Homeodomain-Interacting Pr

Current Biology 22, 1582–1586, September 11, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.06.074

Reportotein Kinase

Regulates Yorkie Activityto Promote Tissue Growth

Joanna Chen1 and Esther M. Verheyen1,*1Department of Molecular Biology and Biochemistry, SimonFraser University, Burnaby, British Columbia V5A 1S6, Canada

Summary

The Hippo (Hpo) tumor suppressor pathway regulates tissuesize by inhibiting cell proliferation and promoting apoptosis

[1–5]. The core components of the pathway, Hpo, Salvador,Warts (Wts), and Mats, form a kinase cascade to inhibit the

activity of Yorkie (Yki), the transcriptional effector of thepathway [6, 7]. Homeodomain-interacting protein kinases

(Hipks) are a family of conserved serine/threonine kinasesthat function as regulators of various transcription factors

to regulate developmental processes including prolifera-tion, differentiation, and apoptosis [8]. Hipk can induce

tissue overgrowth in Drosophila [9, 10]. We demonstratethat Hipk is required to promote Yki activity. Hipk affects

neither Yki stability nor its subcellular localization. More-over, hipk knockdown suppresses the overgrowth and

target gene expression caused by hyperactive Yki. Hipkphosphorylates Yki and in vivo analyses show that Hipk’s

regulation of Yki is kinase-dependent. To our knowledge,this is the first kinase identified to positively regulate Yki.

Results and Discussion

Hipk PromotesDrosophilaWingGrowth by Regulating CellProliferation and Apoptosis

We have previously shown that excess homeodomain-inter-acting protein kinase (Hipk) caused overgrowth in Drosophilaadult tissues [9, 10]. In the third-instar wing imaginal disc, ex-pressing one or two copies of UAS-hipk using omb-Gal4 ledto dose-dependent overgrowth (Figures 1B and 1C), whichstrikingly resembled the phenotype obtained when inhibitingHippo (Hpo) signaling by reducing warts (wts) or by overex-pressing yorkie (yki) [6, 11, 12]. Conversely, reduction of Hipkthrough mutation or double-stranded RNA-mediated knock-down (hipkRNAi) leads to decrease of adult eye and wing size[9, 10]. However, the cellular mechanisms that underlie Hipk’srole in growth are unknown. Tissue overgrowth can result fromenhanced cell proliferation, decreased apoptosis, or both. Weperformed immunohistochemistry with anti-phosphohistone3 (PH3) and anti-cleaved Caspase 3 (Casp3) antibodies,markers for mitosis and apoptosis, respectively. EctopicHipk induced an increase in number of PH3-positive cells(see Figures S1B and S1B’ available online). In contrast,knockdown of hipk led to ectopic Casp3 staining (FiguresS1D and S1D’). Thus, Hipk can promote cell proliferation andinhibits apoptosis to control the wing size.

Hipk Regulates the Expression of Yki Target GenesIn the absence of Hpo signaling, the TEAD/TEF family DNA-binding transcription factor Scalloped (Sd) interacts with Yki

*Correspondence: everheye@sfu.ca

[13, 14] to initiate expression of the target genes, includingDrosophila inhibitor of apoptosis protein 1 (Diap1), cyclinE (CycE), wingless (wg), and the Hpo agonist Expanded (ex)[15–18]. yki overexpression leads to elevated levels of thesetranscriptional targets and organomegaly [19]. Therefore, wesought to examine whether Hipk regulates Yki-mediatedtranscription.Overexpressing hipk along the anterior-posterior (A/P)

boundary of the wing disc using patched (ptc)-Gal4 led toectopic expression of both Diap1-lacZ (Figure 1E) and CycE(Figure 1G). In hipk4 mutant somatic clones, ex-lacZ ex-pression was lost (arrows in Figures 1I and 1I’) and wg-lacZexpression was reduced in the proximal region of the wingpouch (arrows in Figures 1K and 1K’) where its expression isregulated by the Hpo pathway [18], but not at the dorsal/ventral boundary where its expression is Hpo-independent(marked by an asterisk in Figures 1K and 1K’). These resultsshow that hipk can regulate the expression of several Ykitranscriptional targets.

Hipk Counteracts HpoGrowth Inhibition duringDrosophilaWing Development

Because Yki is negatively regulated by the immediateupstream regulator Wts, we examined the role of Hipk onex-lacZ expression in hipk, wts double-knockdown discs.The upregulation of ex-lacZ seen following wts knockdownin the posterior compartment of the wing disc (Figure 2B)was prevented by simultaneous knockdown of hipk (Fig-ure 2C). Thus, in this context, activated Yki requires Hipk forits ability to initiate target gene expression. Further geneticinteraction studies extend and confirm the antagonisticgenetic interaction between hipk and members of the Hpopathway regulating Yki (Figure S2).To assess whether the role of Hipk in regulating Yki target

genes was tissue specific, we examined expression of cyclinE in the eye imaginal disc. In wtsx1 somatic clones (markedby absence of GFP; Figures 2E and 2E’), CycE expressionwas greatly expanded relative to wild-type (WT), due to dere-pression of Yki (Figure 2D). In hipk4, wtsx1/+, wtsx1 loss offunction clones, the expansion of CycE is suppressed due toreduced Hipk expression (Figures 2F and 2F’). This resultindicates that hipk is required for Yki target genes expressionin both wing and eye imaginal discs. Furthermore, coexpress-ing WT yki and hipk promoted massive synergistic overgrowth(Figure 2I) and induction of ex-lacZ (Figure 2I’) in comparisonto expressing yki (Figure 2G) or hipk alone (Figures S3M andS3M’), implying that Hipk potentiates Yki activity to inducegrowth. All these results together suggest a model that Hipkpromotes Yki-mediated growth and target gene expression.

Hipk Does Not Alter Yki Subcellular LocalizationWhen Hpo signaling is activated, Wts phosphorylates Yki atSerine 168, as well as two additional sites, Serine 111 and250 [7]. As a result, Yki interacts with protein 14-3-3, leadingto Yki cytoplasmic retention [19, 20]. In the absence of phos-phorylation by Wts and cytoplasmic retention, Yki can interactwith Sd, which promotes Yki nuclear localization and targetgene expression [13, 14, 21]. We speculated that Hipk might

Figure 1. Hipk Promotes Tissue Overgrowth and Regulates Yki Target Gene

Expression

(A) Control wing imaginal disc. One (B) or two (C) copies of HA-tagged

UAS-hipk were expressed using the omb-Gal4 driver.

(B and C) Anti-HA staining revealed the omb-Gal4 expression domain. Hpo

pathway target genes (Control expression patterns shown in D, F, H, and J)

were examined following hipk overexpression (E, E’, G, and G’) or hipk4

somatic clone generation (I, I’, K, and K’).

(D–E’) Diap1-lacZ expression.

(F–G’) Anti-CycE staining. (E, E’, G, and G’) ptc-Gal4was used to ectopically

express hipk:HA in the domain seen with anti-HA staining in (E’) and (G’).

(H–I’) ex-lacZ expression.

(J–K’) wg-lacZ expression. Asterisk indicates unaffected Wg expression at

the D/V boundary.

(I’ and K’) hipk4 loss-of-function clones are marked by the absence of GFP

(arrows). Also refer to Figure S1.

Hipk Promotes Yki Activity and Growth1583

be acting to regulate Yki nuclear localization or activity. Theactivity of Yki, as measured by overgrowth phenotypes andtarget gene expression, is consistently enhanced by Hipk,

yet we found that the subcellular localization of exogenous(Figures 2J and 2J’) or endogenous (Figures 3B and 3B’)Yki following modulation of Hipk is unaltered, in comparisonto ex knockdown, which resulted in nuclear enrichment ofYki (Figure S3B; [20]). In fact, we observe no difference inYki localization or levels despite a dramatic synergy withHipk in promoting growth and target gene expression (Figures2I and 2I’).To further explore the mechanism of Yki regulation, we

considered whether Hipk might act through any of its previ-ously described targets. Hipk has been shown to regulatethe activity of targets including Slimb/b-TrCP, Armadillo(Arm)/ b-catenin, and Groucho [9, 10, 22]. Recently, it wasshown that the Yki homolog Yes-associated protein (Yap) isubiquitinated by the SCFbTrCP E3 ubiquitin ligase, resulting inYap degradation [23]. Although the absence of a bTrCP-recog-nition site in Yki argues against such a mechanism of controlby Hipk, we confirmed this by finding that knockdown of slimbdid not alter Yki protein levels (Figures S3C and S3C’) nor theexpression of Diap1-lacZ or ex-lacZ (Figures S3G and S3I’).

Figure 2. Hipk Antagonizes theHpoPathway and

Promotes Yki Activity

ex-lacZ expression in (A) control, (B) hh >wtsRNAi,

(C) hh > wtsRNAi, hipkRNAi wing discs. hh-Gal4 is

expressed in the posterior compartment marked

with an asterisk, to the right of the dotted line.

CycE (red) expression in (D) control, (E and E’)

single wtsx1 mutant clones, and (F and F’) double

hipk4, wtsx1 mutant clones in eye discs.

(E’ and F’) Mutant clones are marked by the

absence of GFP.

(D, E, and F) Arrowheads mark the location of the

morphogenetic furrow.

(G and G’) hh-Gal4 driving UAS-yki:GFP and (I

and I’) UAS-yki:GFP;UAS-hipk in wing discs,

stained for ex-lacZ and GFP to mark hh expres-

sion domain.

(H–J’) High magnification image of Yki locali-

zation (GFP) and nuclei (DAPI) in (H and H’)

hh > yki:GFP and (J and J’) hh > yki:GFP; hipk.

Also refer to Figure S2.

Figure 3. Hyperactive YkiS168A Requires Hipk to Promote Overgrowth and Target Gene Expression, whereas Yki Localization Is Unaffected by Modulation

of Hipk

(A–B’) Wing discs stained with anti-Yki (red) and DAPI (blue). en-Gal4was used to drive UAS transgenes in the posterior of the disc as indicated by expres-

sion of UAS-GFP (A’ and B’). Control disc is shown in (A) and (A’). en > GFP; hipkwt is shown in (B) and (B’). The arrowhead in (A) and (B) indicates the D/V

boundary.

(C’, D’, H’, and I’) ptc-Gal4 and hh-Gal4 expression domains are indicated byGFP expression. Expressing hyperactive Yki (Yki S168A:GFP) caused increased

expression of Wg (C and C’) and ex-lacZ (H and H’) accompanied by disc overgrowth.

(D, D’, I, and I’) hipk knockdown rescued the Yki S168A:GFP disc phenotype, Wg (D and D’) and ex-lacZ (I and I’) induction. Adult eyes from control (E), ptc >

ykiS168A:GFP (F), and ptc > hipkRNAi; ykiS168A:GFP (G). hipk reduction did not affect localization of YkiS168A shown as GFP expression with nuclear staining by

DAPI (blue) (L and L’). Anti-Yki stain (red in M and M’, detects all cellular Yki) showed that hipk knockdown did no change subcellular localization of either

YkiS168A or endogenous Yki. Noticeably, knocking down hipk (L, L’, M, and M’) decreased the number of cells expressing Yki S168A:GFP in comparison to

the number of cells seen expressing UAS-YkiS168A:GFP alone (J, J’, K, and K’). Also refer to Figure S3.

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Several recent studies have shown an interaction betweenHpo and Wg/Wnt signaling [24–29]. To rule out that Hipk wasregulating Yki through its role in stabilizing Arm protein, weexamined wing phenotypes and ex-lacZ upon modulation ofArm [9, 22]. Although activated Arms10 can induce over-growths, they were not accompanied by ectopic Yki targetgene expression in contract to robust expansion of targetsand overgrowth seen with ectopic Hipk (Figures S3J–S3L’and S3M). Therefore, we conclude that Hipk does not promoteYki activity via its role in stabilizing Arm and promoting theWg pathway.

Lastly, we have found that Hipk can inhibit the transcrip-tional corepressor Groucho (Gro) to promote Notch signalingduring Drosophila eye development [10]. Interestingly, weobserved that loss of hipk does not affect expression of theNotch target Enhancer of split and Delta in the wing disc(data not shown). This finding suggests that hipk does not

regulate the Notch pathway in the Drosophila wing. To ruleout that the effect on Yki was due to Gro inhibition by Hipk,we examined the effect of Gro on ex-lacZ and growth. Expres-sion of gro caused reduction of both ex-lacZ expression andgrowth that cannot be suppressed by coexpression of hipk(Figures S3N–S3O’). Because Hipk cannot inhibit Gro in thiscontext, it is unlikely that Hipk promotes Yki indirectly viaGro, although we cannot rule out completely that Gro maycompete with Yki for binding Hipk.

Hipk Regulates the Nuclear Activity of Yki

Based on the absence of a change in Yki subcellular localiza-tion or protein levels due to Hipk modulation, we speculatedthat Hipk regulates Yki activity at the nuclear level. To testthis model, we used a hyperactive form of Yki carrying aserine-to-alanine mutation on serine 168 (YkiS168A:GFP), whichloses its ability to interact with 14-3-3, thus resulting in

Figure 4. Hipk Interacts with and Phosphorylates Yki and Its Kinase Activity

Is Required to Modulate the Hpo Target Genes In Vivo

(A) Hipk:Myc, Yki:HA, or HA alone were transfected into HEK293T cells.

Coimmunoprecipitation assays were performed with anti-HA antibodies,

and extracts were visualized by western blotting by using anti-Myc or

anti-HA antibodies to detect Hipk or Yki, respectively. For the negative

control, Hipk:Myc was not detected by pulling down HA protein (lane 4 in A).

(B) In an in vitro kinase assay using radiolabeled ATP, Yki:HA is phosphor-

ylated (indicated as p-Yki) in the presence of Hipk:HA (lane 4), but not in

its absence (lane 3) or when incubated with kinase-dead (KD) Hipk (lane

5). Hipk autophosphorylation (p-Hipk) serves as a positive control in lanes

1 and 4.

(C–D’)Diap1-lacZexpression in (C)ptc>hipkwt:HAand (D,D’)ptc>hipkKD:HA

wing discs.

(E–F’) ex-lacZ expression in (E) hh > hipkwt:HA and (F, F’) hh > hipkKD:HA

wing discs. Expression of hipkKD (D, D’, E and E’) is shown by anti-HA stain-

ing. Also refer to Figure S4.

Hipk Promotes Yki Activity and Growth1585

enhanced nuclear localization and activity [19, 20]. Expressionof UAS-ykiS168A: GFP using hh-Gal4 induced massive over-growth and expression of ex-lacZ (Figure 3H). Similarly,ptc-Gal4 driven UAS-ykiS168A: GFP induced overgrowth andWg expression in the proximal region of the wing pouch (arrowin Figure 3C), as well as prompted overgrowth in the wing disc(Figure 3C), eye (Figure 3F), leg, and haltere (data not shown).Remarkably, knocking down hipk in this genetic contextrescued the overgrowth phenotype in all tissues examined(Figures 3D, 3G, 3I), as well as suppressed Wg and ex-lacZinduction (Figures 3D and 3I), suggesting that Hipk may actas a general regulator of Yki. Furthermore, hipk knockdowndid not affect subcellular localization of YkiS168A:GFP (Fig-ure 3L) or total Yki (Figure 3M) but did cause a reduction incell proliferation, as seen by a decrease in number ofYkiS168A:GFP-expressing cells (compare Figures 3J and 3L).Moreover, hipk knockdown rescued the lethality causedby overexpressing Yki S111A, S168A, S250A (data not shown),which carries phosphoresistant mutations on all three Wts

phosphorylation sites [7]. These findings support the modelthat Hipk promotes Yki transcriptional activity within thenucleus.

Hipk Regulation of Yki Activity Is Kinase-Dependent

To investigate a mechanism underlying the regulation of Ykiactivity, we performed coimmunoprecipitation and in vitrokinase assays. In HEK293T cells transfected with Myc-taggedHipk and HA-tagged Yki, Hipk was coimmunoprecipitated in acomplex with Yki (Figure 4A, lane 2). Furthermore, we foundthat Hipk phosphorylates Yki (Figure 4B, lane 4). Yki interactsthrough its WW-domain with a number of components ofthe Hpo pathway including Wts and Ex via binding to a PPxYmotif [30]. Hipk also possesses a conserved PPxY sequencethat may mediate the interaction with Yki.The in vitro kinase assay shows that WT Hipk (Figure 4B,

lane 4; Figure S4, lane 2), but not kinase-dead (KD) Hipk(Figure 4B, lane 5), phosphorylated Yki. To further determinewhether the kinase function of Hipk is required to promoteYki activity, we used a KD form of hipk (UAS-hipkKD) toexamine Yki target expression in wing discs. Compared tothe potent effects seen with overexpressing WT hipk (Figures4C and 4E), hipkKD was not able to induce ex-lacZ nor Diap1-lacZ expression, nor to promote tissue overgrowth (Figures4D, 4D’, 4F, and 4F’). These data indicate that Hipk requiresits kinase function to positively regulate Yki transcriptionalactivity and to counteract the inhibition of Yki caused by Hpopathway members.Our findings indicate that Hipk promotes Yki transcriptional

activity to regulate tissue growth during Drosophila develop-ment. To the best of our knowledge, this is the first kinaseto positively regulate Yki by phosphorylation. Because Hipkfunctions in multiple signaling pathways, we cannot com-pletely eliminate the possibility that Hipk regulates Hposignaling and Yki via multiple pathways; however, we believethat we have ruled out a regulation through known targets.Furthermore, we show that Hipk phosphorylates Yki, implyinga direct regulatory role of Hipk on Yki. Although Hipk promotesYki activity by phosphorylation, the functional significance ofthis modification has yet to be further investigated. In our anal-yses, we have applied the same genetic criteria and obtainedsimilar results as were used to demonstrate that Sd was anessential factor for Yki-mediated growth [13, 14, 21]. Giventhat Hipk exerts its effects on nuclear Yki and is enriched innuclear speckles (data not shown [31]), it may play an impor-tant role to facilitate the interactions between Yki and its tran-scriptional cofactors. Further studies should reveal whetherthis growth regulation byHipk familymembers is evolutionarilyconserved. Trapasso et al. demonstrate that hipk2 mutantmice have a reduced body size and hipk22/2 mouse embryofibroblasts show reduced proliferation rates, suggesting thatHipks may also promote Yap activity to regulate tissue growthin vertebrates [32]. Using mammalian cell culture, we haveobserved that overexpressing WT Hipk2, but not kinase-dead Hipk2, is able to induce elevated Yap levels (data notshown). This finding suggests that vertebrate Hipks may regu-late Hpo signaling through Yap stability and possibly also byregulating Yap nuclear activity.

Supplemental Information

Supplemental Information includes four figures and Supplemental Experi-

mental Procedures and can be found with this article online at http://dx.

doi.org/10.1016/j.cub.2012.06.074.

Current Biology Vol 22 No 171586

Acknowledgments

Weare grateful to the numerous peoplewho provided fly strains, antibodies,

and plasmid constructs: Helen McNeill, Ken Irvine, D.J. Pan, Bloomington

Drosophila Stock Center, Vienna Drosophila RNAi Center, Developmental

Studies Hybridoma Bank. We also thank Wendy Lee and Nick Harden for

comments on the manuscript and Jessica Blaquiere for help in constructing

the hipkKD fly strain. This work was supported by an operating grant from

Canadian Institutes of Health Research.

Received: February 29, 2012

Revised: May 15, 2012

Accepted: June 11, 2012

Published online: July 26, 2012

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