Drosophila Morphogen Dorsal Can Be Established by a Signaling Pathway Involving the Transmemlirane Protein Toll and Protein Kinase A

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Selective nuclear transport of the Drosophila morphogen dorsal can be established by a signaling pathway involving the transmemlirane protein Toll and protein kinase A

Jacqueline L. Norris and James L. Manley Department of Biological Sciences, Columbia University, New York, New York 10027 USA

Establishment of dorsal-ventral polarity in the early Drosophila embryo requires a concentration gradient of the maternal morphogen dorsal (d/). This concentration gradient is established by selective nuclear transport of d/so that dl protein is present only in ventral nuclei. The activity of 11 genes is required for d/nuclear localization. One of these genes, Toll, encodes a transmembrane protein that appears to play the most direct role in regulating dl localization. We have examined the effects of Toll on dl in cotransfected Schneider cells to gain insight into the nature of the interaction between these proteins. We have found that Toll can enhance the nuclear localization of d/and, independently, the ability of d/to activate transcription once in the nucleus. We present evidence that the signaling pathway from Toll to d/involves protein kinase A (PKA) and that nuclear transport and activation of d/results from phosphorylation of d/by PKA. We discuss the significance of these results with respect both to Drosophila embryogenesis and to the regulation of the mammalian transcription factor NF-KB. [Key Words: Drosophila morphogen; dorsal; Toll; protein kinase A] Received December 30, 1991; revised version accepted June 24, 1992.

Establishment of dorsal-ventral (D/V} polarity m the for the proper localization of the dl protein (Anderson Drosophila embryo requires the activities of several ma- and Nfisslein-Volhard 1986). In embryos that carry null ternal genes, including 11 that comprise the dorsal group mutations in any of these genes, ventral cells follow the (for review, see Anderson 1989; Rushlow and Arora developmental pathway of dorsal cells. The dorsalization 1990). One member of this group is the D/V morphogen of these embryos is the result of the absence of the dl dorsal (dl), a protein that shares homology, over the protein in ventral nuclei, which results in the improper amino-terminal 300 amino acids, with the proto-onco- expression of zygotic genes. The function of the dorsal gene c-rel and the mammalian transcription factor NF- group gene products is to establish the dl nuclear protein KB. This region includes a putative DNA-binding do- gradient. Two of these genes, easter and snake, encode main, a nuclear localization sequence (NLS), and a po- serine proteases (DeLotto and Spierer 1986; Chasan and tential protein kinase A (PKA) phosphorylation site Anderson 1989), suggesting that a cascade of interactions (Steward 1987; Ghosh et al. 1990; Kieran et al. 1990}. involving post-translational processing could result in The dl protein is distributed in a concentration gradient the formation of the signal for dl movement into ventral over the embryo, with the highest levels present in ven- nuclei. This signal is most likely localized in the peri- tral regions and progressively decreasing levels in dorsal vitelline space on the ventral side of the embryo and is regions (Steward et al. 1988). This gradient results from transmitted to dl through the product of the Toll gene the selective nuclear transport of the dl protein, which is (Hashimoto et al. 1988; Stein et al. 1991}, which is lo- uniformly cytoplasmic in the early embryo until cleav- calized uniformally in the embryo plasma membrane age cycle 10 when it is transported into ventral but not (Hashimoto et al. 1991). Genetic studies have shown dorsal nuclei (Roth et al. 1989; Rushlow et al. 1989; that Toll is one of the last genes in the D/V cascade, Steward 1989). Following its accumulation in the nu- acting upstream of dl, and cytoplasmic injection experi- cleus, dI functions to influence the expression of several ments have shown that Toll is responsible for establish- zygotic genes required for D/V polarity (for review, see ing polarity in the embryo by defining the position of the Anderson 1987; Rushlow and Arora 1990). D/V axis (Anderson et al. 1985a, b). These observations, The activity of the other dorsal group genes is essential along with the isolation of both dominant-ventralizing

1654 GENES& DEVELOPMENT6:1654-1667 © 1992 by Cold Spring Harbor Laboratory ISSN 0890-9369/92 $3.00 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Nuclear transport of the dorsal protein as well as recessive-dorsalizing alleles of Toll (Anderson ized primarily in the cytoplasm at low concentrations of et al. 1985a; Schneider et al. 1991), suggest that the Toll expression vector, but increasingly in the nucleus at gene product plays a particularly critical role in regulat- higher concentrations. The dl protein was also shown to ing dl localization. However, nothing is known about activate expression of several reporter genes in a highly the mechanism by which Toll enhances nuclear trans- concentration-dependent manner (Rushlow et al. 1989). port of dl. Here, we have used the cotransfection system as an assay The Toll protein has a large extracellular domain that to show that Toll can bring about selective nuclear trans- contains two blocks of leucine-rich repeats with adjacent port of dl in Schneider cells, ruling out an absolute re- cysteine-containing motifs (Hashimoto et al. 1988; quirement of factors unique to the early embryo. Addi- Schneider et al. 1991} and a small cytoplasmic domain tional experiments indicate that the mechanism appears that is similar to the cytoplasmic domain of the inter- to involve phosphorylation of dl by activated PKA. leukin-1 receptor (IL-1R)(Schneider et al. 1991). The overall structure of the Toll protein is most similar to Results the e~ chain of human platelet glycoprotein lb (GPlb), a receptor for yon Willebrand factor and thrombin (Lopez We have used transient cotransfection assays in Droso- et al. 1987; Hashimoto 1988). The leucine-rich repeat phila Schneider cells to study the activity and nuclear and cysteine-containing motif in GPlb are required for transport of the dl protein. Figure 1 describes the princi- thrombin binding, suggesting that these regions in the pal plasmid constructs used in these studies. The cDNAs Toll protein may serve as ligand-binding sites (Keith and encoding dl and Toll, as well as PKA, were each cloned Gay 1990). The importance of these regions in Toll is into an actin 5C expression vector so that their expres- indicated by the finding that three of the strongest Toll- sion was driven by the actin 5C promoter, a strong pro- dominant alleles contain point mutations in one of the moter in Schneider cells (Han et al. 1989). The activity of cysteine-containing motifs (Schneider et al. 1991). the dl protein was measured by its ability to activate To study the mechanism of dl nuclear transport we expression of the chloramphenicol acetyltransferase have used transient cotransfection assays in Drosophila (CAT) gene from a 200-bp fragment ( - 196 to +39) of the Schneider cells. These cells contain no detectable endog- Drosophila zen promoter (zen-CAT200). The dl protein enous dl protein so the localization and activity of dl was shown previously to activate CAT expression from produced from transfected templates can be readily ob- this promoter in a strongly concentration-dependent served (Rushlow et al. 1989}. In this system, exogenously manner, from 2- to >1000-fold (Rushlow et al. 1989). produced dl protein was shown previously to be local- This promotor fragment contains no detectable dl-bind-

dorsal PKA

678

t£) C.-LN GLN poly (A) Figure 1. Structures of plasmid con- NLS structs. The dl protein shares homology with the rel and NF-KB proteins over the amino-terminal 300 amino acids (solid Toll area). This region contains a conserved PKA Cytoplasmic Extracellular Domain phosphorylation site, a NLS, and a putative Domain 166 452 659 729 1097 DNA-binding domain. The carboxyl terminus contains proline (PRO)- and glutamine +1 (GLNJ-rich regions. Numbers refer to Actin 5C signal LEU CYS IFU CYS / / Actin 5C amino acid residues. The Toll protein has a promoter sequence poly (A) large extracellular domain of 804 amino ac- 804 828 ids, with two blocks of leucine-rich repeats Membrane (LEU), adjacent cysteine motifs (CYS), and a Spanning Domain small (269-amino-acid) cytoplasmic domain. Solid vertical rectangles indicate the locations of signal sequence and mem- zen -CAT200 brane-spanning domain. Numbers refer to amino acid residues. The zen-CAT200 reporter plasmid contains -200 bp from the zen promoter (- 196 to + 39) controlling ex- -196 +1 +39 SV40 pression of the CAT gene. The pUC vector poly (A) also contains SV40 poly(A) sequences.

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Norris and Manley ing sites, and the mechanism by which dl mediates activation is not known (see Discussion). However, for the - To II ÷ Toll purposes of the current study, this reporter construct dorsal provided an excellent assay for dl activity.

1 1.1 Activity of the dl protein in the presence of Toll Genetic studies have identified the Toll protein as the 0.1 2.2 3.3 most likely candidate for receiving and transmitting the signal that results in movement of the dl protein into 0.2 4.1 16 ventral nuclei (for review, see Anderson 1989). To investigate the underlying mechanism, we wished to determine whether a functional interaction between these 0.3 3.6 32 two proteins could be observed in a simplified system, that is, cultured cells. To this end, we cotransfected dif- 0.4 5.6 34 ferent concentrations of expression vectors containing Toll (Act-T/) or dl (Act-d/) cDNAs, along with zen- 0.6 16 54 CAT200, into Schneider cells and assayed the resultant CAT activities. All increases in CAT activity are presented relative to that measured from transfections that 1.0 50 168 contained an equal amount of the actin 5C expression vector without a cDNA insert and were also normalized Figure 2. Activity of wild-type dl in the presence of Toll. to an internal-control plasmid (see Materials and meth- Schneider cells were cotransfected with the indicated amount ods). Transfections containing the dl expression vector of dl expression vector (~tg), 3.0 ~g of the zen-CAT200 reporter alone showed that, as observed previously (Rushlow et plasmid, and either pAct5C (-Toll) or Toll expression vector al. 1989), dl activates CAT expression in a concentra- ( + Toil) to bring the final concentration of expression vector to tion-dependent manner, with strong activations ob- 5.0 ~tg. All transfections were normalized for differences in served at concentrations > 1.0 ~g and weaker, more grad- transfection efficiency by using copia fPgal as an internal con- ually increasing activations at concentrations from 0.1 trol. The CAT activities shown are expressed relative to cotransfections containing 5.0 ~xg of actin 5C expression vector to 1.0 ~g (Fig. 2, and results not shown). without an insert. Cotransfection of Act-T/with Act-d/resulted in significant enhancement of CAT expression (Fig. 2). The greatest effects were seen at moderate dl concentrations, where CAT activities were correspondingly modest. tration by Western blotting showed that cotransfection Thus, an activation of approximately ninefold was ob- of Toll did not affect the accumulation of dl protein (data served with 0.3 ~g of Act-d/, and activation was approx- not shown), ruling out the possibility that the effects of imately sixfold with 0.4 ~g. In numerous independent Toll were the result of increased levels of dl. experiments, we have always detected activations between 6- and 12-fold with optimal concentration of Act- Expression of Toll results in increased nuclear d/. At lower amounts of Act-d/, activations induced by localization of the dl protein the expression of Toll were reproducibly weaker, perhaps reflecting a subthreshold concentration of dl protein in To determine whether the increased activation of CAT the transfected cells. At high dl concentrations (>1.0 expression by dl in the presence of Toll reflected in- I~g), no significant activation was observed with cotrans- creased nuclear localization of the d/protein, cells were fection of Act-T/(results not shown), perhaps reflecting transfected with 0.4 ~g of Act-d/alone or 0.4 p,g of Act- in part an inability to introduce enough Act-T/to further dl plus 4.6 p.g of Act-T/, fixed, and stained with anti-d/ enhance the strong activations brought about by Act-d/ antibodies. Representive cells from each transfection are alone. shown in Figure 3a. Cells that received Act-d/alone pro- Several controls indicated that the observed increases duced the dl protein that was localized predominantly in resulted from a specific interaction involving dl and To//. the cytoplasm (Fig. 3a, A). In contrast, dl protein was For example, cotransfection of Act-T1 with zen-CAT200 found in both the nucleus and cytoplasm, or in some alone did not affect CAT activity {Fig. 2). Likewise, cells predominantly in the nucleus, in cells cotrans- cotransfections of Act-T/along with expression vectors fected with Act-d/and Act-T/(Fig. 3a, B). A population encoding other Drosophila transcriptional activators of stained cells that were transfected with 0.4 ~g of Act- (prd, z2, zen), together with their appropriate promoter- dl and increasing amounts of Act-T/were counted for CAT reporter constructs (Han et al. 1989), did not alter cytoplasmic, nuclear and cytoplasmic, or nuclear local- the CAT expression observed with these expression vec- ization, and the results are presented in Figure 3b. As the tors alone {results not shown). Thus, the enhancement of concentration of Act-T/ was increased, the number of CAT activity induced by Toll appears to reflect a specific cells with protein in both the cytoplasm and nucleus, or interaction with d/. Also, analysis of d/protein concen- predominantly in the nucleus, increased by almost

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Nuclear transport o| the dorsal protein a A B b zation CytopLasm Cytoplasm an( Nucleus Nucleus

0 75 20 5

1.0 78 22

3.0 45 33 22

4.0 50 38 12

4.7 32 44 24

I Figure 3. Subcellular localization of dl in the presence of Toll (a) Schneider cells were transfected with 0.3 ~g of dl (A) or 0.3 v-g of dl plus 4.7 v-g of Toll {B) and stained with anti-d/ antibodies and TRITC-conjugated secondary antibodies. The dl protein accumulates predominantly in the cytoplasm of transfected cells in the absence of Toll. The dl protein is found in the nucleus as well as the cytoplasm of cells transfected with Toll. {b) Schneider cells were cotransfected with 0.3 ~g of dl and the indicated amounts of Toll expression vector (~g) and stained with anti-d/ and TRITC-conjugated secondary antibodies. At least 50 dl-expressing cells from each transfection were counted and scored for staining of the cytoplasm, the nucleus, or both the cytoplasm and nucleus. The per- centage of cells in each category is shown.

threefold. These findings suggest that Toll functions to indicating that these sequences are not necessary for cy- aid in relocalization of the dl protein from the cytoplasm toplasmic retention. to the nucleus of Schneider cells, analogous to its role in These mutants were then used to examine regions of the early embryo. dl required for response to Toll. Figure 4b presents the results of cotransfection of several of the dl mutant expression vectors with Act-T/. In cotransfections contain- The effect of Toll on dl mutants ing any of the dl mutants that were inactive by them- Several dl mutants were analyzed to identify regions of selves, no increase in CAT activity was seen when Toll the protein required for activity and regulation of nuclear was present (d14, d15, (:116, d17, d18). These results show, localization. Figure 4a describes these mutants and lists for example, that the NLS is necessary for Toll respon- the activity and localization observed in transfections siveness. Unexpected results were obtained with the d13 containing 1.0 ~,g of Act-d/, but in the absence of Toll. mutant, which lacks 117 carboxy-terminal amino acids One of these mutants, d13, has been described previously but retains some activity (-30%) and is constitutively as dl-561, and transfections with this mutant showed nuclear (Rushlow et al. 1989). Cotransfections of 0.4 ~g that the carboxyl terminus is essential for cytoplasmic of Act-dl3 with 4.6 ~g of Act-T/resulted in enhance- retention of dl (Rushlow et al. 1989). Deletion of (d17), or ment of CAT expression by -15-fold, an activation sig- point mutations in (d18), the nuclear localization signal nificantly greater than that observed for wild-type dl. As (NLS), resulted in restriction of dl to the cytoplasm, d13 is tightly localized in the nucleus in the absence of showing that the NLS is required for nuclear localiza- Toll, this finding indicates that Toll can affect dl in a tion. Deletion of the carboxy-terminal unique region manner independent of its ability to direct d/to the nu- (dI4), or part of the rel homology region (d16), resulted in cleus, and raises the possibility that Toll expression can a loss of dl activity even though these proteins were result in direct modification of d/. As mentioned above, constitutively localized in the nucleus, indicating that further deletion of the carboxyl terminus (d14) resulted these regions are required both for activity and for cyto- in a protein that, by itself, was totally nuclear, inactive, plasmic retention. In contrast, the subcellular localiza- and not influenced by the addition of Toil. However, tion of d/5, which lacks the DNA-binding region (Ip et al. when d14 was fused to the activation domain from the 1991) and is essentially inactive, is similar to wild type, Drosophila z2 homeo box protein (Han et al. 1989), a

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Norris and Manley

a PKA A B 678 wt I 50 cyto

NLS

561 IB I nuc b

379 -Toll +Toll 1.4 nuc dorsal

0.4 4,8 38 146 244 wt 1.0 50 168 2.1 cyto 0.4 2.3 33

245 325 t.0 15 50

| 1.2 nuc 0.4 1.2 1.5

1.0 1.4 2.3 336 341 1.1 cyto 5 1.0 21 2.2 6 1_0 1.2 1.3

0.4 13 2.1 1.1 cyto 7,8 1.0 1.1 1.6

379 0.4 4.4 10 z2 ] 16 nuc 1.0 16 29

Figure 4. Subcellular localization and activity of dl mutants alone and in the presence of Toll. (a} The top line indicates the region of rel homology (solid area), the NLS, the conserved PKA phosphorylation site, and the approximate position of proline (PRO) and glutamine (GLN) regions of the wild-type dl protein. The regions deleted in the mutants are shown. Numbers refer to amino acid residues. Schneider cells were cotransfected with 1.0 lag of the indicated dl expression vector, 4.0 lag of the actin 5C expression vector without the dl-coding region, and 3.0 lag of the zen-CAT200 reporter plasmid. The predominant location of the protein in the cytoplasm {CYTO), the nucleus (NUC), or both is shown in column A. The ability of the protein to activate CAT expression is shown in column B. The CAT activities are expressed relative to cotransfections containing 5.0 lag of actin 5C expression vector without the dl-coding region. (b) Schneider cells were cotransfected with the indicated amount of dl expression vector (lag), 3.0 lag of the zen- CAT200 reporter plasmid, and either pActSC ( - Toll) or Toll expression vector ( + Toll) to bring the final concentration of expression vector to 5.0 t~g. The dl mutants are referred to by the numbers used in a. Mutants 7 and 8 behave essentially identically, and representive values are shown.

protein displaying low but significant activity was pro- deletion of their extracellular domains (see Discussion), duced (dlg; see Fig. 4a). Cotransfection of Act-dl9 with we deleted most of the extracellular domain of Toll Act-T/ enhanced the activity of the fusion protein (T1520) to determine whether this might activate Toll. (which is constitutively nuclear), but only by approxi- However, this deletion had no effect on Toll activity, as mately twofold, suggesting that sequences in the dl approximately equal CAT activities were detected in unique region are in some way required for maximal ac- cotransfections with Act-T1520 and Act-T/. This finding tivation by Toil. suggests that ligand binding is not required for Toll activation in Schneider cells (see Discussion). Both dominant-ventralizing and recessive-dorsalizing Effect of Toll mutations on activation of dl alleles of Toll have been identified {Anderson et al. To begin to determine regions of Toll that are necessary 1985al, and several of these alleles have been cloned and for its function, we constructed and analyzed several grouped into three classes (Schneider et al. 1991). One Toll deletion mutants. Figure 5a details the structures of class consists of recessive alleles that result from point these mutants, and Figure 5b displays the results of mutations in the cytoplasmic domain. This is consistent cotransfections with Act-d/. A deletion of all but the with our observation that an intact cytoplasmic domain amino-terminal 274 amino acids (RV) of Toll resulted in is required for Toll activity. Another class contains dom- a total loss of Toll activity as measured by its ability to inant alleles that are amino-terminal truncations, al- enhance the activation of CAT expression of dl from though these ventralize only in the presence of wild-type zen-CAT200. A small deletion (122 amino acids) into Toll. The final class are dominant alleles that ventralize the carboxyl terminus (StuI) also abolished Toll activity, the embryo by facilitating nuclear transport of dl indicating that the intracytoplasmic domain is essential throughout the embryo (Roth et al. 1989; Steward 1989). for activity. Because some receptors can be activated by The strongest of these is T11°b (Erd61yi and Szabad 1989),

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Nuclear transport of the dorsal protein

a Extracellular Domain Cytoplasmic DomairT 452 729 804 1097 b w t I I ,o LEU G'~ LEU ~S

RV I 274 30 Stul I ~~ ~~ 1,7o

20 fold 520 I 273 7, 1 ] ac,,v.,,o.

10 lOb I

/ Cys -'~T y r~ Toll RV Stul 520 lOb

Figure 5. The effect of Toll mutants on dl activity. (a) The extracellular and cytoplasmic domains, as well as the leucine {LEU)- and cysteine (CYS}-rich regions are indicated on wt Toll, and regions deleted in the mutants are shown. Numbers refer to amino acid residues. The site of the T1 l°b mutation is at residue 779, a change of cysteine to tyrosine. {b) Schneider cells were cotransfected with 0.4 p.g of dl expression vector, 3.0 p.g of the zen-CAT200 reporter plasmid, and either 4.6 ~g of pActSC (-Toll) or 4.6 ~g of the indicated Toll expression vector ( + Toll). Fold activation is presented as the increase in CAT activity relative to the value from the cotransfection lacking a Toll expression vector.

which contains a single amino acid change of a cysteine IKB, suggesting that it activates NF-KB by a different residue to tyrosine at position 779 (Schneider et al. 1991). mechanism. Given these observations, the conservation Although the mechanism by which this single residue of the PKA site in all tel family members, and our finding change results in constitutive nuclear localization of dl that Toll enhances dl activity independent of subcellular in the embryo is not known, its existence offers a test of localization, we wished to determine whether PKA plays the physiological relevance of the cell culture assay that a role in dl activation by Toll, perhaps by direct phos- we have used to study Toll function. To address this, we phorylation of dl. cotransfected a construct expressing T1 l°b together with We first asked whether dl activity could be influenced Act-d/and zen-CAT200. Expression of T1 l°b resulted in by the expression of exogenous PKA. To this end, a a 40-fold increase in dl activity, or approximately five- cDNA encoding the Drosophila PKA catalytic subunit fold greater than that obtained with wild-type Toll (Fig. (Kalderon and Rubin 1988) was cloned into the actin 5C 5b), providing strong evidence that the mechanism by expression vector (Act-PKA), and this construct was which Toll activates dl in Schneider cells is similar to cotransfected into Schneider cells together with Act-d/ the mechanism operative in the early embryo. and the zen-CAT200 reporter. Figure 6a displays the CAT activities obtained for Act-d/concentrations from 0.1 to 1.0 ~g with Act-PKA. As with Act-T/, expression Expression of PKA results in the enhancement of Act-PKA enhanced the ability of dl to activate CAT of dl activity and nuclear localization expression. Although the responses obtained with both The dl, NF-~B, and rel proteins all contain a conserved proteins were similar, PKA activated dl somewhat more PICA phosphorylation site directly upstream of their efficiently than Toll. Activations > 15-fold were obtained NLSs (Steward 1987; Ghosh et al. 1990; Kieran et al. with PKA, compared with -8ofold with Toll. 1990). Mutations in the v-re/ PKA site can reduce or To investigate the regions of d~ required for PKA acti- abolish the ability of the protein to transform chicken vation, we tested the d~ mutants shown in Figure 4a in spleen cells while mutations in the c-rel PKA site can cotransfections with Act-PICA and the results are pre- alter the subceUular localization of the protein (Mosialos sented in Figure 6b. As with Act-T/, those mutants that 1991). A mutational analysis of the NF-KB PKA site has lacked activity by themselves were not affected by the not been reported, but the effects of several protein kio expression of PKA. However, the constitutively nuclear nases on NF-KB activity in vitro has been tested (Ghosh d/3 was activated by PICA, showing that PKA, like Toll, and Baltimore 1990). Protein kinase C (PKC) was shown affects both dl activity in the nucleus and d/ nuclear to be most effective in activating NF-KB DNA-binding, localization (see below). Act-PKA was also cotransfected by disrupting NF-KB/IKB complexes, and PKA was also with Act-prd, Act-z2, and Act-zen, along with their ap- shown to activate NF-KB, although not as effectively as propriate promoter-CAT constructs, and found not to PKC. While the mechanism of PKC activation was affect the activations detected with these proteins alone shown to involve direct phosphorylation of IKB, inhibit- (results not shown). Therefore, the effect of PICA. is spe- ing its ability to bind NF-KB, PKA did not phosphorylate cific for d/and requires a d/protein that retains some

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Norris and Manley

a with Toll. In the presence of PKA, -50% of the transfected cells showed strong nuclear staining compared -PKA +PKA dorsal with 25% showing predominantly nuclear staining in the presence of Toll. 1 1.3 Expression of a protein kinase inhibitor prevents activation of dl by Toll 0.1 1.6 16 The above results support the hypothesis that Toll acti- 0.2 2.4 20 vates dl by a mechanism involving PKA. To address this idea further, we tested the effect of the inhibitor protein of PKA (PKi) on dl activity in the presence of Toil. PKi 0.3 2.7 47 has been analyzed in some detail, and peptide fragments with inhibitory activity have been isolated (Cheng et al. 0.4 3.5 37 1985; for review, see Kemp et al. 1988). The PKi peptides specifically inhibit cAMP-dependent protein kinase by binding to the catalytic subunit (for review, see Walsh 0.6 18 65 and Glass 1991). An actin 5C construct containing 26 amino acid residues of PKi fused to lacZ-coding se- 1.0 35 140 quences, Act-lacZPKi, was cotransfected with Act-d/ and Act-T/, and the resultant CAT activities were measured (see Materials and methods). This PKi construct b encodes a fusion protein containing the 20-amino-acid -PKA +PKA dorsal peptide that has been shown to be the most potent inhibitor of PKA (Cheng et al. 1985). Coexpression of Act- wt 35 140 lacZPKi nearly abolished the enhancement of d~ activity 3 8.7 71 brought about by Toll (Fig. 8). This inhibition was specific for d~ as the CAT activities induced by z2 or prd 4 2.5 2.1 were not altered by the addition of PKi (results not shown). Inhibition was the result of the PKi moiety in 5 0.5 1 5 the lacZ fusion protein, as identical amounts of an Act- 7,8 1.1 1 2 lacZ expression vector did not affect d~ activity (Fig. 8). These results provide strong evidence that enhancement 9 12 13 of dl activity induced by Toll involves activation of PKA.

Figure 6. The effect of PKA on wild-type and mutant d] activity. (a) Schneider cells were cotransfected with the indicated Mutations in the PKA phosphorylation site amount of dl expression vector, 3.0 ~g of the zen-CAT200 re- of dl affect dl protein localization and activity porter plasmid, and either pActSC (-PKA) or PKA expression in the presence of Toll and PKA vector (+ PKA) to bring the final concentration of expression vector to 5.0 p~g. The activation values are expressed relative to To determine whether activation of d~ by Toll and PKA cotransfections containing the actin 5C expression vector with- is dependent on the dI PKA site, we mutated the pre- out an insert. (bl Schneider ceils were transfected with 1.0 ~g of sumed site of phosphorylation, the serine (S) residue at dl, 3.0 ~g of the zen-CAT200 reporter plasmid, and either 4.0 }ag position 312. We first changed this S residue to glu- of pActSC ( - PKA) or 4.0 ~g of PKA expression vector ( + PKA). tamine (Q) to create a site that should not be phospho- The dl mutants are referred to by the numbers used in Fig. 4a. rylated by PKA. This mutant, Act-d/Q, was then cotransfected with Act-T/or Act-PKA, and the resulting CAT activities are shown in Figure 9a. Mutation of the PKA phosphorylation site resulted in only a slight reduc- activity and has an intact rel homology region, that is, tion (less than twofold) in the activity of d~ alone. How- the requirements for dl activation by PICA and Toll ap- ever, the activation of CAT expression by d/Q in the pear to be identical. presence of either Toll or PKA was reduced significantly To determine whether expression of PKA also in- relative to wild-type dl, in each case by as much as a creased the nuclear localization of d/, cells were trans- factor of 10, providing strong evidence that the d~ PKA fected with 0.4 ~g of Act-d/alone (Fig. 7, panel A) or 0.4 site plays an important roll in Toll-mediated regulation ~g of Act-d/plus 4.6 ~g of Act-PKA (panel B), fixed, and of d~ activity. stained with anti-d/ antibodies. Figure 7 presents pic- To determine whether the decreased activity of d/Q in tures of representive cells from each transfection and the presence of PKA or Toll reflected an inability of the shows that the expression of PKA resulted in an in- mutant protein to be translocated to the nucleus, cells creased nuclear localization of d/. d~ nuclear localization transfected with Act-d/Q alone (Fig. 9b, panel A), or induced by PKA was more substantial than that observed with Act-T/(not shown) or Act-PKA (panel B) were fixed

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Nuclear transport of the dorsal protein

A B

O o

Figure 7. Subcellular localization of dl in the presence of PICA. Schneider cells were transfected with 0.3 ~g of dl (A) or 0.3 ~g of dl plus 4.7 tag of PKA (B} and stained with anti-d/and TRITC-conjugated secondary antibodies. The dl protein accumulates predominantly in the cytoplasm of transfected cells in the absence of PKA (A}. The dl protein is found predominantly in the nucleus of cells transfected with PKA (B).

and stained with anti-d/antibodies, and pictures of rep- negative charge at residue 312. Might this be sufficient resentative cells are shown. These experiments show to enhance nuclear localization in the absence of Toll? that dlQ, unlike wild-type dl, was not transported effec- To address this, the S residue was changed to aspartic tively to the nucleus in the presence of Toll or PKA acid (D), creating the mutant Act-diD. A similar muta- (panel B), indicating that the dl PKA site is critical for tion has been tested in the rel protein and was found to regulated nuclear transport of the dl protein. increase nuclear localization (Mosialos et al. 1991). We then wished to determine the effect of placing a Schneider cells were transfected with 0.4 Ixg of Act-d/D, fixed, and stained with anti-d/ antibodies as described above. In contrast to cells transfected with Act-d/, which had primarily cytoplasmic staining, cells trans-

10- fected with Act-diD displayed diffuse staining of both the cytoplasm and the nucleus (Fig. 9b, panel C). This fold pattern was unaffected by cotransfection with PICA or activation Toll (results not shown). We also assayed the activity of the diD protein both alone and in the presence of Toll or 5- PICA. The CAT activities presented in Figure 9a show that this mutation results in a protein that was not ac- 2- tivated by Toll or PKA and whose activity alone was / | substantially reduced. This latter result is consistent dl dl+Toll dl+Toll+ dl+Toll+ with that observed with v-rel, where the corresponding lacZ PKi lacZ S --* D change also reduced the activity of the protein in Figure 8. Activity of dl in the presence of Toll and PKi. Schnei- a CAT assay (Mosialos et al. 1991). These findings der cells were cotransfected with the indicated amounts of dl suggset that a negative charge at position 312 can en- and Toll expression vectors (ixg), 3.0 lag of the zen-CAT200 hance nuclear localization, and strengthen the view that reporter plasmid, and either 3.6 lag of lacZPKi or 3.6 txg of actin lacZ. The final concentration of expression vector was adjusted Toll (and PKA)function through serine-312. to 7.0 lag with the actin 5C expression vector. Fold activation is Finally, to rule out the possibility that any change presented as the increase in CAT activity relative to a cotrans- made in this region might alter d/activity, the proline (P) fection containing 7.0 lag of actin 5C expression vector without residue at position 311 was mutated to lysine (K). In the an insert. PKA consensus sequence this position can be any amino

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Norris and Manley

a ., ]

dl Q Ser -~ Gin dl D Ser ~ Asp

- TO II + Toll +PKA dorsal

0.4 5.6 40 wt 1.o 40 121 130

0.4 6.1 Q 2.8 51 1.o 16 35

D o.4 2,1 1.6 2.4 lO 2.3 2.5 3.2 b A B C

Figure 9. Activity and subcellular localization of a dl phosphorylation site mutant in the presence of Toll and PKA. (a) Schneider cells were cotransfected with the indicated amount of wild-type and mutant dl expression vectors (lag), 3.0 lag of the zen-CAT200 reporter plasmid, and pAct5C, Toll expression vector ( + Toil), or PKA expression vector ( + PKA) to bring the final concentration of expression vector to 5.0 Izg. The activation values are expressed relative to cotransfections containing the actin 5C vector without an insert. (b) Schneider cells were transfected with 0.4 ~g of dlQ CA), 0.4 lag of dlQ plus 4.6 lag of PKA (B), or 0.4 lag of diD {C) and stained with anti-d/ antibodies and TRITC-conjugated secondary antibodies. The dlQ protein accumulates predominantly in the cytoplasm of transfected cells in the presence or absence of Toll or PKA; the diD protein accumulates in both the cytoplasm and nucleus. acid {Kemp and Pearson 1990) so changing it should not tivities were determined. These results (not shown) re- affect the localization or activity of dl if phosphorylation vealed that the activity of the dlRRKS protein was iden- is the underlying mechanism. This mutant, Act- tical to wild-type dl protein, providing additional sup- d/RRKS, was cotransfected with zen-CAT200 with or port for the idea that phosphorylation of serine-312 by without Act-T/or Act-PKA, and the resulting CAT ac- PKA regulates dl.

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Nuclear transport o[ the dorsal protein

Discussion activation. For example, some oncogenic forms of the neu (erbB-2) proto-oncogene contain point mutations in We have described a cell culture system in which the the extracellular domain, and at least one has been regulated nuclear transport and activity of the dl protein shown to lead to an increased aggregation of neu recep- can be examined. We found that expression of either Toll tors (Weiner et al. 1989). Overexpression has also been or PKA together with dl resulted in an increased nuclear shown to activate erbB-2 (DiFiore et al. 1987). We sug- localization of dl and an enhancement of the ability of dl gest that in Schneider cells, Toll is also activated by over- to activate CAT expression. We also observed that dl is expression and subsequent aggregation. somewhat more active, and more cells have strong nu- Once activated, Toll signals the nuclear localization clear dl staining when cotransfected with dl plus PKA and activation of dl through PKA. Toll most likely uses than when cotransfected with d/plus Toll. This obser- cAMP as a second messenger to activate PICA. Although vation supports the notion, discussed below, that PKA is we have no direct evidence for this, work with IL-1R a downstream step in a signal transduction pathway that supports this view. The IL-1R protein is a receptor for the requires Toll to transmit the signal for dl nuclear local- interleukin 1 (IL-1) hormone, which is involved in me- ization to d/through PKA. diating immune and inflammatory responses (Sims et al. 1989). The IL-1R and Toll cytoplasmic domains share extensive sequence similarity (Schneider et al. 1991), A model for nuclear localization and activation of dl suggesting that they could transmit their signals by a Our results support the model shown in Figure 10 for dl similar mechanism. IL-1 has been shown to induce the nuclear localization and activation. In this model Toll expression of interleukin-2 receptors (IL-2R) and to in- receives an extracellular signal that is ventrally localized duce thymocyte proliferation, in cells that express IL-1 R, in the perivitelline space of the embryo (Stein et al. through stimulation of cAMP production (Shirakawa et 1991). Although the identity of this signal is unknown, it al. 1988). IL-1, as well as cAMP and cAMP analogs, has most likely functions by binding to or cleavage of the also been shown to activate K light-chain expression by extracellular domain. Deletion of extracellular domains activation of a NF-KB like DNA-binding protein is a mechanism that can activate some receptors. The (Shirakawa et al. 1989). Together, these observations epidermal growth factor (EGF) receptor (c-erbB) can be suggest that the signaling pathway from Toll to dl also activated by deletion of its extracellular domain and the involves stimulation of cAMP production, leading to the oncogene v-erbB encodes only the transmembrane and activation of PKA and the phosphorylation of dl. Phos- cytoplasmic domain of c-erbB (Downward et al. 1984; phorylated dl is free to move into the nucleus and has an Nilsen et al. 1985). Two of the Drosophila dorsal group enhanced ability to activate transcription. genes, snake and easter, encode serine proteases that act Genetic studies have placed two dorsal group genes, upstream of Toll (DeLotto and Spierer 1986; Chasan and tube and pelle, downstream of Toll (Govind and Steward Anderson 1989), and either could activate Toll by cleav- 1991). The proteins encoded by these two genes may be ing the extracellular domain. However, the fact that used to help transmit the signal from Toll to dl. The tube many Toll dominant alleles contain point mutations in gene has been cloned, but its sequence reveals nothing the extracellular domain (Schneider et al. 1991) perhaps about its possible function (Letsou et al. 1991). Because supports ligand binding as the mechanism for Toll acti- we do not know whether tube and/or pelle are expressed vation. Point mutations may mimic ligand binding by in Schneider cells, we cannot say whether they are ab- inducing the aggregation of receptors, resulting in their solutely required for this signaling pathway. No genetic

AFTER ACTIVATION

BEFORE ACTIVATION Extracellular Signal C ~ Toll (75 PKA pc,u, (,1

Figure 10. A model for the activation of dl by Toll. Before activation, dl is held in the cytoplasm, and is therefore inactive, through an association with cactus. After Toll receives an extracellular signal, dl is freed from the cytoplasm and activated as a result of phosphorylation by PKA.

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Norris and Manley evidence exists implicating PKA in D/V patterning, al- gous to IKB, anchoring dl in the cytoplasm by forming a though this is not surprising given the multiple func- dl-cactus complex. This idea is further supported by the tions of this protein. finding that cactus, like IKB, contains ankyrin repeats (S. Kidd, pets. comm.). In Schneider cells, dl protein is localized in the cyto- Anchoring of dl in the cytoplasm plasm at low concentrations but is increasingly found in Like dl, NF-KB is regulated by its subcellular localiza- the nucleus as the amount of transfected dl is increased tion, inactive when in the cytoplasm and active when (Rushlow et al. 1989). This observation can be explained localized in the nucleus (Baeuerle and Baltimore 1988a). in terms of an interaction between dl and cactus because NF-KB is retained in the cytoplasm by association with cactus is known to be expressed in Schneider cells (S. IKB; and upon disruption of the complex, NF-KB moves Kidd, pers. comm.). Increasing the amount of dl protein into the nucleus (Baeuerle and Baltimore 1988b). Pro- expressed in these cells could saturate the endogenous teins that belong to the IKB family contain ankyrin re- cactus so that at low dl concentrations all dl is bound by peats (Haskill et al. 1991), and members of the ankyrin cactus. As the amount of dl is increased, there is no free family can regulate interactions between membrane and cactus to interact with dl, so dl is free to move into the cytoskeletal elements (Lux et al. 1990). Therefore, NF-KB nucleus. In our model for Toll-mediated activation of dl, could be held in the cytoplasm owing to its ability to phosphorylation of dl by PKA disrupts the dl-cactus interact with IKB, which could be anchored in the cyto- complexes so that dl is free to move into the nucleus plasm through association with the cytoskeleton. The even at low concentrations. NF-KB/IKB complex can be destabilized by the phosphorylation of IKB by PKC (Ghosh and Baltimore 1990), in- Activation of transcription by dl dicating that phosphorylation can disrupt this protein- protein interaction, resulting in the activation of NF-KB In the early embryo, dl influences the expression of sev- in vitro. Such an interaction with a protein anchored in eral zygotic genes (for review, see Anderson 1987; the cytoplasm may also be used to retain dl in the cyto- Rushlow and Arora 1990). Functional binding sites, sim- plasm. ilar to NF-KB consensus sites, have been identified up- NF-KB can be activated by a variety of agents including stream of zen (Ip et al. 1991} and twist (Jiang et al. 1991; viruses, T-cell mitogens, cytokines, bacterial lipo- Thisse et al. 1991) genes. Expression of zen is repressed polysaccharides, and DNA-damaging agents (for review, in ventral regions of the embryo (Rushlow et al. 1987) see Baeuerle and Baltimore 1990). NF-KB is involved in while twist is activated in the ventral-most regions of activating the expression of a variety of genes that are the embryo (Thisse et al. 19871, suggesting that dl can act required for immune, infection, inflammatory, and acute as both an activator and repressor. In Schneider cells dl phase responses in a variety of cell types (Lenardo and can activate expression from a variety of promoters that Baltimore 1989; Baeuerle and Baltimore 1990; Liber- do not appear to contain dl-binding sites, including, par- mann and Baltimore 1990). Therefore, NF-KB should be adoxically, the zen-CAT200 reporter used here [note responsive to a number of signaling pathways, including that the dl sites in zen are located far upstream of the PKC. In contrast, dl activity is only required once, for the basal promoter (Ip et al. 1991)]. One reason for this could establishment of polarity in the early embryo; therefore, be the presence of dl-binding sites in the CAT reporter dl activation probably depends on only one signaling plasmid. A possible all-binding site has been found in the pathway, and the data presented here suggest that this pUC vector used here (Thisse et al. 1991). However, a involves phosphorylation of dl by activated PKA. We reporter construct containing a deletion of this region is suggest that a similar activation pathway can be used to still strongly activated by dl (J.L. Norris and J.L. Manley, activate NF-KB under certain conditions, for example, in unpubl.). A possible explanation for the ability of dl to response to IL-1. For dl, phosphorylation of dt itself is activate a wide variety of promoters is that the function required and alone may be responsible for activating dl of dl involves a strong interaction with its target, per- by leading to its release from its inhibitor, presumably haps a component of the general transcription machin- the cactus protein (see below). However, we cannot rule ery. This could conceivably allow dl to activate tran- out that modification of cactus also occurs and plays a scription in the absence of d/-binding sites, especially role, as we found that the dlQ mutant was still weakly when expressed at high levels by transfection. In support activated by PKA or Toll. The exact mechanism of acti- of this idea, we have observed a strong and specific func- vation of dl by PKA will not be known until the inter- tional interaction involving TFIID and dl in cotransfec- action between dl and cactus is better understood. tion experiments (J.L. Norris, J. Colgan, and J.L. Manley, The cactus gene is one that is required for establish- unpubl.). ment of D/V polarity in the early embryo tAnderson Whatever the mechanism by which dl activates tran- 1987). Unlike the dorsal group genes that give rise to scription in cultured cells, it is intriguing that both PKA dorsalized embryos when mutated, cactus mutations re- and Toll can enhance this activity independent of their sult in partially ventralized embryos, suggesting that effects on localization. This suggests that phosphoryla o cactus is in some way responsible for inhibiting dl action of dl increases the ability of the protein to interact tivity {Roth et al. 1989, 1991; Steward 1989). Therefore, either with DNA or with other factors required to acti- cactus has been proposed to perform a function analo- vate transcription. Determining the precise effects of

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Nuclear transport of the dorsal protein phosphorylation on dl will require further investigation with a BglII-StuI fragment, containing Toll-coding sequence for into the proteins with which d/interacts, in the cyto- amino acids 323-969, from the T/lob eDNA clone (kindly pro- plasm as well as the nucleus. vided by K. Anderson) to replace the wild-type sequence with the sequence encoding a cysteine to tyrosine change at amino acid 779. The pActPKA expression vector was constructed by cleaving Materials and methods the PKA cDNA clone (kindly provided by D. Kalderon (Colum- Recom bin an t plasmids bia University) with XbaI, filling in the 5' overhang with Kle- now, and digesting with KpnI. A 1.1-kb KpnI-XbaI fragment, All expression vectors were derived from a plasmid that con- containing the PKA-coding sequence, was inserted into the tains the Drosophila actin 5C promoter and poly(A) site, pActSC polylinker by digesting pActSC with KpnI and EcoRV. pAct5CSRS (pActSC), which has been described in detail (Han The pAct-lacZPKi expression vector was provided by M.E. Lane et al. 1989}. The zen-CAT200 reporter plasmid, pAct-d/, and (Columbia University). pAct-dl3 (dl-561) have been described previously by Rushlow et al. (1989). The above constructs and pAct-all4, pAct-diS, pact- d16, and pAct-all9 were provided by K. Han (Columbia Univer- DNA transfection and transient expression assay sity, New York). The dl NTS mutants, pAct-all7 and pAct-d/& Drosophila Schneider L2 cells were grown and transfected as were provided by S. Small. The dlQ, diD, and dlRRKS mutants described previously (Han et al. 1989). Each transfection con- were constructed by site-directed mutagenesis. A 0.65-kb SacI- tained 5.0 ~g of expression vector consisting of the indicated EcoRI fragment was isolated from pAct-d/and cloned into pB- amounts of dl, Toil, or PKA expression vectors and variable luescript SK{ + ) (Stratagene), which had been cleaved with SacI amounts of pAct5C to bring the total amount of expression and EcoRI. The following oligonucleotides were made: 5'-GC- vector to 5.0 ~g. A total of 10 lag of DNA was used for each GACGTCCCCAGGATGGAG-3' for dlQ; 5'-CGACGTCCC- transfection, so the remaining 5.0 lag of DNA consisted of 2.0 ~g GATGATGGAGTTACC-3' for diD; 5'-CTGCGACG- copia long terminal repeat (LTR}-IacZ as an internal control TAAATCGGATGGA-3' for dlRRKS. The oligonucleotides and 3.0 p-g of the zen-CAT200 reporter plasmid. All transfec- were annealed to uracil containing single-stranded DNA, and tions were performed in duplicate, and ~-galactosidase and CAT synthesis of the second strand was done with T4 DNA poly- activities were measured as described previously {Han et al. merase. The resulting clones were screened for sequence that 1989). The CAT activities presented represent the average of encoded the appropriate amino acid change. Clones containing several independent transfections. the desired mutations were isolated and digested with StuI and Transfections that included the lacZPKi expression vector BstXI to generate a fragment, containing the mutated region, contained 0.4 ~g of dl expression vector, 3.0 ~g of Toll expres- that was used to replace the corresponding wild-type fragment sion vector, and 2.0-3.6 lag of lacZPKi expression vector. The in pAct-d/. total amount of expression vector was adjusted to 7.0 ~g with The pAct-T/expression vector was constructed by isolating a pActSC. A total of 12.0 ~g of DNA, including 2.0 lag of copia NsiI-KpnI fragment, containing the Toll-coding region, from LTR-lacZ and 3.0 lag of zen-CAT200, was used in each trans- the Toll cDNA clone [kindly provided by K. Anderson (Schnei- fection. Cells were harvested and protein extracts were prepared der et al. 1991)]. The 3' overhang that resulted from cleavage as described (Han et al. 1989). The same amount of extract from with NsiI was digested with the Klenow fragment of DNA poly- each sample was used in a CAT assay. A ~-galactosidase assay merase (Klenow) before cleavage by KpnI. This fragment was was performed to ensure that protein was being expressed in inserted into the pActSC polylinker, adjacent to the actin 5C approximately equal amounts. Three independent transfections promoter, by a filled-in BamHI site and a KpnI site. The pact- were performed in duplicate, and all gave similar results. T1RV mutant was constructed by digesting pAct-T/ with EcoRV, which cleaves at one site within Toll and another in the pAct5C polylinker, and removing the 3.6-kb EcoRV-EcoRV Staining of cells fragment to create an in-frame stop after amino acid 274. To Schneider cells were transfected as described above, and 48 hr construct pAct-T/520, pAct-T/RV was digested with EcoRV after transfection the cells were fixed on plates with formalde- and BglII and ligated with a 2.1-kb SalI-BglII fragment from hyde. The cells were blocked with 10% BSA, stained with anti ° pAct-T/to restore the Toll membrane-spanning and cytoplas- dl primary antibodies [provided by C. Rushlow and M. Levine mic domains. The 5' overhang generated by digestion with SalI (Rushlow et al. 1989)[, washed twice with washing and dilution was filled in with Klenow to generate an in-frame deletion of buffer (1% BSA, 0.5 M NaC1, and 0.1% Tween 80 in PBS), and amino acids 274-793. The pAct-TIStuI mutant was constructed stained with TRITC-conjugated secondary antibodies. by digesting pAct-T/with BglII, NcoI, and StuI to generate a 1.4-kb NcoI-BglII fragment, containing actin 5C promoter sequences and Toll-coding sequence for amino acids 1-322, and a Acknowledgments 1.9-kb BglII-StuI fragment that contains Toll-coding sequence for amino acids 323-970. These two fragments were ligated We are grateful to K. Anderson, K. Han, D. Kalderon, M.E. Lane, with a 7.0-kb BglII-NcoI fragment from pAct5C, containing ac- and S. Small for providing plasmids, and to M. Levine and C. tin 5C promoter, actin 5C poly(A), and pBR322 sequences, Rushlow for providing antibodies. We thank D. Kaldron, M. which had been cleaved with BglII. The 5' overhang generated Levine, K. Han, J. Colgan, D. Read, M.E. Lane, and J. Wu for by cleavage with BglII was filled-in with Klenow before diges- advice and discussion, D. Kalderon for comments on the manu- tion with NcoI. The pAct-T1 ~°b mutant was constructed by di- script, and S. Kidd for communicating results prior to publica- gesting pAct-T/with BglII and StuI to generate a 7.3-kb BglII- tion. This work was supported by a predoctoral training grant BglII fragment, containing actin 5C promoter and poly(A) se- from the National Institutes of Health (NIH) to J.L.N. and NIH quences, as well as Toll-coding sequence for amino acids 1-322, grant GM37971 to J.L.M. and a 1.6-kb BglII-StuI fragment that contains Toll-coding se- The publication costs of this article were defrayed in part by quence for amino acids 970-1097. These fragments were ligated payment of page charges. This article must therefore be hereby

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Norris and Manley marked "advertisement" in accordance with 18 USC section gene of Drosophila, required for dorsal-ventral embryonic 1734 solely to indicate this fact. polarity, appears to encode a transmembrane protein. Cell 52: 269-279. Hashimoto, C., S. Gerttula, and K.V. Anderson. 1991. Plasma References membrane localization of the Toll protein in the syncytial Drosophila embryo: Importance of transmembrane signaling Anderson, K.V. 1987. Dorsal-ventral embryonic pattern genes of for dorsal-ventral pattern formation. Development 111: Drosophila. Trends Genet. 3: 91-97. 1021-1028. • 1989. Drosophila: The maternal contribution. In Genes Haskill, S., A.A. Beg, S.M. Tompkins, J.S. Morris, A.D. Yuro- and embryos (ed. D.M. Glover and B.D. Hanes), pp. 1-37. chko, A. Johannes-Sampson, K. Mondal, P. Ralph, and A.S. IRL Press, Oxford, England. Baldwin. 1991. Characterization of an immediate-early gene Anderson, K.V. and C. Nfisslein-Volhard. 1986. Dorsal-group induced in adherent monocytes that encodes IKB-like activ- genes of Drosophila. In Gametogenesis and the early em- ity. Cell 65: 1281-1289. bryo (ed. J.Gall), pp. 177-194. Alan R. Liss, New York. Ip, Y.T., R. Kraut, M. Levine, and C.A. Rushlow. 1991. The Anderson, K.V., G. Jiirgens, and C. N~sslein-Volhard. 1985a. dorsal morphogen is a sequence-specific DNA-binding pro- Establishment of dorsal-ventral polarity in the Drosophila tein that interacts with a long-range repression element in embryo: Genetic studies on the role of the Toll gene product. Drosophila. Cell 64: 439--446. Cell 42: 779-789. Jiang, J., D. Kosman, Y.T. Ip, and M. Levine. 1991. The dorsal Anderson, K.V., L. Bokla, and C. Ntisslein-Volhard. 1985b. Es- morphogen gradient regulates the mesoderm determinant tablishment of dorsal-ventral polarity in the Drosophila em- twist in early Drosophila embryos. Genes & Dev. 5: 1881- bryo: The induction of polarity by the Toll gene product. 1891. Cell 42: 791-798. Kalderon, D. and G.M. Rubin. 1988. Isolation and characteriza- Baeuerle, P.A. and D. Baltimore. 1988a. Activation of DNA- tion of Drosophila cAMP-dependent protein kinase genes. binding activity in an apparently cytoplasmic precursor of Genes & Dev. 2: 1539-1556. the NF-KB transcription factor. Cell 53:211-217. Keith, F.J. and N.J. Gay. 1990. The Drosophila membrane re- ~. 1988b. IKB: A specific inhibitor of the NF-KB transcrip- ceptor Toll can function to promote cellular adhesion. tion factor• Science 242: 540-546. EMBO J. 9: 4299--4306. ~. 1990. The physiology of the NF-KB transcription factor. Kemp, B.E. and R.B. Pearson. 1990. Protein kinase recognition Hormonal regulation of transcription. Mol. Aspects Cell. sequence motifs. Trends Biochem. Sci. 15: 342-346. Regul. 6: 409-432. Kemp, B.E., H. Cheng, and D.A. Walsh. 1988. Peptide inhibitors Chasan, R. and K.V. Anderson. 1989. The role of easter, an of cAMP-dependent protein kinase. Methods Enzymol. apparent serine protease, in organizing the dorsal-ventral 159: 173-185. pattern of the Drosophila embryo. Cell 56:391-400. Kieran, M., V. Blank, F. Logeat, I. Vandekerckhove, F. Lott- Cheng, H.C., S.M. Van Pattern, A.J. Smith, and D.A. Walsh. speich, O. Le Bail, M.B. Urban, P. Kourilsky, P.A. Baeuerle, 1985. A active twenty-amino-acid-residue peptide derived and A. IsraEl. 1990. The DNA binding subunit of NF-KB is form the inhibitor protein of the cyclic AMP-dependent pro- identical to factor KBF 1 and homologous to the tel oncogene tein kinase. Biochem. J. 231: 655-661. product. Cell 62: 1007-1018. DeLotto, R. and P. Spierer. 1986. A gene required for the spec- Lenardo, M.J. and D. Baltimore. 1989. NF-KB: A pleiotropic me- ification of dorsal-ventral pattern in Drosophila appears to diator of inducible and tissue-specific gene control. Cell encode a serine protease. Nature 323: 688-692. 58: 227-229. DiFiore, P.P., J.H. Pierce, M.H. Kraus, O. Segatto, C.R. King, and Letsou, A., S. Alexander, K. Orth, and S.A. Wasserman. 1991. S.A. Aaronson. 1987. erbB-2 is a potent oncogene when over- Genetic and molecular characterization of tube, a Droso- expressed in NIH/3T3 cells. Science 237:178-182. phila gene maternally required for embryonic dorsoventral Downward, J., Y. Yorden, E. Mayes, G. Scrace, N. Totly, P. polarity. Proc. Natl. Acad. Sci. 88: 810-814. Stockwell, A. Ullrich, J. Schlessinger, and M.D. Waterfield. Libermann, T.A. and D. Baltimore. 1990. Activation of inter- 1984. Close similarity of epidermal growth factor receptor leukin-6 gene expression through the NF-KB transcription and v-erb-B oncogene protein sequences. Nature 307: 521- factor. MoI. Cell. Biol. 10: 2327-2334. 527. Lopez, J.A., D.W. Chung, K. Fujikawa, F.S. Hagen, T. Papayan- Erd41yi, M. and J. Szabad. 1989. Isolation and characterization of nopoulou, and G.J. Roth. 1987. Cloning of the a chain of dominant female sterile mutations of Drosophila melano- human platelet glycoprotein Ib: A transmembrane protein gaster I. Mutations on the third chromosome. Genetics with homology to leucine-rich ~2-glycoprotein. Proc. Natl. 122: 111-127. Acad. Sci. 84: 5615-5619. Ghosh, S. and D. Baltimore. 1990. Activation in vitro of NF-KB Lux, S.E., K.M. John, and V. Bennett. 1990. Analysis of cDNA by phosphorylation of its inhibitor IKB. Nature 344: 678- for human erythrocyte ankyrin indicates a repeated struc- 682. ture with homology to tissue-differentiation and cell-cycle Ghosh, S., A.M. Gifford, L.R. Riviere, P. Tempst, G.P. Nolan, control proteins. Nature 344: 36-42. and D. Baltimore. 1990. Cloning of the p50 DNA binding Mosialos, G., P. Hamer, A.J. Capobianco, R.A. Laursen, and subunit of NF-KB: Homology to rel and dorsal. Cell T.D. Gilmore. 1991. A protein kinase-A recognition se- 62: 1019-1029. quence is structurally linked to transformation by p59 v-r~i Govind, S. and R. Steward. 1991. Dorsoventral pattern forma- and cytoplasmic retention of p68 crel. Mol. Cell. Biol. tion in Drosophila: Signal transduction and nuclear target- 11: 5867-5877. ing. Trends Genet. 7: 119-125. Nilsen, T.W., P.A. Maroney, R.G. Goodwin, F.W. Rottman, L.B. Han, K., M.S. Levine, and J.L. Manley. 1989. Synergistic activa- Crittenden, M.A. Raines, and H. Kung. 1985. c-erbB activation and repression of transcription by Drosophila ho- tion in ALV-induced erythroblastosis: Novel RNA process- meobox proteins. Cell 56: 573-583. ing and promotor insertion result in expression of an amino- Hashimoto, C., K.L. Hudson, and K.V. Anderson. 1988. The Toll truncated EGF receptor. Cell 41: 719-726.

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Nuclear transport of the dorsal protein

Roth, S., D. Stein, and C. Niisslein-Volhard. 1989. A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell 59:1189- 1202. Roth, S., Y. Hiromi, D. Godt, and C. Niisslein-Volhard. 1991. cactus a maternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophila embryos. Development 112: 371-388. Rushlow, C. and K. Arora. 1990. Dorsal-ventral polarity and pattern formation in Drosophila. Sere. Cell Biol. 1: 173-184. Rushlow, C., M. Frasch, H. Doyle, and M. Levine. 1987. Mater- nal regulation of zenknffflt: A homeobox gene controlling differentiation of dorsal tissues in Drosophila. Nature 330: 583-586. Rushlow, C.A., K. Han, J.L. Manley, and M. Levine. 1989. The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila. Cell 59: 1165- 1177. Schneider, D.S., K.L. Hudson, T. Lin, and K.V. Anderson. 1991. Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal- ventral polarity in the Drosophila embryo. Genes & Dev. 5: 797-807. Shirakawa, F., U. Yamashita, M. Chedid, and S.B. Mizel. 1988. Cyclic AMP-an intracellular second messenger for interleukin 1. Proc. Natl. Acad. Sci. 85: 8201-8205. Shirakawa, F., M. Chedid, I. Suttleo, B.A. Pollok, and S.B. Mizel. 1989. Interleukin 1 and cyclic AMP induce K immunoglob- ulin light-chain expression via activation of an NF-KB-like DNA binding protein. Mol. Cell. Biol. 9: 959-964. Sims, J.E., B. Acres, C.E. Grubin, C.J. McMahan, J.M. Wignall, C.J. March, and S.K. Dower. 1989. Cloning the interleukin 1 receptor form human T cells. Proc. Natl. Acad. Sci. 86: 8946-8950. Stein, D., R. Siegfried, E. Vogelsang, and C. Ntisslein-Volhard. 1991. The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal. Cell 65: 725- 735. Steward, R. 1987. Dorsal, an embryonic polarity gene in Droso- phila, is homologous to the vertebrate proto-oncogene, c-tel. Science 238: 692-694. ~. 1989. Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function. Cell 59: 1179-1188. Steward, R., S.B. Zusman, L.H. Huang, and P. Schedl. 1988. The dorsal protein is distributed in a gradient in early Drosophila embryos. Cell 55: 487-495. Thisse, B., C. Stoetzel, M. E1 Messal, and F. Perrin-Schmitt. 1987. Genes of the Drosophila matemal dorsal group control the specific expression of the zygotic gene twist in presump- tive mesodermal cells. Genes & Dev. 1: 709-715. Thisse, C., F. Perrin-Schmitt, C. Stoetzel, and B. Thisse. 1991. Sequence-specific transactivation of the Drosophila twist gene by the dorsal gene product. Cell 65:1191-1201. Walsh, D.A. and D.B. Glass. 1991. Utilization of the inhibitor protein of adenosine cyclic monophosphate-dependent protein kinase, and peptides derived from it, as tools to study adenosine cyclic monophosphate mediated cellular pro- cesses. Methods Enzymol. 201: 304-317. Weiner, D.B., J. Liu, J.A. Cohen, W.V. Williams, and M.I. Green. 1989. A point mutation in the neu oncogene mimics ligand induction of receptor aggregation. Nature 339: 230-231.

GENES & DEVELOPMENT 1667 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Selective nuclear transport of the Drosophila morphogen dorsal can be established by a signaling pathway involving the transmembrane protein Toll and protein kinase A.

J L Norris and J L Manley

Genes Dev. 1992, 6: Access the most recent version at doi:10.1101/gad.6.9.1654

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