alleles as suppressors of signalling from an activated torso RTK 1221. Similarly, mutations in sem-5 were recovered along with let-23 and let-60 alleles in a screen for mutations affecting C. elegans vulva1 development 13O”l. The current model for Drk and SOS function, based on these genetic data and biochemical experiments per- formed with both the Drosophila proteins [28”,29”1 and their vertebrate homologues 133-371, postulates

that Drk binds, via its SH2 domain, to phosphotyro- sine residues in the cytoplasmic domain of the activated RTK, and, via one or both SH3 domains, to proline-rich sequences in the carboxy-terminal tail of SOS. This interaction, perhaps by inducing a conforma- tional change or allowing phosphorylation of the SOS protein, or simply by recruiting it to the plasma membrane, stimulates SOS GNRF activity, and thereby the exchange of inactive Ras-GDP for active Ras-GTI?

In contrast to the Ras homologues, no gain-of-function alleles that bypass the requirement for RTK stimulation have been recovered for D&&m-Sor for SOS. This may simply be due to experimental difficulties in isolating such alleles, but could also be an indication of the fact that this pathway alone is not sufficient to induce Ras activation. Indeed, there is circumstantial evidence that parallel pathways to Ras may exist and, furthermore, that in contrast to the Drk-Sos route, these pathways may to some extent be RTK-specific. In the sev pathway, Rasl appears to be subject to negative regulation via the putative GTPase stimulating protein Gap1 1381, and also via the raplA/Krev homologue encoded by the Roughened gene 1391. Both of these potentially offer additional means of regulating Rasl activity in response to sev stimulation. In the torso pathway, loss-of-function co&screw (csw) mutations produce a phenotype similar to that observed with partial loss of torso function [40’1. This defect can be rescued by activated p21C-‘a, suggesting that csw functions between torso and Rasl 124’1. The csw gene encodes a protein tyrosine phosphatase linked to tandem SH2 domains 140’1. Its mammalian homologue, known variously as SH-WI’2 1411, PTPlD 1421, or Syp [431, has been shown to bind activated RTKs and become phosphorylated on tyrosine residues, consistent with it having a role in mediating signal transduction between RTKs and Ras. No invertebrate homologues of vertebrate RTK substrates such as SHC (Src homology containing), IRS-l (insulin

66 Oncogenes and cell proliferation

receptor substrate-l), PI3-K (phosphatiolylinositol 3-kinase) and PLC-y (phospholipase C-r> [441 have yet been recovered in such genetic screens. However, several loci identified in screens for additional components of the sev, torso and let-23 signalling pathways still await molecular analysis, and it would not be surprising if at least some of these turn out to encode homologues of these pioteins.

A common kinase cascade

Signal transduction from the plasma membrane to the nucleus appears to involve a highly conserved cascade of serine/threonine and threonine/tyrosine kinases. Genetic evidence is consistent with a single, linear cascade being responsible for all aspects of signalling initiated with RTK activation; as yet, there is no indication of any branching or of the existence of parallel signalling pathways.

This single cascade involves the consecutive actions of the kinases Raf, MEK and MAPK. The Drosophila Raf-1 homologue, encoded by the gene variously known as r-al Draf or i(l)polehole, has been shown to be required for signalling in both the torso and sev pathways [45,46’1, and the C. elegans homologue h-45 is required for let-23 signalling 1471. A MEK OVIAPK or ERK kinase) homologue has been identified only (so far) in Drosophila. Mutations in this gene, Dsorl, were recovered as second-site suppressors of a weak ruf allele, suggesting that activation of MEK can compensate for a partial loss of Raf function [48*1. An involvement of Dsorl in the torso pathway has been clearly demon- strated, but a possible role in the sev pathway awaits further investigation. The third kinase in this cascade is MAPK (mitogen-activated protein kinase), encoded in Drosophila by the gene rolled. Like Raf, the MAPK encoded by rolled has been shown to be required for signalling from both torso and sev [49**1.

Biochemical investigations of the vertebrate Raf, MEK and MAPK kinases have led to a model for signal transduction along this cascade (see reviews by Marshall (pp 82-W) and Ammerer (pp 90-951, and references therein). Activated Ras-GTP interacts directly with the amino-terminal regulatory region of Raf-1 . Raf-1 phosphorylates and activates MEK, which in turn phosphorylates both the threonine and tyrosine residues in the conserved TEY motif (in the one letter amino acid code) of MAPK. Activated MAPK then enters the nucleus, where it phosphorylates, and thereby regulates, the transcriptional activities of proteins such as c-Jun and Elk-l. Genetic analysis of the sev pathway has es- tablished the same order of action for Rasl, Raf and the MAPK encoded by rolled; both raf and rolled mutations interfere with signalling from an activated Rasl protein, and rolled, but not Rasl, mutations suppress signalling from an activated Raf kinase [46’,49”1.

The most important contribution of invertebrate genetics to our understanding of this kinase cascade,

however, is the indication that it is a linear cascade mediating all aspects of signal transduction. Analysis of loss-of-function phenotypes is somewhat difficult in this regard, since, due to the more general requirement for these intracellular signal transduction components, a complete loss of gene function is lethal. Neverthe- less, certain viable allelic combinations of these genes, or homozygous mutant clones generated within a het- erozygous, and therefore viable, animal, show phenotypes similar to those seen in torso, sa, or let-23 mutants, consistent with a model in which they mediate the full response to RTK activation.

More convincing evidence for a single pathway, however, is provided by the ability of constitutively activated versions of these signal transduction components to mimic gain-of-function RTK mutations. For Drosophila Rasl and Raf, such constitutively activated proteins have been generated by in vitro mutagenesis, and either injected as mRNA into embryos or reintro- duced into transgenic flies under the control of foreign regulatory sequences (namely those of the sev RTK). Injected into syncitial embryos, they produce phenotypes characteristic of gain-of-function torso alleles (124.1; F Sprenger, personal communication), and when expressed in the developing eye, they mimic constitutive activation of the sev RTK [23’,46’1. These results argue strongly against the existence of parallel pathways required to transduce either the torso or the sev signal. They are, however, subject to the caveat that the artificially generated activity of these components may be so high as to overcome the need for signalling by any parallel pathway. This objection is much harder to make for the gain-of-function Dsorl and rolled alleles, which have been generated by in vivo mutagenesis and are therefore expressed under the control of the endogenous regulatory elements [48*,49”]. Genetic evidence suggests that both are only weakly activating mutations (stronger mutations would presumably have been lethal, an obvious hindrance to their in vivo isolation), yet they are still able to mimic constitutive activation of either the torso RTK (for Dsorl) or both the torso and sev RTKs (for rolled).

Divergence

Biochemical studies have led to the identification of an increasing number of MAPK substrates, including transcription factors such as Elk-l and c-Myc, and other kinases such as p74nk and MAPKAP2, which may also enter the nucleus and phosphorylate additional transcription factors. In addition, several of these transcription factors are known to be substrates for other kinases, such as c-Jun which is phosphorylated by protein kinase A and casein kinase II. The picture that emerges from these studies is a complex network of reactions in which MAPK regulates the DNA-binding properties and/or transcriptional activities of a number of proteins, which are also responsive to additional regulatory inputs. Events downstream from MAPK thus


represent a point of divergence from a common signal transduction cascade to cell-specific, and probably also cell-cycle-specific, effecters that ultimately determine the nature and timing of any response to stimulation.

Invertebrate studies have yet to provide a con- firmed MAPK substrate, though likely candidates have emerged for both the torso and sev pathways. Pattern- ing along the anterior-posterior axis of the Drosophila embryo is controlled by three independent systems. A morphogenetic gradient of the bicoid homeobox protein regulates pattern formation in the anterior half of the embryo, and a gradient of nanos mRNA directs pattern formation in the posterior half. Activation of the torso signal transduction pathway imposes a third level of pattern formation upon these two systems, allowing formation of the unsegmented head and tail regions of the embryo [501. The differentiation of head and tail structures at opposite ends of the embryo in response to torso activation illustrates how a common signal can elicit both general and context-specific responses. The response common to both anterior and posterior poles is the expression of the genes tailless <tlO and htrcke- kin (hkb) (51,521. Expression of tlf can also be induced in the central regions of the embryo by local, ectopic activation of the torso pathway [531. These observations argue for the existence of an as yet unidentified, ubiq- uitously expressed transcription factor responsible for initiating tll and hkh expression upon stimulation of the torso pathway. In the simplest model, this would be a direct result of its phosphorylation by the rolled MARK.

While tll and hkh expression is a response common to both poles of the embryo, there are additional responses that are observed only at the anterior pole, such as the repression of the genes ortbodenticle

and hunchback [54,55”1. These responses have been shown to be due to down-regulation of bicoid activity by the torso signal transduction pathway, and correlate with the phosphorylation of bicoid [%“I. Regulation of bicoid at the anterior pole thus represents a branch point within the torso pathway, which, on the basis of genetic evidence, must lie downstream of Raf but upstream of the activation of tll and hk&. The presence of consensus MARK phosphorylation sites in the bicoid protein suggests that this branch point occurs immedi- ately downstream of the rolled MARK.

A likely MARK substrate during eye development is the ets-like protein encoded by the gene yan l56.1, which contains no less than 19 potential MARK phosphorylation sites. Loss of yan function produces a phenotype reminiscent of constitutive activation of the sev pathway, indicating that the normal role of the yan protein might be to inhibit photoreceptor development, and that phosphorylation by the MARK encoded by rolled releases this inhibition. R7 photoreceptor development also requires the presence of the nuclear protein encoded by the gene seven in absentia Gina), irrespective of whether it is induced by activation of the sev signal transduction pathway or ectopically in yan mutants [56.,571. The putative sina protein contains neither a recognizable DNA-binding domain nor consensus MARK phosphorylation sites, but both its expression pattern and mutant phenotype suggest that it plays a key role in the initiation of R7 development in response to sev activation.

It is still premature to speculate on events occurring in the nuclei of the vulva1 precursor cells during C. elqgans vulva1 development, since the most downstream component of the let-23 pathway yet to have

Caenorhabditis Drosophila

let-23 torso sev

-&fyfL ZL

SH2 adaptors sem-5 Drk Drk

Ras CNRFs ? SOS SOS

Ras let-60 Rasl Rasl

CAPS ? ? CAP1

Raf lin-45 Raf Raf

MAPKK ? Dsorl Dsorl ?

MAPK ? rolled rolled

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Nuclear factors ? bicoidj? sina/yan

;~Jg;“e

0 1994 Current Opinion in Generics and Developm?nl

Fig. 2. Comparison of the signal transduction components identified genetically in the let-23, torso and sev pathways. Note the striking similarity in the cytoplasmic components used in the three different systems and their homology to signal transduction components identified bio- chemically in vertebrates.


been characterized molecularly is the I&f homologue lin-45. However, the rapid progress in the cloning of genes previously identified in screens for defects in vulval development is certain to ensure that this sit- uation is only temporary. One of the more intriguing developmental defects in these collections occurs in lin-32 mutants, where the vulval precursor cells se- lect their fates apparently at random. This gene has recently been cloned and found to encode a member of the I-DE-$/forked head family of transcription factors [581, though in the absence of information on its expression pattern or site of action it is difficult to const.rWt models for its normal function.

Coriclusions and future prospects

The genetic analysis of RTK-activated signal transduction pathways in invertebrates has provided strong evidence that the biochemical properties of signalling components observed in vitro accurately reflect their function in vivo. These studies have also revealed the high degree of evolutionary conservation of this pathway (Fig. 2), suggesting that it represents a fundamen- tal mechanism by which cell growth and differentiation are regulated in response to external stimuli. While the remarkable consistency in the results of vertebrate and invertebrate studies inspires confidence in the essen- tial details of this pathway, many aspects still remain to be clarified. In addition, it will be important to doc- ument to what extent this basic pathway is subject to local, cell-specific variations, and to unravel the complex events by which the incoming signal is translated into specific patterns of gene transcription, and ultimately cell differentiation, The results presented in this review are based on the detailed genetic and molecular analysis of only a handful of the genes identified in screens for mutations affecting signal transduction from either the torso, sev or let-23 RTKs. Many more still await further analysis. Invertebrate genetics thus holds the promise of providing further insights into the molecular mechanisms involved in intracellular signal transduction.

References and recommended reading 15.

Papers of particular interest, published within the annual period of review, have been highlighted as: . of spcclal interest . . of outstanding interest

1. HILL RJ, STERNBERG PW: The Gene fh3 Encodes an Induc- . tlvc Signal for Vulval Dcvclopment in C. e&gans Nahrre

1992, 358:470-476. The gene fin-3, encoding a novel member of the EGF family of growth factors, ls,identificd as the likely l&and for the ler-23 EGF- receptor homologue during C. eAtguns vulval development; /in-3 is expressed ln the anchor cell and acts genetically upstream of ler-23. Overexpression of ffn-3 produces a dominant, multivulva phenotype dependent on &r-.25 function.

2. Aaolrur RV, KOGA M. MENDEL JE, OHSHIMA Y, STERNBERG PW: The let-23 Gcnc Ncccssary for GaetwrbabdltLs eiegans Vulval induction Encodes a Tyrosine Kinasc of the EGF Receptor Subfamily. Nanrre 1990, 348693699.

3. KLINGLER M, EHD~LYI M, S~A~AD J, N~JSSLEIN-VOLHARD C: Function of rorsu in Dctcrmining the Terminal Anlagcn of the Dtusopbika Embryo. Nature 1988, 335275277.

4. SPRENGER F, STEVENS LM, N&SLEIN-VOLHARD C: The Dmsopbika Gcnc torso Encodes a Putative Receptor Ty- mslne Kinase. Nahm 1989, 33847-3.

5. CASANOVA J, STRUHL G: Locallzcd Surface Activity of rorso, a Receptor Tyrosinc Klnase, Spccilics Terminal Body Pattern in Dmsopbfla. Genes L&u 1989, 3:2025-2038.

6. SPRENGER F, N~SSI.EIN-VOLHARD C: torso Receptor Activity is . Regulated by a Diisiblc Ligand Produced at the Extraccl-

htlar Terminal Regions of the Lkxwpbfla Egg. CeU 1992, 71987-1001.

Injection of rorso mRNAs into Dmsopbf&a embryos reveals that torso activity is regulated by a ligand produced at the terminal regions of the egg during embryogenesis. Binding of the ligand (present in limiting amounts) to the receptor (present at saturating levels) is proposed to establish a gradient of ligand distribution, and thus, torso tyrosine kinase activity. Therefore, torso also serves to local- ize its own ligand!

7.

8.

9.

10.

11.

12.

13.

14.

16.

17.

18.

CASANOVA J, SIRUBL G: The torso Rcccptor Localizes as Well as Transduces the Spatial Signal SpccQing Terminal Body Pattern in Dmsopbffa Nature 1993, 362:152-155.

SI-EVENS LM, FROHNH~)FEH HG, KLINGLEH M, N~JSSLEIN- VOLHARD C: Localized Requirement for rorso-&e Expression in Follicle Cells for Dcvclopmcnt of Terminal Anlagcn of the Dmsopbfla Embryo. Nahrre 1991, 346660-663.

TOMLINSON A, RWDY DF: seuenless: a Cell-Specific Homcotic Mutation of the Lkwopbfla Eye. Scfence 1986, 231:400402.

HAFEN E, BA!XER K, EDSTROEM JE, RUUIN GM: sevenless, a Cell-Spcclflc Homcotic Gene of LWosopbffa. Encodes a Putative Transmcmbranc Receptor with a Tyrosinc Kinasc Domain. Scfence 1987, 23655-63.

BASLER K, HAFEN E: Control of Photoreceptor Cell Fate by the scvcnlcss Protein Rcquircs a Functional l)msinc Kinasc Domain. Cell 1988, 54:299-312.

BOWTELL D, SIMON MA, RUMN GM: Nucleotidc Sequence and Structure of the smfess Gene of Drosopbifa mekwwgasm Genes L&u 1988, 2:62C-634.

TOMLIN~ON A, BOW~HLL DDL, HAFEN E, RUI~IN GM: Localiza- tion of the Scvcnlcss Protein, a Putative Receptor for Posi- tional Information in the Eye lmaginal Disc of Dmsopbffa. Cell 1987, 51:143150.

BANERJEE U, KENFRANZ PJ, HINIDN DR, U~IN BA, BENZER S: The scvcnlcss Protein is Exprcsscd Apically in Cell Mcm brancs of Dcvcloping Lhnsopbffa Retina; it is Not Restricted to CcU R7. Cell 1387, 51:151-15%

DICKXIN B, SPRENGER F, HAFEN E: Prcpattem in the Develop ing Dnxopbffa Eye Rcvcalcd by an Activated torso-scvcnless Chimcric Receptor. Genes L%v 1992, 6:2327-2339.

HART AC, KR~~MER H, VAN-VACI’OR DLJ, PAIDHLINGA~’ M, 2lPURsKY SL: Induction of Cell Fate in the Llnxopbila Retina: The bride of scvenlcss Protein is Prcdictcd to Con- tain a Large Extracellular Domain and Seven Transmcmbranc Segments. Genes Deu 1990, 4:1835-1847.

KI&~MEH H, CAGAN RL, ZIPUR~KY SL: Interaction of bride of scvcnlcss Membrane-Bound Ligand and the sevenless TyrosincKinasc Rcccptor. Nuhrre 1991, 352:207-212.

HART AC, KRAMER H, ZIPUR~KY SL: Extracellular Domain of the boss Transmcmbranc L&and Acts as an Antagonist of the scv Receptor. Nature 1993, 3613732-736.


19. NEUMANN-SILBERBERG FS, SCHEJ’IXR E, HOFFMANN FM, SHILO 30. CLARK SG, STERN MJ, HORVITZ HR: C e&zgans CeU-SignaRing B: The Lhwopbfka ras Oncogenes: Structure and Nucleotide . . Sequence. Cell 1984, 37:1027-1033.

Gene send Encodes a Proqin with SH2 and SH3 Domains. Nature 1992, 356:34&344.

20. HAN M, STERNBERG PW: lerdo, a Gene that Specifies Cell Fates during C. ekgans Vulval Induction, Encodes a ras Protein. Cell 1990, 63:921-931.

21. SIMON MA, BOWIXLL D, DODSON GS, ~VER’I~ TR, RUBIN GM: Rasl and a Putative Guanine Nuclcotidc &change Factor Perform Crucial Steps in Signaling by the sevenless Protein ‘Qrosinc Kinase. Cell 1991, 67:701-716.

Genetic studies provide the first clear evidence that SH2 and SH3 domains participate directly in signal transduction from receptor ty- mine kinascs. The C. ekgans gene sent-5 is shown to encode a protein consisting of nothing more than a single SH2 domain flanked by SH3 domains. Genetic epistatis experiments indicate that Sem-5 acts between the let-23 RTK and the let-60 Ras protein.

31.

22. DOYLE HJ, BISHOP JM: torso, a Rcccptor ‘Qrosinc Kinase Re- quired for Embryonic Pattern Formation, Shares Substrates with the sevenless and EGF-R Pathways in Lhsopblla. Genes Dw 1993, 7~633-646.

LOWENSIEIN EJ, DALY RJ, BATZER AG, LI W, MARGOLIS B, lAMMERS R, ULLRICH A, SKOLNIK EY, BAR SD, SCHLWINGER J: The SH2 and SH3 Domain-Containing Protein GRB2 Links Receptor Tyrosinc Kinases to ras Signaling. CeU 1992, 70~431-442.

32.

23. FORI~NI ME, SIMON MA, RUI~IN GM: SignaIling by the seven- . less Protein Tyrosinc Kinase is Mimicked by Rasl Activation.

Nature 1992, 355:559-561.

ROGGE R D, KAR~OVICH C A, BANEWEE U: Genetic Diic- tion of a Ncurodevelopmcntal Pathway Son of sevenless Functions Downstream of the scvenlcss and EGF Rcccptor Tyrosiic Kinascs. Cell 1991, 64:3-B.

Constitutive activation of Rasl both mimics the response to, and by- passes the need for, sev activation in the induction of R7 cell development, suggesting that the sole function of sev activation is to set the Rasl switch.

24. Lu x, CHOIJ 723, WILLIAMS NG, HORERIS T, PERRIMON N: . Control of Cell Fate Dctcrmination bypZ1ras/Rasl. an Es

scntial Component of torso Signaling in Lhsopbfla. Genes Dev 1993, 7:621-632.

Just as ectopic activation of Rasl mimics sev activation, injection of pZl”mS protein into embryos produces phenotypes chamcteristic of torso gain-of-function alleles. By injecting into embryos of various maternal genetic constitution, Ras is placed downstream of tomo and co&screw, but upstream of ra/

25. BEITFL GJ, CLARK SG, HORVI’IZ K: CaenorbatdtUs elegans rus Gene lerdo Acts as a Switch in the Pathway of Vulva1 Induction. Nature 1990, 348:503-509.

26. DIAZ-BENJUMEA F, HAFEN E: The scvenlcss SignaIling Cas- sette Mediates Lhsopblla EGF Receptor Function during Epidcrmal Development. Development 1994, in press.

27. REICHMAN-FRIED M, DICKSON B, HAFEN E, SHILO B-Z: Ehtcida- tion of the Role of halbless, a Lhsopbtla FGF Receptor Homolog. in Tracheal Cell Wigration. Genes Dev 1994, in press.

2n. OLI~I~R JP, RMBE T, HENKEMEYER M, DICKSON B, MBAMALU . . G, MARGOLIS B, %ZHL~%SINCEH J, HA~XN E, PAWSON T: A

Lhsopbfla SHZ-SH3 Adaptor Protein Implicated in Cou- pling the sevenless ‘Qrosinc Kinasc to an Activator of Ras Guaninc Nuclcotidc Exchange, Sos. Ceil 1993, 73:179-191.

A h-osophih homologue of the sem-5/Grb2 SH3-SHZ-SH3 proteins was identified simultaneously by both by biochemical and genetic approaches: as a protein capable of binding an activated RTK, and as a mutation which suppressed the phenotype caused by the constitutive activation of the sev RTK. This protein, called Drk, is shown to bind not only to activated RTKs, but also to the SOS guanine nucleotide releasing factor, suggesting for the first time a means by which Ras activation is coupled to RTK stimulation. Whereas wild-type Drk protein is located at the plasma membridne, mutant Drk proteins, with defective SH2 domains, are shown to be cy- toplasmicdlly localized, providing a good explanation of why this pathway breaks down in Drk mutant...

29. SIMON MA, LIODSON GS, WUIMN GM: An SH3-SHZ-SH3 . . Protein is Required for p21Rasl Activation and Binds to

sevenless and SOS Proteins In vllm. Cd 1993, 73:169-177. In an earlier paper [Zll, the authors describe an elegant genetic screen in which a partially functional sttv kinase (constructed In olrro) was used to identify rate-limiting components in the scv signal transduction cascade. Mutations were isoktted that dominantly enhance this weak SW phenotype. Seven such lz@u) loci were identified, including Rash and SOS. Here a third E&u) locus, Drk, is described. Similar conclusions concerning Drk function are rcdched as in [28”1.

33.

34.

35.

36.

37.

38.

39.

40. .

EGAN SE, GIDDINGS BW, BROOKS MW, BUDAY L, SIZELAND AM, WEINBERG RA: Association of Sos Ras Exchange Protein with Grb2 is Implicated in Tyrosiic Kinase Signal Transduc- tion and Transformation. Nattrre 1993, 363:45-51.

ROUKIS AM, FERNLEY R, WADE J, PAWSON T, BOWTELL D: The SH2 and SH3 Domains of Mammalian Grb2 Couple the EGF Receptor to the Ras Activator mSos1’. Nature 1993, 363:8%85.

LI N, BATZER A, DALY R, YAJNIK V, SKOLNIK E, CHARDIN P, BAR SD, MARGOWS B, SCHLESSINGER J: Guaninc-Nuclcotide- Releasing Factor hSos1 Binds to Grb2 and Links Receptor mine Kinascs to Ras SignaIling. Nature 1993, 363:85&l.

GALE NW. KAPLAN S, LOWENS~FIN EJ, %ZHLE%SINGER J, BAR SD: Grb2 Mediates the EGF-Dependent Activation of Guanine Nuclcotidc Exchange on Ras. Nature 1993, 363:88+2.

BUDAY L, DOWNWARD J: Epidermal Growth Factor Regulates pllras Through the Formation of a Compla of Receptor, Grb2 Adapter Protein, and SOS Nuclcotidc Exchange Factor. Cell 1993, 73:611620.

GAUL U, MARDON G, RUBIN GM: A Putative Ras GTPase Activating Protein Acts as a Negative Regulator of Sii ing by the smtdcss Receptor Tyrosinc Kinase. Ceil 1992, 68:1007-1019. ’

HAUIHARAN IK. CARTHEW RW, RUBIN GM: The Dmsopbila Roughened Mutation: Activation of a Rap Homolog Diirupts Eye Development and Interferes with Cell Determination. Cell 1991, 67~717-722.

PERKINS LA, LARSEN I, PERRIMON N: co&scn?tu Encodes a Pu- tative Protein Tyrosinc Phosphatase that Functions to Trans duct the Terminal Sii from the Receptor Qrosine Kinase torso. Ceil 1992, 70:225-236.

A somewhat enigmatic component of the torso pathway, co&screw, encodes a cytoplasmic tyrosine phosphatase with two SH2 domains required for efficient signalliig from torso. Mammaliin homologues have since been isolated and shown to bind RTKs. conbscrew pro- vides a unique genetic means to investigate the function of thii class of proteins in signal transduction.

41.

42.

43.

44.

FREEMAN RJ, PLUIZKY J, NEEL BG: Identification of a Human Src Homology Z-Containing ProtcirrTyrosinc-Phosphatase: A Putative Homolog of Dmsopbila ctdscn?w. Pmc Narl Acad Scf USA 1992, 89~11239-11243.

VOGEL W, LAMMERS R, HUANG J, ULWIICH A: Activation of a Phosphotyrosinc Phosphatase by Tyrosine Phosphorylation. Scitnce 1993, 259:1611-1614.

FENG GS, HUI CC, PAWSON T: SHZ-Containing Phosphotyro- sine Phosphatase as a Target of Protein-Tyrosiic Kinases. Scfence 1993, 259:1607-1611.

SCHLE~~INGER J, ULLRICH A: Growth Factor Signaling by Rc- ccptor Tyrosine Kinascs. Neuron 1992, 9:383-391.


45. AMBROSIO L, MAHOWALD A P, PERRIMON I$ Requirement of the Lkwopblla taf Homologue for torso Function. Nature 1989, 342:2-291.

46. DICKSON B, SPRENGER F, MORRISON D, HAFEN E: Raf Func- . tions Downstream of Rasl in the sevenless SignaI Transduc-

tion Pathway. Nature 1992, 360600603. Loss-of-function and gaInof-function aiieles Implicate Raf as a component of the sev signal tmnsduction cascade that acts after Rasl but before the nuclear factor sina. Raf activation mimics both sev and Rasl activation,’ suggesting a linear pathway from sev to Raf.

47. HAN M, GOLDEN A, HAN Y, SIFI~NUERG PW; C elegans llrr45 ruf Gene Pa&cipatcs in Ier-60 m&Stimulated VuIvaI DItTcr- cntiation. Naftrre 1993, 363:133-140.

48. TSUDA L, INOUE YH, Yoo MA, MIZUNO M, HATA M, LIM . YM, ADACHI YT, RYO H, MASAMUNE Y, NISHIDA Y: A Protein

Kinase Similar to MAP KInase Activator Acts Downstream of the raf Kinasc in Dmsopbila. Cell 1993, 72:407-414.

Dominant mutations that partially restore the viability of mf mutants leadsto the identification of L&w-l a Drosophila MEK homologue. Dsorl is required for signaliing in the torso pathway.

49. BRUNNER D, OELLERS N, S~BAD J, BIGGS III WH, ZI~UR~KY . . SL, HAFEN E: A Gain of Function Mutation in Lhxsopbila

MAP Kinase Activates Multiple Receptor Tyrosinc Kinasc SignaIIing Pathways. Cell 1994, in press.

A dominant mutation In the ?oIlti MAPK is recovered In a screen for mutations that can restore signaiiing in the sev pathway in the absence of the Inducing signal, the boss protein. This mutation is also found to activate the torso pathway, suggesting that activation of MAPK is both necessary and sufficient for signal tmnsduction in both pathways.

50. PEKRIMON N: The torso Receptor Protein-‘Iyrosinc KInase SignaIing Pathway An Endless Story. Cell 1993, 74:219-222.

51. PICNONI F, BALDAREU RM, SIFINCRIMS!N E, DIAZ FJ, PATAPOUWW A, MEKHIAM JR, &NGYEL JA: The Drosopbfla Gene tah7es.s is Expressed at the Embryonic Termini and is a Member of the Steroid Receptor Superfamily. Cell 1990, 62:151-163.

52. WEIGEL D, J~RGENS G, KLINGLER M. JACKI.E H: tie Gap Genes Mediate Maternal Terminal Pattern Information in LhwopbUa. Sclence 1990, 248:495-498.

53. STEINGRIMSSON E, PIGNONI F, LIAW GJ, LENGYEL JA: Dual Role of the Llmsopblka Pattern Gene tailless in Embryonic Tcr- mini. Sctence 1991, 254:4X%421.

54. FINKEUIXIN R, PERRIMON N: The ododetzticle Gene is Re& ulated by bicoid and torso and Specifies Dmsopbih Head Development. Nattrre 1990, 346:485-W.

55. RONCHI E, TREISMAN J, D~STAINI N, STRUHL G, DESPLAN C: . . Down-ReguIation of the Dmsopbfla Morphogcn bicoid by

the torso Receptor-Mediated Signal Transduction Cascade. Cell 1993, 74347-355.

The bicoid protein, a transcription Factor that acts as a morphogen in the patterning of Dmsopbtla embryos along the anterior-posterior axis, is found to be one of the targets of the torso signal tmnsduction cascade. The tnmscriptional activity of bicoid is down-regulated in response to activation of the torso pathway, correlating with the phosphotyiation of bicoid In vlvo. These observations demonstrdte how the input from a signal transduction cascade can be integrated with other Inputs (e.g. from a morphogenetic grddient) to produce different, context-specific responses to the same signal.

56. LAI Z-C, RUBIN GM: Negative Control of Photoreceptor De- . velopmcnt in Dmsopbila by the Product of the yun Gcnc,

an ets Domain Protein. Cell 1992, 70609620. Genetic experiments show that the product of the yan gene is required cell autonomously to inhibit photoreceptor development. The yan gene encodes a putative DNA-binding protein with an ets domain, suggesting a model in which activation of the sev pathway might serve to release certain genes required for photoreceptor differentiation from yan-mediated repression.

57. CAR~HEW RW, RUBIN GM: seven In absentia, a Gene Rc- quircd for Specification of R7 CeU Fate in the Drosophila Eye. CclI 1990, 63561-577.

58. MILLER LM. GALLEGOS ME, MORI~XAU DA, KIM SK: Iii-31, a Caenorbabditfs elegans HNF-3/fork bead Transcription Factor Homolog, Specifics Three Alternative Cell Fates in VuIvaI Development. Genes Pro 1993, 7:933-947.

1~ Dickson and E Iiafen, Zoologisches Institut, Universitiit Zurich, Winterthurerstrdsse 190, 8057 Ziirich, Switzerland.

Genetics of signal transduction in invertebrates

Documents

Transcript of Genetics of signal transduction in invertebrates