Genetic Analysis of rolled, Which Encodes a Drosophila Mitogen-Activated Protein Kinase

Corresponding author: Yasuyoshi Nishida, Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan., nishida{at}bio.nagoya-u.ac.jp (E-mail)

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic and molecular characterization of the dominant suppressors of D-raf^C110 on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rl^Su23, was found to bear the same molecular lesion as rl^Sem, which has been reported to be dominant female sterile. However, rl^Su23 and the current stock of rl^Sem showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.

MITOGEN-activated protein kinase (MAP kinase or MAPK) plays essential roles in the transduction of diverse extracellular signals regulating cellular proliferation and differentiation. Its activity is closely regulated by phosphorylation of both threonine and tyrosine residues in its activation loop by a dual-specificity kinase MAP kinase kinase (MAPKK or MEK). MAPKK is also regulated by phosphorylation of two adjacent serine/threonine residues in its activation loop by MAP kinase kinase kinase (MAPKKK or MEKK). This cascade of protein kinases, known as the MAPK cascade, is highly conserved during evolution and found ubiquitously among eukaryotes (NISHIDA and GOTOH 1993 ; DAVIS 1994 ).

Drosophila also contains the MAPK cascade, and rolled (rl) (BIGGS and ZIPURSKY 1992 ; BIGGS et al. 1994 ; BRUNNER et al. 1994 ), Dsor1 (TSUDA et al. 1993 ; LU et al. 1994 ), and D-raf (NISHIDA et al. 1988 ; AMBROSIO et al. 1989 ) have been identified as encoding the components of the cascade, MAPK, MAPKK, and MAPKKK, respectively. Genetic analyses revealed the involvement of these genes in the transduction of signals from the receptor tyrosine kinases (RTKs) encoded by sevenless (sev), torso (tor), and Drosophila EGF receptor homolog (DER; AMBROSIO et al. 1989 ; DICKSON et al. 1992 , DICKSON et al. 1996 ; TSUDA et al. 1993 ; BIGGS et al. 1994 ; BRAND and PERRIMON 1994 ; BRUNNER et al. 1994 ; HATA et al. 1994 ; HSU and PERRIMON 1994 ; NISHIDA et al. 1996 ; LIM et al. 1997 ). The RTKs encoded by sev and tor are responsible for the determination of the R7 photoreceptor cell fate in the eye disc and of the cell fates at the embryonic termini, respectively (HAFEN et al. 1993 ; DUFFY and PERRIMON 1994 ). DER has multiple functions, such as the determination of dorso-ventral polarity of the ovarian follicle, the embryonic ectodermal differentiation, and the differentiation of the compound eye and wing veins (SHILO and RAZ 1991 ). In addition, D-raf and Dsor1 have been demonstrated to be involved in the regulation of cellular proliferation (PERRIMON et al. 1985 ; NISHIDA et al. 1988 , NISHIDA et al. 1996 ; TSUDA et al. 1993 ; HATA et al. 1994 ). Thus, the ubiquitous MAPK cascade receives diverse extracellular signals and generates responses specific to each RTK. Elucidation of the molecular mechanism by which signal specificity is generated will be crucial for understanding the molecular mechanisms of development.

It has been demonstrated that the transduction of the signals generated by different RTKs is mediated through a cassette of pathways composed of multifunctional factors encoded by drk, Sos, Ras1, D-raf, Dsor1, and rl (AMBROSIO et al. 1989 ; DOYLE and BISHOP 1993 ; LU et al. 1993A ; TSUDA et al. 1993 ; BRUNNER et al. 1994 ; DIAZ-BENJUMEA and HAFEN 1994 ). However, the cassette does not seem to be a simple, straightforward pathway, and the signal specificity in each RTK pathway may be provided by branching and integration of signals as well as the differential modulation of some components. For example, it has been proposed that a Ras1-independent pathway activates D-raf in the Tor pathway from the observation that a loss of Ras1 activity did not completely abolish Tor signaling (HOU et al. 1995 ). Genetic analysis of gain-of-function mutations of Dsor1 demonstrated that Dsor1 transduces signals less efficiently in the DER pathway than in the Tor or Sev pathways, suggesting that differential modulation of the Dsor1 activity may be involved in generating signal specificity (LIM et al. 1997 ). We herein characterize gain-of-function mutations of rl and their activities in Tor signaling and in imaginal cell proliferation. The results obtained in this study suggest novel points for the integration or branching of signals in the MAPK cascade.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetics:
Fly cultures and crosses were performed at 25° unless otherwise described. Fly stocks used in this study were provided as follows: fs(1)ph¹⁹⁰¹/FM6 and tor^RL3 cn px sp/CyO from G. Struhl; D-raf^C110; rl^Sem from D. Brunner and E. Hafen; w; l(2)rl^EMS64 Pin/SM1 from D. Yamamoto; and rl¹, Df(2R)rl^10a, lt rl^10a cn/SM1 and Df(2R) rl^10b, lt rl^10b cn/SM1 from the Bloomington Stock Center. For descriptions of the genetic markers and balancers, see LINDSLEY and ZIMM 1992 . D-raf^C110/Y; rl^Sem/rl^Sem males were crossed with Canton-S (wild-type) females and their male progeny were further crossed with Canton-S females to remove D-raf^C110. Females heterozygous for rl^Sem (+/+;rl^Sem/+) were selected by the dominant eye/wing phenotypes and tested for dominant sterility.

Clonal or twin-spot analysis was performed with Dp(1; Y; 3)M2', mwh⁺ ve⁺ FR1, y cv v f^36a/C(1)RX, y f/B^SY; mwh ve h as described earlier (LAWRENCE et al. 1986 ; TSUDA et al. 1993 ). Females with relevant mutations that were also homozygous for mwh were crossed with males of the above strain, and their progeny were irradiated with X ray (1500 R) during early third instar (~72 hr after eggs were laid). Clones formed in wing blades were analyzed, and only those twin-spots with >16 f^36a (control) cells were considered.

Molecular procedures:
RNA was extracted from homogenized adult flies homozygous for either rl^Su14 or rl^Su23, and cDNAs were synthesized using oligo(dT) primers and Superscript reverse transcriptase (Boehringer, Indianapolis). The mutant rl cDNAs were cloned as two overlapping fragments synthesized by RT-PCR using sets of sense and antisense primers synthesized according to the rl coding sequence (BIGGS and ZIPURSKY 1992 ). The sense primers were 5'-GAGGATTCCGACAAGTGAATTTATTCTATTTCACCC-3' and 5'-GAGGATTCCATGTCACAAACTACCTCAGAC-3', and the antisense primers were 5'-GAGGATCCTCCCGATGCAAGACGTTTGCGGAATG-3' and 5'-GAGGATTCGCAAAATGGAGAAGTCCAGC-3'. Each primer contained a BamHI restriction site (underlined) to facilitate ligation into the BamHI cloning site in pBluescript (Stratagene, La Jolla, CA). Three independent clones were sequenced on each strand using synthetic oligonucleotide primers.

Whole-mount in situ hybridizations:
Digoxigenin-labeled antisense and sense RNA probes were made from linearized plasmid DNAs containing tll, hkb, or ftz cDNA fragments using the DIG RNA labeling kit (Boehringer). Probes were treated with alkali to reduce their sizes to an average of 100 nucleotides long and were used for in situ hybridization with whole-mount embryos following the method of TAUTZ and PFEIFLE 1989 , with slight modifications.

Other procedures:
Histological sections for electron microscopy were prepared as previously described (BASLER et al. 1991 ). Cuticle preparations of embryos were made as described by WIESCHAUS and NUSSLEIN-VOLHARD 1986 , and viewed with dark-field optics.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic and molecular characterization of gain-of-function mutations of rolled:
To identify factors acting downstream of D-raf, we screened for dominant suppressors of a hypomorphic allele of D-raf, D-raf^C110, and obtained 19 such mutants (TSUDA et al. 1993 ; LIM et al. 1997 ). In this article, the two second chromosomal suppressors, Su14 and Su23, are described. These mutations fully suppressed the phenotypes of D-raf^C110 in terms of the viability and the morphologies of the compound eye and wing veins (data not shown). Both mutations mapped genetically at 55.0 in the centromeric heterochromatic region of the second chromosome, where rolled (rl), encoding a Drosophila homolog of MAPK, is located (BIGGS et al. 1994 ).

In the D-raf⁺ background, Su23 caused a dominant phenotype, producing a mild rough eye and extra wing veins (Fig 1B and Fig 2B). Observation of ultra-thin sections of the compound eye revealed multiple R7-like cells in each ommatidium (Fig 1D). Flies heterozygous for Su14 showed no apparent rough eye phenotype, but observations of their eye sections revealed extra R7-like cells in a small fraction of ommatidia. Flies homozygous for Su14 showed a mild rough eye phenotype with multiple R7-like cells in most of the ommatidia (Fig 1E). Both mutations strongly suppressed the loss-of-function mutation of sev, and extra R7-like cells were produced in Su23 even with the genetic background of a null sev mutation (data not shown).

View larger version (137K): In this window In a new window Download PPT slide   Figure 1. Scanning (A and B) and transmission (C–F) electron microscopy of compound eyes. The compound eye of a wild-type fly is composed of a regular array of ommatidia (A). Within each ommatidium, a centrally located smaller rhabdomere derived from the R7 photoreceptor cell, and six large outer rhabdomeres from the outer photoreceptor cells (R1 to R6) with surrounding pigment cells, can be seen in a tangential section of the eye (C). In a rlSu23/+ fly, the eye surface is rough (B) and each ommatidium contains multiple R7-like cells that are marked with small rhabdomeres (D). A rlSu14/rlSu14 fly has a mild rough eye phenotype (not shown) with multiple R7-like cells formed in most of the ommatidia (E). The eye phenotype of rlSu23/+ was significantly enhanced in rlSu23/Df(2R)rl10b (F).

View larger version (67K): In this window In a new window Download PPT slide   Figure 2. Wing phenotypes with the gain-of-function mutations in rl and their enhancement by loss-of-function mutations in rl. (A) A wing blade of a wild-type fly. Extra wing vein materials are formed in the wing of rlSu23/+ (B), and the phenotype was significantly enhanced in rlSu23/Df(2R)rl10b (D). Wings of rlSu14/+ are normal, but extra wing veins (arrowheads) were occasionally formed in rlSu14/Df(2R)rl10b (C).

A gain-of-function mutation of rl, rl^Sevenmaker (rl^Sem) resulted in a similar phenotype that was significantly enhanced by loss-of-function mutations of rl (BRUNNER et al. 1994 ). We observed that rl mutations significantly enhanced the wing phenotype seen in Su23, and that more wing vein materials were produced in Su23/rl¹ than in Su23/+ (Fig 2D). The eye phenotype was also enhanced and more R7-like cells were produced in Su23/rl¹ and Su23/Df(2R)rl^10b flies [3.19 ± 1.30 (N = 97) and 3.18 ± 1.23 (N = 90) R7-like cells per ommatidium, respectively; Fig 1F] than in Su23/+ flies (2.75 ± 1.33 R7-like cells per ommatidium, N = 122). Although Su14/+ showed no extra wing veins, flies transheterozygous for rl¹ and Su14 did (Fig 2C). The enhancement of the dominant activities may be due to lack of competition from the normal product, and the results may suggest that they are the alleles of rl.

To confirm the allelism further, we cloned the rl cDNA fragments by RT-PCR with template RNAs extracted from flies homozygous for the suppressor mutations, and sequenced them as described in MATERIALS AND METHODS. Comparison of the nucleotide sequences with those reported (BIGGS and ZIPURSKY 1992 ) revealed guanine-to-adenine substitutions at nucleotide position 737 in Su14 and 1214 in Su23, respectively, causing changes from aspartic acid to asparagine at amino acid residue 185 in kinase subdomain VII in Su14 and at 334 in kinase subdomain XI in Su23, respectively (Fig 3). Both residues are highly conserved among MAPK family members, and the alterations are likely the cause of the suppressor mutations. Hereafter, we refer to them as rl^Su14 and rl^Su23. To our surprise, the molecular lesion associated with rl^Su23 was identical to that found in rl^Sem (BRUNNER et al. 1994 ).

View larger version (91K): In this window In a new window Download PPT slide   Figure 3. An alignment of the amino acid sequence of Rl with its homologs and molecular lesions associated with rlSu14 and rlSu23. The amino acid sequence of Rl (Dm-Rl; BIGGS et al. 1994 ) was aligned with those of MAP kinases from human (h-ERK1 and h-ERK2; OWAKI et al. 1992 ), rat (rat-ERK2; BOULTON et al. 1991 ), Xenopus (Xl-MAPK; GOTOH et al. 1991 ), budding yeast (Sc-FUS3; ELION et al. 1990 ), and fission yeast (Sp-spk1; TODA et al. 1991 ). Dashes indicate insertions introduced to optimize similarities, and the residues identical to those of Rl are shaded. Alterations found in RlSu14 and RlSu23 are indicated with bold letters above the Rl sequence. A guanine-to-adenine alteration in each allele caused an amino acid substitution of aspartic acid to asparagine at residue 185 in RlSu14 and at residue 334 in RlSu23.

Activity of rolled in the Torso pathway:
It has been reported that flies heterozygous for rl^Sem are almost invariably dominant female sterile (BRUNNER et al. 1994 ). On the contrary, rl^Su23/+ females are fertile, and a detailed analysis revealed a weak dominant sterility. A fraction of the eggs (10.9%, N = 2688) laid by rl^Su23/+ females crossed with normal males failed to hatch, and observation of the cuticular pattern of the nonviable embryos revealed that 15.6% (N = 205) of them were associated with reduction in the number of abdominal segments (data not shown). This phenotype is similar to that observed in embryos produced by females carrying gain-of-function muations in tor (KLINGLER et al. 1988 ). The remaining nonviable embryos showed no obvious aberration. Reexamination of rl^Sem, kindly provided by D. Brunner and E. Hafen, also demonstrated a weak dominant sterility similar to rl^Su23. The rl^Sem/+ flies produced nonviable embryos (10.2%, N = 2066), a fraction of which (20.0%, N = 184) showed defects similar to those associated with the gain-of-function alleles of tor. Thus, both rl^Su23 and the current stock of rl^Sem are only weakly dominant female sterile. Females homozygous for rl^Su23 are essentially sterile due to defects in vitellogenesis (data not shown). Due to this discrepancy, we analyzed the effects of the rl mutations in the terminal system in more detail.

The effects of the gain-of-function mutations of rl on the expression of tll and hkb, target genes in the Tor signaling pathway (PIGNONI et al. 1990 ; WEIGEL et al. 1990 ; TSUDA et al. 1993 ), were examined. In normal blastoderm embryos, tll is expressed in a pattern having a posterior cap and an anterior dorsal-lateral stripe (PIGNONI et al. 1990 ; TSUDA et al. 1993 ; Fig 4A). In the embryos derived from the tor dominant females, the tll expression region is greatly expanded into the central region, and the expression pattern of the pair-rule gene fushitarazu (ftz) is severely affected so as to produce only three stripes instead of the normal seven stripes (STEINGRIMSSON et al. 1991 ; STRECKER et al. 1991 ). In the embryos laid by rl^Su23/+ or rl^Su14/+ females (Fig 4B and Fig C), no gross alteration of the tll expression pattern was observed, although the tll-expressing regions were significantly expanded. The anterior border of the posterior tll expression region was shifted to 19.3% egg length (EL) and 18.8% EL in the embryos laid by rl^Su23/+ or rl^Su14/+ females, respectively, as compared to 14.6% EL in normal embryos. There was also a significant posterior shift in the anterior border of the anterior stripe in these embryos (Fig 4B and Fig C). Another target gene, hkb, is expressed at both termini in blastoderm embryos (WEIGEL et al. 1990 ; Fig 4F), and its expression was also significantly expanded at both termini in these embryos (Fig 4G and Fig H). No gross alteration of the expression pattern of ftz was observed, although the seven stripes were considerably condensed toward the central region (Fig 4L). As most of the embryos develop normally, the altered pattern of development would thus be regulated during later stages. Similar results were obtained with the embryos laid by rl^Sem/+ females (data not shown).

View larger version (135K): In this window In a new window Download PPT slide   Figure 4. The gain-of-function mutations of rl strengthened the signal from the Tor receptor tyrosine kinase but failed to suppress terminal class mutations. Expression patterns of tll (A–E), hkb (F–J), and ftz (K and L) in the cellular blastoderm-stage embryos laid by females with the following genotypes: Canton-S (normal) (A, F, and K), rlSu23/+ (B, G, L), rlSu14/+ (C and H), fs(1)ph1901 Dsor1Su1/fs(1)ph1901 + (D and I), and fs(1)ph1901/fs(1)ph1901; rlSu23/+ (E and J). Expression of tll, hkb, and ftz was visualized by in situ hybridization of whole-mount embryos (TAUTZ and PFEIFLE 1989 ). Cuticular preparations of embryos (M–O). The terminal structures posterior to the seventh abdominal segment (arrowheads) are missing in the embryo laid by a fs(1)ph1901/fs(1)ph1901 female (M). The posterior defect was significantly suppressed in the embryo laid by a fs(1)ph1901 Dsor1Su1/fs(1)ph1901 + female, and an eighth abdominal segment (arrow) was formed (N). On the other hand, the defect was preserved in the embryo laid by a fs(1)ph1901/fs(1)ph1901; rlSu23/+ female (O).

We also analyzed the genetic interactions of rl with the terminal class maternal-effect mutations. Embryos produced by females homozygous for terminal class maternal-effect mutations such as fs(1)ph fail to develop structures posterior to the seventh abdominal segment as well as anterior-most structures, including the head skeleton (Fig 4M and Table 1). Expression of tll and hkb is severely affected in these embryos (WEIGEL et al. 1990 ; TSUDA et al. 1993 ; not shown, but essentially identical to expression shown in Fig 4E and Fig J). Dsor1 has been demonstrated to act downstream of D-raf in the Tor pathway, and the gain-of-function mutations of Dsor1 suppress the posterior defects in embryos devoid of terminal class gene maternal activities (TSUDA et al. 1993 ; LIM et al. 1997 ; Table 1; Fig 4D, Fig I, and Fig N). The loss-of-function mutations of rl significantly reduced the suppressor activity of Dsor1^Su1 (Table 1), demonstrating that Rl acts downstream of Dsor1 in the Tor pathway.

A temperature-sensitive gain-of-function allele of tor, tor^RL3 (KLINGLER et al. 1988 ), was significantly enhanced by rl^Su23 and rl^Su14. As shown in Table 2, considerable numbers of embryos produced by females heterozygous for tor^RL3 and also for either rl^Su23 or rl^Su14 failed to hatch at 28°. Most of the nonviable embryos had a reduced number of abdominal segments (data not shown).

The above results demonstrate the involvement of Rl in the Tor signaling pathway, and it is likely that the gain-of-function mutations of rl suppress the terminal defects of the terminal class mutant embryos similar to the gain-of-function mutations of Dsor1 (TSUDA et al. 1993 ; LIM et al. 1997 ). Contrary to expectations, rl^Su23 and rl^Su14 did not suppress fs(1)ph at all: Neither the cuticular pattern nor the expression patterns of tll and hkb in the embryos lacking maternal fs(1)ph were affected by rl^Su23 or rl^Su14 (Table 1; Fig 4E, Fig J, and Fig O).

Functions of rolled and Dsor1 in cell proliferation:
Loss-of-function rl mutants die as third instar larvae that lack imaginal discs (HILLIKER 1976 ), suggesting the involvement of rl in the proliferation of imaginal disc cells. Animals hemizygous for loss-of-function mutations of D-raf showed a similar phenotype (PERRIMON et al. 1985 ; NISHIDA et al. 1988 ; TSUDA et al. 1993 ; HATA et al. 1994 ). Clonal analysis demonstrated that the rate of proliferation was reduced ~40% in clones homozygous for null D-raf¹ (TSUDA et al. 1993 ; HATA et al. 1994 ; Table 3). A clonal analysis in the present study demonstrated that proliferation was much more severely affected in null Dsor1^Gp158 than in null D-raf¹ (Table 3). The rate of proliferation in Dsor1^Gp158 was greatly reduced, and only one or two doubling events took place in most of the Dsor1^Gp158 clones, while more than six doublings occurred in their sibling clones. Proliferation rates in the clones homozygous for the Dsor1 hypomorphs, Dsor1^r1 and Dsor1^r2, were affected in a similar manner to those observed in D-raf¹ (TSUDA et al. 1993 ; Table 3).

To elucidate the function of rl in cell proliferation, we tested whether rl^Su23 suppresses the proliferation defects in the D-raf and Dsor1 mutant clones. As shown in Table 3, rl^Su23 suppressed the proliferation defects in the D-raf and Dsor1 mutant clones, although it did not restore the viability of the flies hemizygous for D-raf¹, Dsor1^r1, or Dsor1^r2. This indicates that Rl acts downstream of Dsor1 in the signaling pathway regulating the imaginal cell proliferation. It should also be noted that the proliferation defects in null Dsor1^Gp158 were significantly suppressed by rl^Su23, suggesting considerable basal level activity by rl^Su23.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Function of rolled in the Torso signaling pathway:
Genetic and molecular characterization of dominant suppressors of D-raf on the second chromosome identified two gain-of-function alleles of rl with different dominant activities: rl^Su14 (weak) and rl^Su23 (strong). Surprisingly, the molecular lesion associated with rl^Su23 was found to be identical to that of rl^Sem (BRUNNER et al. 1994 ). In fact, the dominant activity of rl^Su23 on the eye and wing morphology was similar to that observed in rl^Sem. It has been reported that rl^Sem is invariably dominant female sterile with a dominant tor-like phenotype (BRUNNER et al. 1994 ), whereas both rl^Su23 and rl^Sem showed only a weak dominant female sterility in this study. This discrepancy would be due to a difference in the genetic background: either the presence of a mutation(s) that enhances Tor signaling in the original rl^Sem stock or the occurrence of a suppressor mutation(s) in the current stocks of both rl^Sem and rl^Su23. So far, outcrossings of the current stocks have shown no evidence of suppressor mutations.

The cell fates at the anterior and posterior termini of the early embryo are determined by the Tor signaling pathway (NUSSLEIN-VOLHARD et al. 1987 ; LU et al. 1993B ). Unfortunately, the significance of rl in the pathway could not be directly assessed with germline clones because of its proximity to the centromeric heterochromatin (BERGHELLA and DIMITRI 1996 ). However, a half-reduction in the gene dosage of rl significantly reduced the suppressor activity of the dominant Dsor1 mutation on the posterior defect in the embryos lacking terminal class gene maternal activities (Table 1). This indicates that Rl is required in Tor signaling and that it acts downstream of Dsor1 (Fig 5). A detailed analysis of the gain-of-function mutations of rl demonstrated that they significantly strengthened the signals from the Tor receptor tyrosine kinase. The dominant activity is rather moderate and explains the observed weak dominant sterility (Fig 4 and Table 2). It also should be noted that the dominant rl mutations exhibited no suppressor activity on the terminal class mutations, in contrast to the dominant Dsor1 mutations (Fig 4; Table 1). The significance of this observation is discussed in a later section.

View larger version (16K): In this window In a new window Download PPT slide   Figure 5. Models for the Tor signaling pathway at the embryonic posterior end (A and B) and for the cascade regulating cell proliferation (C). The Tor receptor activation at the posterior end of the early embryo causes local activation of tll and hkb by antagonizing repressor activities of Grainyhead (Grh) and/or Groucho (Gro; LIAW et al. 1995 ; PAROUSH et al. 1997 ). It has been suggested that Ras1 mediates only a part of the signals from the Tor receptor and that D-raf integrates signals from Ras1 and another yet-unidentified factor (HOU et al. 1995 ; A and B). Loss-of-function mutations of rl markedly reduced the suppressing activity of Dsor1Su1 on fs(1)ph1901, and this clearly indicates that Rl acts downstream of Dsor1 in this pathway. However, the inability of gain-of-function mutations in rl to suppress the terminal class mutations (Fig 4E, Fig J, and Fig O) suggests that activation of Rl is not sufficient for Tor signaling. One possible explanation would be that Rl transduces only a portion of the signals from Dsor1 and that another unknown factor participates in the signaling in addition to Rl (A). It is also possible that Dsor1 relieves Rl from inhibition by an unidentified antagonizing factor in addition to activating Rl (B). Clonal analysis demonstrated that the proliferation defect in null Dsor1 mutant clones is much more severe than in null D-raf clones, and this can be explained if D-raf mediates only a part of signals and Dsor1 integrates signals from other unknown factors as well as from D-raf (C).

MAPK cascade and cell proliferation:
The clonal analysis demonstrated that D-raf and Dsor1 encode the essential components of the signaling pathway regulating proliferation of imaginal disc cells (TSUDA et al. 1993 ). The proliferation defects of loss-of-function mutations of D-raf and Dsor1 were significantly suppressed by rl^Su23, indicating that Rl acts downstream of Dsor1. It should also be noted that the proliferation defect in the null Dsor1^Gp158 clones is much more severe than in the null D-raf¹ clones (Table 3). This may suggest that D-raf mediates only a portion of the signals for proliferation to Dsor1 and that Dsor1 integrates growth-stimulating signals from other unknown factor(s) as well (Fig 5C).

Signal branching and integration in the MAPK cascade:
As described above, the dominant rl mutations exhibited no suppressor activity on the terminal class mutations. This could be explained if the rl gain-of-function mutations were devoid of constitutive activity, and if the expression of their dominant activity were strictly dependent on upstream signals. However, the significant suppressor activity of rl^Su23 in the proliferation defects in the null Dsor1^Gp158 clones and in the R7 cell fate decision in the null sev mutants may run counter to this assumption.

It has been reported that the increased signal sensitivity of the mammalian ERK2^D319N protein that has a mutation analogous to Rl^Sem is due to a decreased sensitivity to dual-specificity MAPK phosphatases such as PAC1, CL100/MKP-1, MKP-2, and MKP-3 rather than to an increased kinase activity (BOTT et al. 1994 ; CHU et al. 1996 ; CAMPS et al. 1998 ). On the other hand, an in vitro kinase assay of the recombinant Rl^Sem mutant protein produced in bacteria demonstrated significant activity for the phosphorylation of Yan, a native substrate of Rl, in the absence of activating MAPKK, while the normal recombinant Rl did not (OELLERS and HAFEN 1996 ). In the presence of activated mammalian MAPKK, Rl^Sem exhibited a higher kinase activity than Rl⁺. The latter observations suggest an increased basal level activity of Rl^Sem in addition to an increased sensitivity to the activator. The constitutive activity observed in vitro is consistent with the significant suppressor activity of rl^Su23 in the proliferation defect in the null Dsor1^Gp158 clones (Table 3). Taking these observations into account, it is most likely that the dominant activity of the Rl^Sem and Rl^Su23 mutant proteins is due to both an increased basal level activity and a decreased sensitivity to inactivating phosphatases.

On the basis of the above considerations, we propose that the activation of Rl is necessary but not sufficient for Tor signaling, and that Dsor1 may provide yet another branching point in the Tor signaling pathway. One possible model would be that Dsor1 activates another unknown factor in addition to Rl in the Tor pathway, and that both are required for the transcriptional activation of tll and hkb (Fig 5A). It would also be possible that an inactivation of a factor that antagonizes the Rl function by Dsor1 would be required for the activation of the pathway (Fig 5B). Defects of varying degrees were seen in mitoses in the syncytial blastoderm embryos devoid of the maternal Dsor1 activity (L. TSUDA, H.-Y. HA and Y. NISHIDA, unpublished observations), suggesting that Dsor1 participates in the regulation of mitosis and is activated throughout the embryo during cleavage divisions. Bifurcation of the Tor signals downstream of Dsor1 may constitute a mechanism for preventing Dsor1 from activating the target genes in regions other than the terminal regions of the embryo. As discussed above, integration of signals for imaginal cell proliferation would then take place at some other point in the MAPK cascade (Fig 5C). The differential branching and integration of signals may contribute to the functional diversification of the ubiquitous MAPK cascade.

	FOOTNOTES

¹ Present address: Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, CA 90024.

	ACKNOWLEDGMENTS

We are grateful to G. Struhl, D. Brunner, E. Hafen, D. Yamamoto, and the Bloomington Stock Center for fly stocks. We are also greatly indebted to S. Tokumasu, T. Tsuboi, and K. Dohmoto for technical assistance. This work was supported by grants from the Mitsubishi Foundation, the Ministry of Education, Science, Sport, and Culture of Japan, and the Japan Science and Technology Corporation.

Manuscript received April 3, 1999; Accepted for publication June 8, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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