Ectopic Expression of Inhibitors of Protein Phosphatase Type 1 (PP1) Can Be Used to Analyze Roles of PP1 in Drosophila Development

, Balázs Ször

Corresponding author: Luke Alphey, Oxford University, South Parks Rd., Oxford OX1 3PS, United Kingdom., luke.alphey{at}zoo.ox.ac.uk (E-mail)

Communicating editor: K. GOLIC

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGFß superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.

REVERSIBLE protein phosphorylation is an important mechanism of post-translational regulation controlling cellular and developmental processes (HUNTER 1995 ). Phosphorylation is controlled by the relative activity of protein kinases, which add phosphate, and protein phosphatases, which remove phosphate. Protein phosphatase type 1 (PP1) is one of the major classes of protein serine/threonine phosphatases and has been found in all eukaryotic cells examined to date (DOMBRADI 1997 ; LIN et al. 1999 ). Together with PP2A, PP1 contributes 90% of the serine/threonine protein phosphatase activity in mammalian cells. The importance of PP1 in glycogen metabolism and cell cycle control is well established. PP1 is also involved in the regulation of gene expression, muscle contraction, memory, learning, and a host of other processes (BOLLEN and STALMANS 1992 ; SHENOLIKAR 1994 ; DOMBRADI 2002 ). While earlier studies focused on protein kinases, it is now clear that protein phosphatases play an equally tightly regulated and important role in the control of cellular phosphoproteins (BOLLEN 2001 ; COHEN 2002 ).

The catalytic subunit of PP1 (PP1c) is associated in vivo with regulatory subunits that target it to specific locations and modify its substrate specificity and activity (BOLLEN 2001 ; COHEN 2002 ). The isolation of novel targeting subunits has revealed important roles for PP1 in developmental signal transduction pathways. For instance, we recently showed that PP1c binds the anchor protein Sara (Smad anchor for receptor activation), a component of the TGFß superfamily receptor complex (BENNETT and ALPHEY 2002 ). In TGFß/Bone Morphogenic Protein (BMP)/Activin superfamily signaling, the ligand induces the formation of a stable complex between a type II serine/threonine receptor kinase and a type I serine/threonine receptor kinase. The type II receptor phosphorylates and activates the type I receptor, which then propagates the signal (reviewed by HELDIN et al. 1997 ; MASSAGUE 1998 ; MASSAGUE and WOTTON 2000 ). Experiments with mutant Sara protein designed to reduce the level of PP1 at the Sara-receptor complex in mammalian cells showed that the role of Sara-bound PP1 is to regulate the phosphorylation state of the type I receptor (BENNETT and ALPHEY 2002 ). Although the specificity for different ligands and type I receptors was not clear, genetic analysis revealed that PP1 also functions in TGFß superfamily signaling in Drosophila to control growth and patterning of the wing (BENNETT and ALPHEY 2002 ).

Sara is known to be involved in TGFß signaling, but in many cases the physiological role of the PP1c-targeting subunits is unknown. In addition, the exact number and variety of these regulatory subunits has not been determined (CEULEMANS et al. 2002 ). Much can yet be learned from analysis of PP1c loss-of-function phenotypes. However, metazoans from Drosophila to humans have multiple genes encoding PP1c isoforms (DOMBRADI et al. 1989 , DOMBRADI et al. 1993 ; DOMBRADI 1997 ; LIN et al. 1999 ) and partial redundancy and perdurance of the different PP1c isoforms has made it difficult to fully analyze the developmental roles of PP1. There are four PP1c genes in Drosophila, which are named according to their cytological location and isotype: PP113C, PP187B, PP196A, and PP1ß9C (DOMBRADI et al. 1990B , DOMBRADI et al. 1993 ). Of these, PP187B contributes 80% of the total PP1 activity. Mutant alleles of PP187B show lethality, aberrant mitosis, suppression of position effect variegation, and reduced levels of protein phosphatase activity (AXTON et al. 1990 ; BAKSA et al. 1993 ). PP1ß9C, which is the fly homolog of mammalian PP1, is required for maintenance of muscle attachments (RAGHAVAN et al. 2000 ). PP113C is not essential for either viability or fertility (CLYNE et al. 1999 ). No mutants are available for PP196A. Clonal analysis, which is a powerful tool for examining the role of genes in developmental processes (ST. JOHNSTON 2002 ), cannot easily be applied to the PP1c isoforms because of the need to simultaneously test multiple loci. Moreover, a sensitized genetic background is often necessary to analyze a specific effect of such pleiotropic genes, which further complicates genetic analysis (ST. JOHNSTON 2002 ).

As well as binding various noncatalytic targeting subunits that regulate PP1 activity, PP1c also associates with endogenous inhibitors of PP1, such as Inhibitor 2 (I-2) and Nuclear Inhibitor of PP1 (NIPP1), which may help to prevent inappropriate dephosphorylation of nonphysiological targets (SHENOLIKAR 1995 ; BOLLEN 2001 ; COHEN 2002 ). Various endogenous and nonendogenous inhibitors of PP1 have long been used in vitro as a means of distinguishing between different phosphatase activities. For example, I-2 is often used in combination with okadaic acid (OA), a polyether fatty acid from dinoflagellates, to distinguish between PP1 and PP2A activity or to detect PP2C in the absence of the other major protein phosphatases in cell extracts (SHENOLIKAR 1995 ; WERA and HEMMINGS 1995 ). However, the use of inhibitors on live cells has been limited by poor penetration into cells and slow accumulation, which makes it difficult to control their concentration (WERA and HEMMINGS 1995 ).

To dissect the roles of PP1c in Drosophila we have sought to control PP1c activity by overexpression of Drosophila I-2 and NIPP1 in a cell- and developmental-specific manner. We find that these ectopic inhibitors specifically reduce PP1c activity in vivo and result in phenotypes resembling those of PP1c mutants. Using this approach, we have extended the analysis of PP1's role in TGFß signaling in the wing by demonstrating that reduction of PP1 in cells overexpressing Punt is sufficient for wing overgrowth phenotypes and by identifying Thick-veins (Tkv) as the relevant type I receptor pathway affected by PP1 in the wing.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

I-2Dm, NIPP1Dm, and PP1c transgene construction:
The translation starts of PP113C, PP187B, PP196A, PP1ß9C, and NIPP1Dm were modified to NdeI sites and the coding regions were subcloned as NdeI/NotI fragments into pUASHA (PARKER et al. 2001 ), using NdeI and NotI restriction sites in the polylinker. I-2Dm was subcloned in a similar way into the NdeI/NotI sites of pUASHM (PARKER et al. 2001 ). Drosophila carrying UAS PP113C, UAS PP187B, UAS PP196A, UAS PP1ß9C, UAS NIPP1Dm, and UAS I-2Dm were generated by P-element-mediated germline transformation of a y w strain.

Fly strains and crosses:
For measurement of PP1c activity, UAS I-2Dm on the second chromosome was combined with arm-GAL4 on the third chromosome. For the effect of ectopic I-2Dm and NIPP1Dm together, UAS I-2Dm/CyO; arm-GAL4/TM6B flies were crossed to UAS NIPP1Dm flies. For rescue experiments with PP1c, UAS I-2Dm on the second chromosome was combined with UAS-NIPP1Dm on the third chromosome. arm-GAL4 on the third chromosome was recombined individually with UAS PP113C, UAS PP187B, UAS PP196A, UAS PP1ß9C, and UAS-GFP (YEH et al. 1995 ). These arm-GAL4 UAS-PP1c (various isoforms) flies were crossed to UAS NIPP1Dm or UAS NIPP1Dm UAS I-2Dm flies. The progeny of these crosses were screened for phenotypic abnormalities and reduced viability. For interactions with Punt, vg-GAL4 on the second chromosome, obtained from T. Tabata, was recombined with UAS I-2Dm and separately combined with UAS Punt on the third chromosome, which was obtained from M. O'Connor. vg-GAL4 UAS I-2Dm was then combined with UAS-Punt. vg-GAL4/CyO; UAS-Punt/TM6B or vg-GAL4 UAS I-2Dm/CyO; UAS-Punt/TM6B flies were then crossed to the following stocks [as described in FlyBase (http://flybase.bio.indiana.edu) unless otherwise stated] to look for genetic interaction: UAS PP113C/TM6B, UAS PP187B/TM6B, UAS PP196A/TM6B, and UAS PP1ß9C/TM6B (all this study); UAS-GFP (YEH et al. 1995 ); UAS-babo, UAS-sax, and UAS-tkv (HAERRY et al. 1998 ; BRUMMEL et al. 1999 ); and babo³²/CyO, babo^K16912/CyO, sax⁴/SM6a, tkv¹/CyO, tkv⁷/CyO, tkv⁰⁴⁴¹⁵/CyO, dpp^d6/CyO, dpp^d12/CyO, and dpp^hr4/CyO.

Preparation of protein extracts from flies:
Adult females were collected and either stored at -80^° or used directly. Flies were homogenized at 4° in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1% ß-mercaptoethanol (0.1 ml/fly) containing EDTA-free protease inhibitor mix (Roche, Indianapolis). The homogenates were clarified by centrifugation (5 min at 6000 x g, 4°) and the supernatants were used in phosphorylase a phosphatase assays.

Phosphatase assays:
Phosphatase assays were performed essentially as in BENNETT et al. 1999 and RAGHAVAN et al. 2000 . Phosphorylase b was converted to phosphorylase a with phosphorylase kinase using [-³²P]ATP with the protein phosphatase assay system (GIBCO BRL, Gaithersburg, MD). Protein phosphatase activities were assayed in duplicate after 25- to 50-fold dilutions at 0.1 mg/ml concentration. PP2A activity was taken as the difference of the total protein phosphatase activity and the activity measured in the presence of 1 nM okadaic acid. PP1 activity was taken as the difference in activity between extracts treated with or without 100 units of I-2Dm (BENNETT et al. 1999 ). A unit of phosphorylase phosphatase is defined as that amount of enzyme that releases 1 µmol of [³²P]phosphate/min at 30° from ³²P-labeled phosphorylase a.

I-2 assays and GSK3ß reactivation of I-2:PP1c complexes:
I-2Dm was assayed as in PARK and DEPAOLI-ROACH (1994). For GSK3ß phosphorylation, 50-µl reactions containing adult fly extract in reactivation buffer (20 mM Tris-HCl, 10 mM MgCl₂, 5 mM dithiothreitol, 0.2 mM ATP with or without 0.5 units GSK3ß) were incubated at 30° for 30 min after the addition of 0.2 mM ATP. After 30 min 10-µl samples were withdrawn and assayed for phosphatase activity. GSK3ß kinase assays were performed as in PARK and DEPAOLI-ROACH (1994) and PARK et al. 1994 . One unit is defined as the amount of GSK3ß required to catalyze the transfer of 1 pmol of phosphate to protein substrate I-2 in 1 min at 30°.

Neuroblast squashes:
arm-GAL4/arm-GAL4 or UAS I-2Dm/UAS I-2Dm; arm-GAL4/arm-GAL4 flies were crossed to UAS NIPP1Dm/TM3, actin5C-GFP flies. arm-GAL4/UAS NIPP1Dm [arm>NIPP1Dm] or +/UAS I-2Dm; arm-GAL4/UAS NIPP1Dm [arm>(I-2Dm, NIPP1Dm)] larvae were selected on the basis of nonfluorescence using an Olympus SZX12 fluorescence microscope. Squashes were prepared by the method of GONZALEZ et al. 1988 and examined using an Olympus BX50 microscope with a x100 phase contrast objective. A field of nuclei was defined as the area covered by the viewfinder at x100 magnification.

LacZ/ß-gal staining and microscopy:
Fixation and histochemical staining was as in BRAND and PERRIMON 1993 . Stained wing imaginal discs and adult wings were mounted in Canada Balsam or glycerol and examined by light microscopy using an Olympus BX50 microscope.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Identification of Drosophila PP1 inhibitors:
To identify Drosophila inhibitors of PP1c we previously screened for PP1ß9C-binding proteins using the two-hybrid system (ALPHEY et al. 1997 ; BENNETT et al. 1999 ). We isolated Drosophila homologs of Inhibitor-2 (I-2Dm) and Nuclear Inhibitor of PP1c (NIPP1Dm) (BENNETT et al. 1999 ; PARKER et al. 2002 ). Both I-2Dm and NIPP1Dm are capable of binding to all four Drosophila PP1c isoforms in the two-hybrid system. I-2Dm and NIPP1Dm are predicted to encode proteins with significant identity to their mammalian counterparts (>35% and 45%, respectively) and in vitro specifically inhibit PP1c activity against phosphorylase a (with IC₅₀, the concentration of protein required for 50% inhibition, 1 nM and 0.2 nM, respectively), similar to their mammalian homologs.

Ectopic I-2Dm and NIPP1Dm reduce PP1 activity in fly extracts:
To examine the effect of expressing these PP1c-binding proteins in vivo, we expressed I-2Dm and NIPP1Dm in Drosophila using the bipartite Gal4-UAS system (BRAND et al. 1994 ). Extracts from flies expressing I-2Dm or NIPP1Dm at moderate levels (arm-GAL4 with UAS I-2Dm or UAS NIPP1Dm, henceforth referred to as arm>I-2Dm and arm>NIPP1Dm, respectively) showed a significant reduction in PP1 activity. Complexes with NIPP1Dm are unstable because NIPP1Dm is rapidly degraded in tissue extracts, as has previously been reported for its mammalian homolog (JAGIELLO et al. 1995 ). Nevertheless, we also observed a significant reduction in PP1 activity in extracts expressing NIPP1Dm (Table 1, line 2). Extracts expressing I-2Dm, which forms a stable complex with PP1c, showed up to a 30% reduction in PP1 activity (Table 1, lines 4–7). The level of PP2A activity was unaffected in these extracts, showing that I-2Dm and NIPP1Dm are specific for PP1c in vivo as well as in vitro (Table 1). We also measured the PP1 activity from flies with different numbers of Gal4 and UAS elements (Table 1, lines 4–7). These data show that PP1 activity is reduced in a dose-dependent manner.

Ectopic I-2Dm inhibits PP1 by titrating PP1:
PP1c isolated as a complex with I-2 from tissue is mostly inactive but can be reactivated by incubation with MgATP (MACKINTOSH et al. 1996 ). The mechanism by which this occurs is controversial but is likely to involve a conformational change of PP1c, accompanied by phosphorylation and dephosphorylation of I-2 by GSK3 and PP1, respectively (PARK and DEPAOLI-ROACH 1994 ; PARK et al. 1994 ). Drosophila I-2 has the same properties in this respect as the mammalian protein (BENNETT et al. 1999 ); therefore we examined the extent to which we could increase PP1 activity from extracts overexpressing I-2Dm by addition of GSK3ß and MgATP. PP1 activity was restored to 98% of wild type in arm>I-2Dm fly extracts by this treatment (Table 2). We saw no increase in the reactivated PP1 activity of arm>I-2Dm extracts relative to wild type, suggesting that overexpression of I-2Dm does not lead to a compensating increase in total PP1c. Unfortunately, lack of a suitable antibody meant that we were unable to examine PP1 protein levels directly. Together, these data suggest that ectopically expressed I-2Dm and NIPP1Dm inhibit PP1c activity in vivo by sequestering PP1.

Ectopic NIPP1Dm and I-2Dm phenotypically resemble PP1c mutants:
To test whether this loss of PP1c activity was associated with loss of PP1c function in vivo we examined arm>I-2Dm and arm>NIPP1Dm flies for phenotypes known to be caused by PP1c mutants. Null mutants in PP187B, which is the major PP1c isoform in Drosophila (DOMBRADI et al. 1990A ), die as late larvae or early pupae and show a high frequency of abnormal mitosis with a high degree of chromosomal condensation (AXTON et al. 1990 ). Similarly, >95% arm>NIPP1Dm flies die either before or just after pupation. When we examined squash preparations of brains from arm>NIPP1Dm third instar larvae, we saw significantly fewer cells per microscope field (119 ± 21 compared to 143 ± 19 in wild type; Table 3) and a significantly higher proportion of cells in mitosis than in wild type (3.6 ± 1.0 compared to 2.1 ± 0.9 in wild type; Table 3), indicating failure of diploid cell proliferation (GATTI and BAKER 1989 ). There were also high levels of overcondensed chromosomes (Table 3). These phenotypes are very similar to, but somewhat less severe than, those of strong PP187B mutants (Table 3). I-2Dm, which is a weaker inhibitor than NIPP1Dm in vitro, had no effect when expressed on its own with arm-GAL4, but did enhance the effect of ectopic NIPP1Dm (Table 3). Therefore ectopic expression of NIPP1Dm and I-2Dm phenotypically resembles PP187B mutants.

PP1ß9C is responsible for no more than 10% of the total PP1c activity, yet mutations in PP1ß9C show lethality and defective locomotion and have cell adhesion defects especially in muscles (RAGHAVAN et al. 2000 ). Formation of the indirect flight muscles (IFMs) is normal in these mutants, but later the muscles detach from their attachment sites. Weak mutations in PP1ß9C are viable, but are flightless due to defects in the indirect flight muscles (RAGHAVAN et al. 2000 ). This prompted us to examine the IFMs from the few arm>NIPP1Dm survivors. We found that the IFMs were not present or in complete disarray in the few arm>NIPP1Dm flies (Fig 1C), although the jump muscle (tergal depressor of trochanter muscle) was mostly normal, as is the case for PP1ß9C mutants (Fig 1). Therefore arm>NIPP1Dm flies show defects characteristic of loss-of-function mutants of both PP1ß9C and PP187B.

View larger version (21K): In this window In a new window Download PPT slide   Figure 1. Polarized light micrographs showing muscle defects in flies expressing NIPP1Dm. Anterior is to the left, dorsal is up. (A) Wild type (Oregon-R), showing fully developed indirect flight muscles. The dorsal longitudinal muscles (indicated by arrowhead) are clearly visible. (B) The IFMs are completely absent in PP1ß9C mutants (RAGHAVAN et al. 2000 ) and (C) in UAS NIPP1Dm/+; arm-GAL4/+ flies, which ectopically express NIPP1Dm. (D) The IFMs are restored in UAS NIPP1Dm/+; arm-GAL4, UAS PP1ß9C/+ flies, which overexpress both NIPP1Dm and PP1ß9C. For clarity, the outline of each fly is indicated by a dashed blue line.

Ectopic PP1 suppresses the effect of ectopic NIPP1Dm and I-2Dm:
To confirm that the dominant phenotypes caused by overexpression of I-2Dm and NIPP1Dm were due to decreased levels of PP1c activity we examined whether the phenotypes could be suppressed by coexpression of UAS-PP1c. Flies expressing only UAS-PP1c (any isoform) under the control of arm-GAL4 resembled wild type (not shown). The effects of arm>NIPP1Dm and arm>(I-2Dm, NIPP1Dm) were completely suppressed by coexpression of UAS-PP113C, UAS-PP187B, UAS-PP196A, or UAS-PP1ß9C (Fig 1; data not shown), indicating that I-2Dm and NIPP1Dm are not specific for particular isoforms. Coexpression of UAS I-2Dm or UAS NIPP1Dm with UAS-GFP did not modify the effect of I-2Dm or NIPP1Dm, showing that suppression by UAS-PP1c was not simply due to titration of GAL4. Therefore, the dominant phenotypes caused by overexpression of I-2Dm and NIPP1Dm are due to decreased levels of available PP1c.

I-2Dm and NIPP1Dm have the same effect as PP1c mutants on TGFß superfamily signaling:
Reduction of PP187B levels in the wing imaginal disc enhances the growth stimulatory effects of ectopic type II TGFß superfamily receptor Punt (BENNETT and ALPHEY 2002 ). To determine the effect of I-2Dm and NIPP1Dm on TGFß signaling, we crossed the UAS inhibitors to flies expressing punt along the presumptive wing margin under the control of vg-GAL4. Flies expressing UAS I-2Dm or UAS-NIPP1Dm alone using vg-GAL4 resembled wild type (not shown). Expression of UAS-punt using vg-GAL4 (henceforth referred to as vg>punt) resulted in a modest wing overgrowth phenotype (Fig 2B). UAS I-2Dm or UAS-NIPP1Dm dominantly enhanced this phenotype (Fig 2C and Fig D), with NIPP1Dm being the more potent enhancer. The effect of ectopic I-2Dm was remarkably similar to that of PP187B¹, a hypomorphic PP187B allele, while the effect of ectopic NIPP1Dm resembled that of PP187B^87Bg-6, a protein null allele (Fig 2E and Fig F). Therefore I-2Dm and NIPP1Dm induce TGFß signaling in a background of ectopic Punt in a way similar to that of PP1c mutants. The effects of UAS I-2Dm and UAS NIPP1Dm were additive, indicating that the severity of the effect depends on the level of PP1c (Fig 2G). The effects of ectopic I-2Dm or NIPP1Dm were completely suppressed by coexpression of PP1c and were enhanced by a reduction in PP187B gene dosage, showing that the effect was indeed due to loss of PP1 activity rather than to any neomorphic effect of ectopic NIPP1Dm or I-2Dm (Fig 2H and data not shown). Taken together, these data show that ectopic expression of I-2Dm or NIPP1Dm in cells also expressing Punt has the same effect as PP1c loss-of-function mutants on TGFß superfamily signaling in the wing.

View larger version (66K): In this window In a new window Download PPT slide   Figure 2. I-2Dm and NIPP1Dm have the same effect as PP1 mutants on TGFß signaling in the wing. (A) Wild-type wing. A, anterior; P, posterior. (B) Wings from vg>punt flies are somewhat overgrown. The wing phenotypes in vg>punt flies are enhanced by UAS I-2Dm (C) and UAS NIPP1Dm (D). In particular, there is an increase in the number of anterior bristles (arrowhead) and enlargement of the posterior wing compartment (arrow). These phenotypes resemble the effects of (E) PP187B1/+ and (F) PP187B87Bg-6/+ on vg>punt. (G) vg>punt flies expressing both I-2Dm and NIPP1Dm have very overgrown, crumpled wings. Coexpression of UAS-PP187B (H) or any of the other PP1c isoforms (data not shown) completely suppresses the effects of I-2Dm and NIPP1Dm, showing that they are specific to PP1. UAS-PP1c (any isoform) also suppresses the effects of I-2Dm and NIPP1Dm individually (data not shown). (I) Quantitative representation of wing area from different genotypes. Values are mean ± SD (n = 4). Wings from vg>(I-2Dm, NIPP1Dm, punt) flies, which are severely crumpled, could not be included in this analysis.

PP1 is involved in Dpp/Tkv signaling in the Drosophila wing:
Studies in vertebrates and Drosophila indicate that there are at least two distinct signal transduction pathways for TGFß superfamily signaling (RAFTERY and SUTHERLAND 1999 ; WRANA 2000 ). Bone Morphogenic Protein (BMP)-type ligands (e.g., Decapentaplegic, Dpp, the Drosophila homolog of BMP2/4) activate one pathway and Activin or TGFß-like proteins activate another. These ligands bind to distinct receptor complexes in which the type I receptor appears to define the specificity of the biological response (RAFTERY and SUTHERLAND 1999 ; WRANA 2000 ). The type II receptor Punt is required for BMP-type signaling but can bind either Activin or BMP ligands when associated with the appropriate type I receptor, suggesting that it can function as the type II receptor for both Activin and BMP pathways (RAFTERY and SUTHERLAND 1999 ). To examine whether the Punt-dependent wing overgrowth phenotypes we observed as a result of reduced PP1 were due to an effect specifically on the Activin or BMP pathways we coexpressed different type I receptors in addition to punt and I-2Dm. Additional overexpression of the Activin receptor baboon (babo) and BMP receptor saxophone (sax) had little or no effect on the wing phenotypes (data not shown; indistinguishable from Fig 2C and Fig 3A). In contrast, additional overexpression of another BMP-type receptor, thick-veins (tkv), strongly enhanced the phenotype of vg>(punt, I-2Dm) (Fig 3B). In addition to expansion of the wing area we also found notching of the wing margins (Fig 3B, arrowed) and modification of the bristle morphology. The observed phenotypes closely resemble the effect of a constitutively active form of Tkv (LECUIT et al. 1996 ; NELLEN et al. 1996 ) and indicate elevated levels of signaling by the ligand Dpp (ADACHI-YAMADA et al. 1999 ). This phenotype was suppressed by extra PP1, showing that the effect was due to specific loss of PP1 (Fig 3C).

View larger version (81K): In this window In a new window Download PPT slide   Figure 3. Wing overgrowth phenotypes in flies with reduced levels of PP1 are caused by elevation of Tkv signaling. (A) The wing overgrowth phenotype of vg>(I-2Dm, punt) flies is enhanced (B) by UAS-tkv. Increase in number of bristles along the anterior margin, partial suppression of L5 vein formation, and notching of anterior and posterior wing margins (indicated by arrows), which is a consequence of Dpp-induced DJNK-mediated apoptosis (ADACHI-YAMADA et al. 1999 ), are all clearly visible. These effects are suppressed by UAS-PP187B (C) or any of the other PP1c isoforms (data not shown). (D) Wings from flies expressing UAS-tkv alone resemble wild type. (E) This is enhanced by UAS-punt and resembles the overgrowth phenotype in A, showing that increasing the level of Tkv has the same effect as reducing PP1c activity on ectopic Punt and (C) showing that increasing the level of PP1c counters the effect of I-2Dm. (F) The effect of vg>(I-2Dm, punt) is suppressed by tkv mutants. In particular, the bristle morphology at the anterior margin and the formation of the L5 vein are restored and the enlargement of anterior and posterior compartments is suppressed. (G) Quantitative representation of wing area from different genotypes. Different genotypes are grouped into different experimental categories: effect of ectopic I-2Dm on ectopic Punt, effect of ectopic type I receptor or heterozygous type I receptor mutations on vg>(I-2Dm, punt), effect of ectopic type I receptors on vg>punt, and effect of heterozygous dpp mutations on vg>(I-2Dm, punt). Values are mean ± SD (n = 4). Wings from vg>(I-2Dm, tkv, punt), which have wing margin deletions, were not included in this analysis.

As a control, we also examined the effect of expressing tkv alone or together with punt in a PP1^+/+ background. Wings from vg-GAL4 UAS tkv (vg>tkv) flies were essentially wild type (Fig 3D). When we tested UAS-tkv in combination with vg>punt, we found that extra tkv dominantly enhanced the effect of punt and phenotypically resembled the effect of I-2Dm on vg>punt, shown in Fig 3E. In particular there was a similar enlargement of the posterior compartment and increase in the number of double row bristles. Development of the L5 vein appeared normal and both anterior and posterior margins were intact. Coexpression of UAS-sax or UAS-babo with UAS-punt had little or no effect on size of the wing or on bristle morphology (Fig 3). Therefore the effect of reducing PP1c activity was the same as increasing the level of the type I receptor kinase Tkv.

To investigate whether the type I receptor was required for the interaction between I-2Dm and punt we examined the effect of reducing the levels of babo, sax, and tkv on vg>(punt, I-2Dm) flies. We found that mutations in sax or babo showed no suppression of the wing overgrowth caused by ectopic I-2Dm and punt (Fig 3). However, a dominant negative tkv mutation (tkv⁷), which results in loss of expression of Dpp targets (PENTON et al. 1994 ), suppressed the phenotype. Hypomorphic mutations of tkv showed a similar, but weaker, effect (Fig 3F and Fig G). These data show that the interaction between ectopic expression of the type II receptor Punt and PP1c requires the type I receptor Tkv and demonstrates that PP1 antagonizes Dpp/Tkv signaling in the wing.

Reduction of PP1 induces a target of Dpp signaling at a distance from the normal stripe of Dpp expression:
To test whether elevation of Dpp/Tkv signaling in cells with higher levels of Punt and reduced levels of PP1 was sensitive to the levels of Dpp we crossed vg>(I-2Dm, Punt) flies to various dpp mutant alleles. Reduction in the gene dosage of dpp was not effective in suppressing the vg>(I-2Dm, Punt) phenotype (see Fig 3G, last three columns, wings indistinguishable from 3A). Flies heterozygous for these dpp alleles but without vg>(I-2Dm, Punt) have wild-type wings (data not shown). Taken together with the cell-autonomous effect of Sara^F678A on a Dpp-responsive reporter gene (BENNETT and ALPHEY 2002 ), this suggests that reduction of PP1 activity does not affect Dpp production or sensitivity to Dpp per se but instead acts as a downstream component of the Dpp/Tkv signaling pathway.

To further investigate the involvement of PP1 in Dpp/Tkv signaling in the wing we examined the effect of I-2Dm on a downstream target of Dpp/Tkv signaling. In wild-type wing discs, Bifid (Bi, also known as optomotor-blind), a T-box family transcription factor, is expressed in broad domain along the A-P boundary (GRIMM and PFLUGFELDER 1996 ; LECUIT et al. 1996 ; NELLEN et al. 1996 ; Fig 4B). Punt and Tkv mediate the responses downstream of Dpp that lead to Bi expression (PENTON et al. 1994 ; RUBERTE et al. 1995 ). Ectopic expression of Punt along the presumptive wing margin showed little or no effect on the level or spatial distribution of Bi-lacZ expression (Fig 4C). When we coexpressed I-2Dm and Punt, we observed clear induction of Bi-lacZ in the pattern of ectopic I-2Dm and Punt expression (Fig 4D). Coexpression of NIPP1Dm and Punt had a similar, but stronger, effect on Bi-lacZ induction than ectopic I-2Dm Punt did (Fig 4E). Significantly, we observed induction of Bi-lacZ expression at a distance from the stripe of Dpp expression, which lies along the AP boundary. Coexpression of PP1c with I-2Dm and Punt suppressed induction of Bi-lacZ, demonstrating that the effect was specific for PP1 (Fig 4F). These data show that reduction of PP1 activity also stimulates the expression of a downstream target of Dpp/Tkv signaling even in cells that receive the lowest level of the Dpp morphogen.

View larger version (84K): In this window In a new window Download PPT slide   Figure 4. Reduction in PP1c levels induces ectopic expression of Bi. Wing discs from third instar larvae were stained with X-gal. A, anterior; P, posterior; D, dorsal; V, ventral. All wing discs are at the same magnification. (A) Expression pattern of vg-GAL4 visualized with UAS-lacZ. vg-GAL4 is expressed along the D-V boundary and presumptive wing margin (dashed line). The approximate location of the Dpp-expressing cells along the A-P boundary is indicated with a bold line. (B) Bi-lacZ expression in the BiP1 enhancer trap line (SUN et al. 1995 ). Strongest expression is seen in a broad domain centered on the stripe of Dpp expression and in two stripes flanking the D-V boundary (GRIMM and PFLUGFELDER 1996 ; arrows). (C) Bi-lacZ expression in vg>punt flies. (D) vg>(I-2Dm, punt) and (E) vg>(NIPP1Dm, punt) wing discs showing enlargement of posterior and anterior domains, with ectopic expression of Bi-lacZ in cells flanking the D-V boundary (arrowheads). (F) vg>(I-2Dm, punt) is suppressed by co-overexpression of PP187B (compare with D) or any of the other PP1c isoforms (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

I-2Dm and NIPP1Dm inhibit PP1c in vivo:
I-2Dm and NIPP1Dm are potent inhibitors of PP1 in vitro. Our observations indicate that ectopic I-2Dm and ectopic NIPP1Dm also inhibit PP1c in vivo, resulting in titration of PP1c from its other functions. First, PP1 activity is reduced in extracts from flies ectopically expressing I-2Dm or NIPP1Dm. NIPP1Dm has a larger effect on PP1 activity than I-2Dm does, consistent with the potent inhibition of PP1c by NIPP1Dm in vitro. NIPP1Dm degradation in tissue extracts releases NIPP1 from PP1c, thereby restoring PP1 activity (JAGIELLO et al. 1995 ). Consequently, the reduction in PP1c activity in ectopic NIPP1Dm flies is probably higher than that measured in our extracts. This is consistent with the phenotypic effect of NIPP1Dm overexpression (see below). Second, ectopic expression of I-2Dm or NIPP1Dm results in phenotypes resembling those of PP1c loss-of-function mutations. In conjunction with arm-GAL4, ectopic NIPP1Dm flies phenotypically resemble PP187B^-/- and PP1ß9C^-/- mutants. In conjunction with vg-GAL4, which expresses only weakly in the wing, ectopic NIPP1Dm resembles strong PP187B^-/+ mutants in combination with type II TGFß superfamily receptor Punt. In contrast, overexpression of I-2Dm in the wing resembles the effect of weak PP187B^-/+ mutants in combination with Punt, indicating that the effects of I-2Dm overexpression are similar to, but weaker than, the effects of ectopic NIPP1Dm. Lastly, PP1 activity is reduced in flies modestly overexpressing I-2Dm, but can be restored by reactivation of I-2Dm:PP1c with GSK3ß + MgATP. This implies that I-2Dm and NIPP1Dm sequester PP1c away from other functions and suggests that there is no compensation for titration of endogenous PP1c by production of additional active PP1c. This might be because PP1 is normally in excess, which would also explain why PP1c overexpression in a wild-type background has no phenotypic effect. However, the effects of ectopic I-2Dm and NIPP1Dm on their own, in combination with each other, or in a sensitized background, can be suppressed by coexpression of PP1c, indicating that exogenous PP1c can restore PP1c levels by titrating additional inhibitor.

We found that co-overexpression of either I-2Dm or NIPP1Dm with PP1 has no phenotypic effect. This implies that neither inhibitor:PP1c complex (I-2Dm:PP1c or NIPP1Dm:PP1c) has any significant function when in excess, and may simply be inactive, as expected if the binding proteins are simply inhibitors of PP1. The ability of I-2 to convert recombinant PP1 to more native-like activity upon phosphorylation of I-2 has led to the suggestion that I-2 may be a molecular chaperone of PP1 as well as an inhibitor of PP1 (ALESSI et al. 1993 ; MACKINTOSH et al. 1996 ). This biochemical property is also conserved in I-2Dm (BENNETT et al. 1999 ). However, the net effect of I-2Dm overexpression in flies is to reduce PP1 activity. This indicates that phosphorylation of I-2Dm and reactivation of PP1 is rate limiting, at least when I-2Dm is in excess. Alternatively, reactivation of PP1 in vivo might serve another purpose, namely to allow a pool of inactive (I-2Dm-bound) PP1 to be recruited by targeting subunits to specific subcellular loci. Indeed there is some precedent for cycling of PP1c from inhibitors to targeting subunits: for instance phosphorylation and inactivation of Inhibitor-1 (I-1) by PKA releases I-1-bound PP1 and might be coordinated with phosphorylation and activation of glycogen-binding subunit of PP1 to recruit PP1 to glycogen particles (BOLLEN 2001 ; COHEN 2002 ). The isolation of loss-of-function mutations in I-2Dm and NIPP1Dm will help to resolve the question of whether these proteins act solely as inhibitors of PP1c.

Ectopic I-2Dm or ectopic NIPP1Dm can be used selectively to disrupt PP1c function during different stages of development or in specific tissues:
To explore the utility of PP1c inhibitors in vivo we examined the effect of I-2Dm and NIPP1Dm on TGFß signaling. We found that ectopic expression of I-2Dm or NIPP1Dm in the cells expressing ectopic Punt had the same effect as PP187B mutants, which reduce PP1 levels across the whole wing disc. Since genetic analysis indicates that both Babo signaling and Tkv/Sax signaling have a role in regulating growth of the wing (HAERRY et al. 1998 ; BRUMMEL et al. 1999 ), we sought to identify the relevant type I receptor responsible for these effects. We found that in a sensitized genetic background in which Punt is not limiting (vg-GAL4, UAS-punt), the wing phenotype is sensitive to the level of Tkv but not the other type I receptors. We found that the overgrowth phenotype is enhanced by extra Tkv (UAS-tkv) and that this closely resembles the phenotype of extra Punt in a background of reduced PP1 (vg-GAL4, UAS-punt PP1^-/+). Furthermore, the wing phenotype of elevated punt in a background of reduced PP1 (vg-GAL4, UAS-punt PP1^-/+) is suppressed by reducing the level of Tkv (tkv^-/+) and enhanced by elevating the level of Tkv (UAS-tkv) but is not affected by similar manipulation of the levels of Sax or Babo. Therefore the overgrowth wing phenotypes are entirely regulated by Tkv.

We have previously shown that displacement of PP1 from Sara induced TGFß superfamily signaling, implying that the role of Sara-bound PP1 may be to prevent inappropriate ligand-independent signaling by the receptors. We have shown here that that the interaction between PP1 and Punt is not sensitive to the levels of the ligand Dpp and that reduction of PP1 activity results in ectopic expression of Bi, a target of Tkv/Dpp signaling, beyond the region in which Dpp is known to elicit signaling (reported to be up to 25 cell diameters from its source; LECUIT et al. 1996 ; NELLEN et al. 1996 ; ENTCHEV et al. 2000 ; TELEMAN and COHEN 2000 ). Since PP1 loss of function has the same effect on TGFß/Dpp signaling as a PP1c-nonbinding mutant of Sara, we can rule out the possibility that free PP1 goes off and does something else, ultimately leading to the increased Dpp signaling by some other mechanism. Taken together, this suggests that reduction of PP1 activity can activate the downstream Dpp signaling pathway in cells that receive low levels or no Dpp and is consistent with a role for PP1 in preventing inappropriate activation of the signaling pathway where the ligand is low or absent.

In summary, we have developed a method of inhibiting PP1 activity in a cell- and tissue-specific manner. I-2Dm and NIPP1Dm do not discriminate between different PP1c isoforms; therefore the role of PP1 in a given pathway can be easily tested without having to test the separate isoforms individually. We have examined the role of PP1c in wing development using ectopic inhibitors of PP1c and shown that reduction of PP1c activity enhances the effect of the TGFß superfamily type II receptor Punt, giving rise to overgrowth of the wing. We have examined the basis of this interaction using I-2Dm and shown that this interaction requires the type I receptor Tkv and is accompanied by induction of a downstream target Bi, suggesting that PP1c negatively regulates Dpp/Tkv signaling during wing morphogenesis. We anticipate that ectopic inhibitors of PP1c can be used in a wide variety of contexts to test for the effect of reducing PP1 activity on specific developmental processes. While these inhibitors are very useful for identifying a role for PP1 in a particular developmental process, they are not able to dissect the role of a specific PP1c species. Our demonstration of the usefulness of ectopic expression of PP1c inhibitors in Drosophila, together with the highly conserved nature of both PP1 and the inhibitors of PP1, suggests that this approach is also applicable to other, less genetically tractable systems.

	ACKNOWLEDGMENTS

We thank J. Gausz, U. Heberlein, M. O'Connor, and T. Tabata for fly strains. This work was supported by grant G117/255 from the UK Medical Research Council (MRC) together with grant 43/G11827 from the UK Biotechnology and Biological Sciences Research Council (BBSRC), with additional support from the Royal Society. L.A. is an MRC Senior Research Fellow.

Manuscript received August 7, 2002; Accepted for publication January 27, 2003.

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