Par-1 Regulates Tissue Growth by Influencing Hippo Phosphorylation Status and Hippo-Salvador Association

Par-1 regulates the Hippo signaling pathway in Drosophila melanogaster by modifying the phosphorylation status of Hippo and also by inhibiting the interaction of Hippo and Salvador.


Introduction
The control of organ size, which requires the delicate coordination of cell growth, cell proliferation, and cell death, is a fascinating biological process. The identification of the Hippo (Hpo) signaling pathway has shed some light on this biological phenomenon. The Hpo pathway has emerged as an evolutionarily conserved pathway that controls organ size during animal development. It regulates tissue growth by balancing cell proliferation and apoptosis and has also been implicated in stem cell maintenance, tissue homeostasis, and repair [1][2][3][4]. In addition, it has been reported to play a role in cell contact-dependent growth inhibition [5]. Accumulating evidence has suggested that mutations and malfunctions of the components of the Hpo pathway result in a wide range of human cancers and diseases [6].
Several recent proteome-wide phosphorylation studies, which have uncovered a large number of previously unknown phosphorylation events in Hpo signaling [39,40], have indicated the involvement of a large number of unknown participants in the Hpo pathway. To identify novel pathway modulators, we performed a gain-of-function EP screen and identified Par-1 as a novel Hpo pathway regulator. Par-1 is a multifunctional serine/ threonine kinase containing an N-terminal conserved catalytic domain, a ubiquitin-associated (UBA) domain adjacent to the catalytic domain, and a kinase associated domain 1 (KA1 domain) within the last 40 amino-acids [41]. Par-1 plays a major role in anterior/posterior (A/P) axis formation and germline determinant polarization, and it also regulates diverse cellular processes, including microtubule dynamics and neuronal polarity [42][43][44][45][46][47]. Although Par-1 is involved in multicellular processes, little is known regarding the function of Par-1 in disease and tumor formation. Hyper-phosphorylation of the microtubule-associated protein Tau by microtubule affinity regulating kinase, the homolog of Drosophila Par-1 (MARK) [48], which is activated by upstream kinases, such as LKB1 [49] and Tao-1 [50], results in microtubule depolymerization and abnormal aggregation of Tau in Alzheimer disease. Additionally, MARK4 has been reported to be involved in hepatocellular carcinogenesis and gliomagenesis [51,52].
In this study, we identified Par-1 as a negative regulator of the Hpo kinase complex. We found that overexpression of Par-1 drove tissue overgrowth and upregulated the expression of Hpo pathway-responsive genes. Moreover, knockdown of Par-1 blocked tissue growth and downregulated the expression of Hpo pathway-responsive genes. We demonstrated that par-1 functioned downstream of ex and ft but upstream of hpo and sav. We also provided evidence that Par-1 associated with the Hpo-Sav complex and regulated the phosphorylation of Hpo at Ser30 to regulate Hpo activity. Furthermore, we found that Par-1 promoted the dissociation of Sav from the Hpo-Sav complex, eventually resulting in Sav dephosphorylation and destabilization. Thus, these results identified Par-1 as a novel regulator of the Hpo signaling pathway and supported a model by which Par-1 regulates Hpo phosphorylation and Hpo-Sav association to control organ growth.

Par-1 EP Lines Promote Growth via Hpo Signaling
To identify novel candidates of the Hpo pathway, we performed an overexpression screen in which flies carrying GMR-Gal4 and UAS-Yki (referred to as GMR-Yki) were crossed with a collection of EP lines. Overexpression of UAS-Yki posterior to the morphogenetic furrow (MF) under the control of the GMR-Gal4 driver (GMR-Yki) resulted in enlarged eyes (compare Figure 1A9 with 1A), providing a sensitive background for a genetic modifier screen [53]. Each EP line was crossed with GMR-Yki flies, and the F1 progeny was screened for an increase in eye size. From more than 10,000 EP lines, we screened numerous lines that enhanced the overgrowth phenotype induced by Yki overexpression. We then analyzed the UAS element insertion sites of these lines and found that the insertion sites of L[484], L[507], and F[727] were all within the 59 UTR region of the par-1 gene ( Figure 1C). Although these three EP lines did not display an overgrowth phenotype when driven by GMR-Gal4 in Drosophila eyes (compare Figure 1B with 1A, and unpublished data), these lines dramatically enhanced the GMR-Yki-induced overgrowth phenotype (compare Figure 1B9 with 1A9, and unpublished data). In addition, the expression of these lines driven under the wing-specific Gal4 driver MS1096 produced enlarged adult wings ( Figure 1D-1D0), indicating that the candidate genes expressed in these lines may play a role in organ size control. To determine whether the UAS element of these lines regulated par-1 gene expression, real-time PCR analysis was performed for the L[484] line. The mRNA level of par-1 was significantly upregulated when the L[484] line was crossed with MS1096, whereas the mRNA level of genes located proximal to par-1, mei-W68, and hpo, demonstrated a slight or no change ( Figure 1E), suggesting that ectopic Par-1 expression could be responsible for the tissue overgrowth phenotype that we observed.
To determine whether overexpression of L[484] promoted tissue growth via Hpo signaling, the L[484] line was expressed under the control of the hh-Gal4 driver, which drives gene expression in the posterior compartment (P-compartment). As shown in Figure 1G-1G9, ex-lacZ (EX-Z), an enhancer trap for ex [27], was increased in the P-compartment of the wing imaginal disc, suggesting an inhibition of Hpo signaling. Furthermore, the Hpo downstream marker diap1-lacZ was also upregulated in the flip-out clones expressing L[484] ( Figure 1H-1H0). Briefly, these observations suggested that the expression of the L[484] line promoted tissue growth via Hpo signaling by controlling the expression of Par-1.

Overexpression of Par-1 Inactivates Hpo Signaling to Induce Tissue Growth in a Kinase-Dependent Manner
To verify the functional relationship between Par-1 and the Hpo pathway, a dual luciferase assay, which reflected Sd-Yki transcriptional activity [20], was performed. As shown in Figure 2A, in S2 cells, coexpression of Yki and Sd activated the luciferase reporter gene (36Sd2-Luc), which was greatly promoted by Par-1, indicating that Par-1 enhanced the activity of the Sd-Yki transcriptional complex in vitro. To further determine the functional relationship between Par-1 and the Hpo pathway in vivo, Myc tagged Par-1 transgenic flies were generated. Consistent with the results in Figure 1A-1B9, overexpression of two copies of UAS-Myc-Par-1, using the GMR-Gal4 driver (referred to as GMR/2*Myc-Par-1), resulted in rough eyes without a discernible overgrowth (compare Figure 2B9 with 2B), while coexpression of UAS-Myc-Par-1 with GMR-Yki enhanced the overgrowth phenotype caused by GMR-Yki

Author Summary
An organism's organ size is determined by cell number, the size of each cell, and the distance between cells. All of these factors are controlled by the coordination of different cell signaling pathways and other mechanisms. The Hippo signaling pathway controls organ size by restricting the number of cells that make up the organ. Malfunction of this pathway leads to abnormal overgrowth, and is involved in a large number of human diseases and cancers. We identify here a component of the Hippo pathway, Par-1, which controls tissue growth by negatively regulating the Hippo pathway. We show that overexpression or depletion of Par-1 influences tissue growth in fruit flies via Hippo signaling. Then, by genetic and biochemical experiments, we show that Par-1 interacts with Hippo, regulating the Hippo Ser30 phosphorylation status to alter Hippo activity. In addition, we found that Par-1 regulates Hippo signaling via inhibition of the Hippo-Salvador association in a kinase-dependent fashion. We predict that Par-1 is a potential oncogene and that its regulatory role in Hippo signaling could be conserved.
(compare Figure 2C9 with 2C). Although GMR/2*Myc-Par-1 did not induce a discernible overgrowth phenotype in the eyes ( Figure 2B9), the expression of two copies of UAS-Myc-Par-1, using the MS1096 driver (referred to as MS1096/2*Myc-Par-1), resulted in enlarged wings and caused a wing bending-down phenotype, which indicated an expansion of the wing ( Figure 2D90 and compare Figure 2D9 with 2D and 2E9 with 2E). We also found that the relative Pcompartment area of the wings expressing UAS-Par-1, using the hh-Gal4 driver, was increased (Figure S1A-S1B). We then examined whether overexpression of Par-1 affected the expression of Hpo pathway-responsive genes. We found that flip-out clones expressing UAS-Myc-Par-1 in the wing imaginal discs showed upregulated expression of EX-Z ( Figure 2F-2F9), diap1-lacZ ( Figure 2H-2H9) and diap1-GFP3.5 (a diap1 enhancer element reporter [20], Figure S1C-S1C0), suggesting compromised Hpo signaling activity. Taken together, these results suggested that overexpression of Par-1 promoted tissue overgrowth by inhibiting Hpo pathway activity.
Considering that the Ser/Thr kinase activity of Par-1 was important for its function in polarity regulation, we speculated that the function of Par-1 in Hpo signaling might also be dependent on its kinase activity. To examine this hypothesis, we first constructed a kinase-dead form of Par-1 (Par-1-KD), which contained the T408A and S412A mutations. Par-1, containing these two mutations, was thought to be a kinase inactive mutant because the activation loop of the catalytic domain was disrupted [54]. This was confirmed by an in vitro kinase assay in which the kinase activity of Par-1-KD was completely abolished ( Figure S1D). Unlike the phenotype observed with the expression of two copies of the Par-1 transgenes, the expression of two copies of the Par-1-KD transgenes had no obvious effect on either eye growth or wing growth and did not dramatically enhance the overgrowth phenotype induced by Yki overexpression (compare Figure 2B0, 2C0 with 2B-2B9, 2C-2C9 and 2D0, 2E0 with 2D-2D9, 2E-2E9). Importantly, to exclude the possibility that the functional variation between Par-1 and Par-1-KD was due to a low expression level of Par-1-KD, we compared the overexpressed Par-1 and Par-1-KD protein levels in both the eye and wing imaginal discs using direct Western blot analysis. We found that the overexpression level of Par-1-KD was higher compared to Par-1, verifying that the functional variation did not result from a low Par-1-KD expression level ( Figure S1E-S1F). Furthermore, Par-1-KD failed to elevate Ex and Diap1 expression in flip-out clones ( Figure 2G-2G9 and 2I-2I9). Taken together, these observations demonstrated that overexpression of Par-1 promoted tissue overgrowth by promoting the activity of the Sd-Yki complex and upregulating the expression of Hpo pathway-responsive genes in a kinase-dependent fashion.

Loss of Par-1 Inhibits Tissue Growth by Downregulating Hpo Pathway Targets
To determine whether Par-1 is necessary for normal growth, we examined the effect of the loss-of-function of Par-1 on Hpo signaling. By expressing UAS-Par-1-RNAi under the control of eyeless-Gal4 (ey-Gal4) or MS1096, adult eye/wing sizes were reduced ( Figure 3A-3B0), suggesting that Par-1 (activity) was required for normal eye and wing development. Par-1 RNAi efficiency was also confirmed by in vivo staining, in which Par-1-RNAi transgenes were expressed under the control of hh-Gal4. As shown in Figure S2B-S2B9, endogenous Par-1 protein levels were efficiently knocked down by expressing Par-1-RNAi in the Pcompartment. In addition, shrinkage of the P-compartment was also observed ( Figure S2B-S2B9). To eliminate the concern regarding Par-1 RNAi off-target effects, a second line of Par-1 RNAi (Par-1-RNAi-2), which targets a different region of Par-1, was generated. Par-1-RNAi-2 also efficiently knocked down endogenous Par-1 expression ( Figure S2C-S2C9) and restricted wing growth when expressed by MS1096 ( Figure S2D-S2D9). Furthermore, the expression of Par-1-RNAi by GMR-Gal4 resulted in the detection of caspase-3 in its active (cleaved) form ( Figure 3D- We further tested whether knockdown of Par-1 resulted in downregulation of Hpo pathway-responsive genes. Expression of either UAS-Par-1-RNAi or UAS-Par-1-RNAi-2 by hh-Gal4 resulted in diminished levels of EX-Z, DIAP1, and diap1-GFP3.5 and a reduced P-compartment size ( Figures 3E-3E9, 3H-3H9, and S2E-S2E9, S2F-S2F0). Consistent with these results, a bantam sensor (mic32-GFP) signal was upregulated in Par-1-RNAi flip-out clones ( Figure 3F-3F9) or wing discs expressing Par-1-RNAi-2 by hh-Gal4 ( Figure S2G-S2G0), suggesting a restriction of microRNA bantam expression by knockdown of Par-1. Thus, this evidence suggested that the inactivation of Par-1 resulted in abnormal growth by antagonizing the expression of Hpo-responsive genes. To further strengthen this conclusion, the expression of Hpo-responsive genes was examined in par-1 w3 mosaic clones. As shown in Figure 3I-3I0 and 3J-3J0, in par-1 null clones, the diap1 transcriptional level was reduced ( Figure 3I-3I0), and bantam-lacZ was decreased ( Figure 3J-3J0). Importantly, the size of the par-1 null clones was significantly reduced compared to their twin spots ( Figure 3C-3C9), indicating a proliferation disadvantage for par-1 null clones. Taken together, these observations demonstrated that par-1 was essential for normal growth and that perturbation of Par-1 expression resulted in growth suppression and apoptosis by stimulating the Hpo pathway. Drosophila third-instar larval eye discs containing par-1 w3 clones were dissected. par-1 mutant clones were marked by the loss of GFP expression, while their twin spots were marked by increased GFP expression. The representative par-1 mutant clone and its twin spot were separately indicated by the white dashed line and red dashed line (C). The total area of the par-1 mutant clones or their twin spots within one eye disc were calculated (C9). The results represented the mean 6 SEM. *** means p,0.001 (n.6) for each genotype. (D-D9) Par-1 knockdown initiates apoptosis. Drosophila third-instar larval control eye discs (D) or discs expressing Par-1 RNAi (D9) under the control of GMR-Gal4 were immunostained with a cleavedcaspase-3 antibody. Note that caspase-3 was cleaved to its active form when Par-1 RNAi was expressed. (E-H9) Par-1 knockdown suppresses the expression of the Hpo pathway-responsive genes. Wing discs expressing Par-1 RNAi with hh-Gal4 (E-E9, H-H9) or with act.CD2.Gal4 (F-F9) were immunostained to demonstrate the protein expression levels of EX-Z (E-E9), mic32-GFP (F-F9), and DIAP1 (G-H9). Cells expressing Par-1 RNAi were labeled either by the lack of Ci or CD2 expression. Note that the expression of EX-Z and DIAP1 was inhibited, whereas the levels of mic32-GFP were increased. The arrows indicate the P-compartment or clone regions. (I-J0) Loss of par-1 disrupts the expression of Hpo pathway-responsive genes. D. melanogaster third-instar larval eye discs containing par-1 W3 clones were dissected and examined to determine the expression of diap1-lacZ (I-I0) and bantam-lacZ (J-J0). par-1 mutant clones were marked by the loss of GFP expression. Note the downregulation of diap1-lacZ and bantam-lacZ in the absence of Par-1. The clone regions are indicated by arrows. doi:10.1371/journal.pbio.1001620.g003 Par-1 Restricts Hippo Signaling PLOS Biology | www.plosbiology.org par-1 Acts Downstream of ex and fat but Upstream of hpo in the Hpo Pathway Given the findings presented in the previous section, we next determined the functional relationship between Par-1 and Hpo pathway components by identifying the genetic interactions of Par-1 in the Hpo pathway. We first examined whether the function of Par-1 was dependent on the activity of the Sd-Yki transcriptional complex because this complex was the main downstream effector of the Hpo pathway. Although expression of two copies of UAS-Myc-Par-1 in flip-out clones increased diap1-lacZ ( Figure S3A-S3A9), no increase in diap1-lacZ was detected when UAS-Yki-RNAi was coexpressed ( Figure S3B-S3B9). Coexpression of UAS-Sd-RNAi suppressed the overgrowth phenotype induced by Par-1 overexpression in Drosophila wings ( Figure S3C-S3C9). In addition, the elevated levels of diap1 transcription caused by Par-1 overexpression were reverted by coexpression of Sd RNAi ( Figure  S3D-S3F9). Furthermore, ectopic Yki expression reverted downregulated DIAP1 levels and the shrunken P-compartment phenotype induced by the expression of Par-1 RNAi ( Figure 4A-4B9). These results indicated that par-1 functioned upstream of the Sd-Yki transcription complex in the Hpo pathway. To strengthen this conclusion, the levels of phosphorylated Yki, which reflected Hpo/Wts activity, were examined. As expected, Par-1, but not Par-1-KD, reduced phosphorylated Yki levels ( Figure 4C). In addition, Par-1 also inhibited the Hpo/Wts signaling-induced Yki mobility shift ( Figure 5I, lanes 2-5). These findings suggested that Par-1 functioned upstream of yki to affect the activity of the Sd-Yki transcription complex. We next examined the genetic epistasis between Par-1 and the upstream components of the Hpo pathway. We found that elevated DIAP1 levels and the enlarged Pcompartment size ( Figure  We then determined whether Par-1 regulated the Hpo pathway via Wts, which phosphorylates Yki at Ser168 to retain Yki in the cytoplasm [19]. By generating Par-1 RNAi clones in eye discs using the mosaic analysis with a repressible cell marker (MARCM) technique, we found that the size of Par-1 RNAi clones was extremely small compared to that of the control clones (compare Figure 4F9 with 4F), indicating adverse development of the Par-1 RNAi clones. Strikingly, we found that knockout of wts rescued the adverse developmental phenotypes of the Par-1 RNAi clones and that the size of the wts mutant clones expressing Par-1 RNAi was more comparable to that of the wts clones, rather than that of Par-1 RNAi clones ( Figure 4G and compare Figure 4F0 with 4F90). These findings indicated that par-1 functioned upstream of wts. Because Wts was activated and phosphorylated by upstream components of the Hpo pathway, we then tested whether Par-1 regulated Wts phosphorylation. Par-1, but not Par-1-KD, reduced the mobility shift of Wts phosphorylation in the presence of Hpo/ Sav/Merlin/Tao-1 ( Figure 4H), suggesting that par-1 functioned upstream of wts and regulated Wts phosphorylation in a kinasedependent manner. We then determined whether Par-1-regulated Hpo signaling was dependent on the activity of Hpo, which restricted tissue growth by phosphorylating Wts. By generating hpo mutant clones in Drosophila compound eyes using the MARCM system, we found that the ablation of Hpo resulted in tumor outgrowth (compare Figure 4I9 with Figure 4I). Furthermore, we found that Par-1 RNAi was incapable of reverting the growth advantage of hpo null clones (compare Figure 4I0 with Figure 4I9), indicating that Par-1 functioned upstream of hpo to regulate Hpo signaling.

Par-1 Interacts with Hpo-Sav and Regulates the Phosphorylation of Hpo at Ser30
Given that Par-1 functions upstream of hpo (Figure 4), we speculated that Par-1 modulated the function of the Hpo-Sav complex. To test our hypothesis, we first examined whether Par-1 bound to the Hpo-Sav complex using a co-immunoprecipitation assay. As expected, full-length Par-1 interacted with both Hpo and Sav, although these associations were weak ( Figure 5A-5B). In addition, we also found that the HA-tagged N-terminal fragment of Par-1 (Par-1-N, Figure S4A) had a strong association with both Hpo and Sav, whereas there was only a weak interaction between the Par-1 C-terminal fragment (Par-1-C, Figure S4A) and Hpo/ Sav had been detected ( Figure 5A-5B). Importantly, the interaction between Par-1 and Hpo/Sav was specific because neither the full-length nor the N-terminal fragment of Par-1 co-immunoprecipitated with Merlin (unpublished data), Wts, or Mats ( Figure  S4B). On the basis of the previous results that Par-1 functioned in a kinase-dependent manner in Hpo signaling, we performed a phosphorylation shift experiment using a phos-tag gel to determine whether Par-1 affected the phosphorylation of Hpo and Sav in vitro. The Phos-Tag is a phosphate binding compound that, when incorporated into polyacrylamide gels, results in an exaggerated mobility shift for phosphorylated proteins, which is dependent upon the degree of phosphorylation [18,55]. We observed that Flag-Hpo cotransfected with Par-1, but not Par-1-KD, in S2 cells that exhibited a mobility shift ( Figure S5A). However, such a mobility shift was not detected for Sav when it was cotransfected with either Par-1 or Par-1-KD ( Figure 6B, compare lanes 3-4 with lane 1). To remove the effect of Hpo auto-phosphorylation, a kinase-dead form of Hpo (Hpo-KD) was also tested in the phosphorylation shift experiment. As shown in Figure 5C, Par-1 also induced Hpo-KD to generate a phosphorylation mobility shift. These results suggested that Par-1 specifically induced Hpo phosphorylation in S2 cells.
To identify which sites on Hpo were affected by Par-1, we cotransfected Flag tagged Hpo with HA tagged Par-1 in S2 cells. Flag-Hpo protein was then immunoprecipitated and separated using SDS-PAGE and harvested for a semi-quantitative mass spectrometric (MS) analysis (details are described in the Materials and Methods). According to the MS results, we identified four potential phosphorylation sites that were affected by Par-1 expression, S30, S66, Y365, and T615. We then generated different Hpo variants by individually mutating these candidate sites and then examined how these mutations affected Par-1induced Hpo phosphorylation. Interestingly, only Hpo (S30A) failed to generate mobility shifts in a direct Western blot analysis compared to wild-type Hpo protein ( Figure S5C), while other Hpo mutations, T615A, S66A, and Y365E, did not affect the mobility shift upon Par-1 induction ( Figure S5B-S5C). Moreover, we also found that Hpo (S30A)-KD could not be shifted by Par-1 ( Figure 5D). These results suggested that Par-1 may regulate the phosphorylation of Hpo at Ser30. To verify our prediction, an antibody that specifically recognized phosphorylated Hpo at Ser30 was generated. As shown in Figures 5E and S5D, Par-1 but not Par-1-KD was able to induce Hpo phosphorylation at Ser30. To validate the phospho-Hpo S30 antibody, a phosphatase treatment was applied. Upon phosphatase treatment, the p-Hpo(S30) band was undetectable ( Figure 5E, compare lane 5 with lane 2). To further test the specificity of this antibody, we transfected Hpo(S30) mutants with Par-1. As shown in Figure 5F and in Figure S5E, in the presence of Par-1, Hpo(S30A) could not be detected by the p-Hpo(Ser 30) antibody, whereas wild-type Hpo(S30) was detected. These findings indicated that Par-1 regulated the phosphorylation of Hpo at Ser30.

Par-1-Induced Hpo Ser30 Phosphorylation Regulates Hpo Activity
In recent proteome-wide phosphorylation studies using Drosophila embryos [40], it was suggested that Hpo was phosphorylated at whether Ser30 phosphorylation state affects Hpo phosphorylation at Thr195, which was important for Hpo activation. As shown in Figure 5G-5H, Par-1, but not Par-1-KD, significantly inhibited Hpo phosphorylation levels at Thr195, whereas this inhibitory effect was abolished when the Ser30 site was mutated. More importantly, phosphorylation at Thr195 was slightly elevated when Ser30 was mutated into an alanine ( Figure 5G, compare lane 4 with lane 1, and Figure 5H). These findings suggested that Par-1 regulated Hpo activity via antagonizing phosphorylation at the Thr195 site by regulating Ser30 phosphorylation. It has been reported that the Hpo Thr195 site is not only auto-phosphorylated but also phosphorylated by Tao-1 [37,38], which is a partner of Par-1 in the regulation of microtubule dynamics. Thus, we asked whether Par-1induced phosphorylation at Ser30 also affected Tao-1-mediated phosphorylation at Thr195. As shown in Figure S5F, consistent with the results shown in Figure 5G-5H, Par-1 suppressed Tao-1mediated phosphorylation at Thr195. The antagonistic effect of Par-1 and Tao-1 on Hpo phosphorylation at Thr195 motivated the examination of the interrelationship of Par-1 and Tao-1 in the Hpo pathway. We found that Tao-1 disrupted Par-1-induced a phosphorylation mobility shift of Hpo-KD ( Figure S5F), suggesting that the function of Par-1 in the Hpo pathway was modulated by upstream signaling.
Because Hpo(S30A) demonstrated a higher activity compared to the Hpo wild type, we examined whether Hpo(S30A) exerted its inhibitory effect on Yki. We found that Hpo(S30A) resulted in a dramatic Yki mobility shift, whereas the Hpo wild type resulted in a moderate phosphorylation of Yki ( Figure 5I, compare lane 2 with lane 6). Furthermore, we found that co-transfection of the Hpo(S30A) mutant blocked Par-1-induced Yki dephosphorylation ( Figure 5I, compare lane 3 with lane 7), which further confirmed our conclusion that Par-1 modulated Hpo activity by regulating Hpo phosphorylation at Ser30.
To further investigate the role of Par-1-induced Hpo phosphorylation at Ser30 in Hpo activity regulation, we generated Figure 7. MARK, the mammalian homologue of Par-1, regulates the Hpo pathway. (A) MARK4 activates YAP transcriptional activity. The indicated plasmids were co-transfected with the 5XUAS-luc reporter, Gal4-TEAD4, Flag-YAP, and CMV-b-galactosidase construct into HEK293T cells. Luciferase activity was measured and normalized against b-galactosidase activity. (B) Ectopic expression of MARK4 inhibits YAP phosphorylation. HEK293T cells were transfected with the indicated constructs, and YAP was immunoprecipitated using the anti-Flag antibody. YAP phosphorylation was detected using Western blot analysis with a phospho-YAP specific antibody and determining its mobility on a Phos-tag-containing SDS-PAGE gel. (C) MARK4 induces the phosphorylation of MST2. HEK293T cells were transfected with the indicated constructs, and MST2 phosphorylation was analyzed by electrophoresis on a Phos-tag-containing gel and through Western blotting with an anti-Flag antibody. (D) MARK1 expression is significantly upregulated in squamous lung cancer biopsy specimens compared with matched control specimens. The expression profiling data were downloaded from the GEO dataset GDS1312. These data were expressed as the mean 6 SEM and were analyzed using the paired t-test. *p,0.05. (E) Prostate cancer progression is accompanied by an increased expression of MARK1. PIN, prostatic intraepithelial neoplasia; PCA, localized prostate cancer; MET, metastatic prostate cancer. The expression data were obtained from GEO dataset GDS3289. All of the results were expressed as the mean 6 SEM. ***p,0.001. doi:10.1371/journal.pbio.1001620.g007 Par-1 Restricts Hippo Signaling PLOS Biology | www.plosbiology.org transgenic flies of attB-UAS-Hpo variants at the 75B1 attP locus, which ensured equal expression of different forms of the Hpo mutant. Because the flies were grown at room temperature, overexpression of Hpo under the control of GMR-Gal4, Ci-Gal4, and hh-Gal4 resulted in adult lethality; therefore, we selected weak drivers, such as C765, which induced gene expression moderately in the Drosophila wing to compare the activity of Hpo variants. Consistent with our in vitro studies, Hpo(S30A) mutants exhibited much smaller wings compared to wild-type Hpo flies ( Figure 5J-5K), indicating that Hpo(S30A) variants demonstrated a higher activity than wild-type Hpo in vivo. To further strengthen this conclusion, we used an inducible Ci-Gal4, which was controlled by a temperature-sensitive Gal80, to drive Hpo expression in order to exclude the early lethality. Because some progeny were viable when Hpo expression was induced after pupa formation, we then compared the activity of Hpo variants by measuring the wing size of the survivors and calculated the mortality rate. We found that survivors with Hpo expression maintained a relatively normal wing size ( Figure S5G-S5H), while survivors with Hpo(S30A) expression demonstrated much smaller wings compared to controls ( Figure S5G-S5H). Meanwhile, Hpo(S30A) flies exhibited a higher mortality rate compared to wild-type Hpo flies ( Figure  S5I). These findings suggested that the activity of Hpo(S30A) mutants was higher compared to wild-type Hpo. Taken together, on the basis of the above biochemical and in vivo evidence, we speculated that Par-1 inhibited Hpo activity via the regulation of Hpo phosphorylation at the Ser30 site.

Par-1 Inhibits Hpo-Sav Association in a Kinase-Dependent Manner
We observed that Par-1 blocked Hpo-induced Sav stabilization in a kinase-dependent manner ( Figure S5C, and Figure 6A, compare lane 3 with lane 2). Thus, we examined whether Par-1 induced Sav destabilization, which was dependent on the phosphorylation of the Hpo Ser30 site. Interestingly, we found that the stabilization of Sav by Hpo was not affected by Par-1induced Hpo phosphorylation at Ser30 because Hpo(S30A) mutants were still able to stabilize Sav, and this stabilization could be reversed by Par-1 ( Figure S6A). In addition to the change in Sav protein levels, Par-1 also decreased the phosphorylation status of Sav, which was induced by Hpo in a kinase-dependent manner ( Figure 6B, compare lanes 5-6 with lane 2). Given that full-length Par-1 weakly interacted with both Hpo and Sav ( Figure 5A-5B), we then determined whether Par-1 affected the association between Hpo and Sav. As shown in Figure 6C, Par-1, but not Par-1-KD, impaired the association of endogenous Hpo and transfected Sav, suggesting that the interaction between Hpo and Sav was disrupted by Par-1 overexpression. To mimic the disruption of the Hpo-Sav complex, we ablated sav using the MARCM system. We found that the growth defect induced by Par-1 RNAi was incapable of inhibiting sav mutant-induced clone growth ( Figure S6B-S6C) and adult eye overgrowth ( Figure 6D--6D0), suggesting that Par-1 functioned upstream of sav. These observations supported the notion that Par-1 kinase activity was important to restrain the function of the Hpo-Sav complex.
On the basis of the evidence provided above, we propose a model of how Par-1 restricts the activity of Hpo pathway ( Figure 6E). Par-1 regulates Hpo phosphorylation at Ser30 to modulate the Hpo kinase activity; simultaneously, Par-1 also promotes the dissociation of Sav from Hpo, resulting in the dephosphorylation and destabilization of Sav, thereby repressing the function of the Hpo-Sav complex.
The Mammalian Homologue of Par-1, MAP/MARK, Regulates the Hpo Pathway An evolutionally conserved function of Par-1 in regulating microtubule dynamics has been reported [41]. To determine whether the function of Par-1 on the Hpo pathway is conserved, the human homologue of Par-1, MARK1 and MARK4 were cloned. The Gal4-tead4 reporter [56] was used to examine the effect of MARK1 and MARK4 on the mammalian Hpo pathway. As expected, both MARK1 and MARK4, but not the kinase-dead form of MARK4, activated the YAP transcription co-activator activity (Figures 7A and S7A). Since MARK4 activated YAP more than MARK1, we investigated whether MARK4 affected YAP phosphorylation. Indeed, the phosphorylation levels of YAP were significantly decreased upon MARK4 overexpression ( Figure 7B), indicating that MARK inhibited Hpo signaling in mammals. Finally, we investigated whether MARK also resulted in MST (human homologue of Hpo) phosphorylation. We found that both MARK4 and MARK1 induced mobility shifts of MST2 ( Figures 7C and S7B). Taken together, these findings suggested that the inhibitory function of Par-1 on Hpo signaling was conserved from Drosophila to mammals. Our study suggested that Par-1 may have a procarcinogenic role because its hyperactivation in Drosophila is sufficient to induce tissue overgrowth, and in mammals, Par-1 is sufficient to activate YAP. Interestingly, MARK4 has been suggested to play a role in hepatocellular carcinogenesis and gliomagenesis [51,52]. However, whether MARK1 is also involved in carcinogenesis is largely unknown. To further elucidate this question, we characterized the mutation and expression of MARK1 in different cancer samples using the COSMIC and GEO databases. Although no mutation or deletion of MARK1 has been reported in the tumors that were surveyed, the transcription levels of MARK1 showed significant upregulation in squamous lung cancer samples and during the progression of prostate cancer ( Figure 7D-7E). Taken together, these findings suggested that Par-1 was a potential oncogene and that its regulatory role in Hpo signaling could be conserved.

Discussion
The Hpo signaling pathway has emerged as a conserved pathway that controls tissue growth and balances tissue homeostasis via the regulation of the downstream Sd-Yki transcription complex. Despite the importance of this pathway in development and carcinogenesis [2,6], many unknown regulators of the Hpo pathway remain to be identified. Here, we identified Par-1 as one such Hpo pathway regulator via a genetic overexpression screen using Drosophila EP lines. In this study, we demonstrated that Par-1 was essential for the restriction of Hpo signaling. We also demonstrated that overexpression of Par-1 promoted tissue growth via the inhibition of the Hpo pathway, whereas loss of Par-1 promoted Hpo signaling to suppress growth and induce apoptosis. Using the Drosophila eye and wing imaginal discs as well as cultured cells, we provide the first genetic and biochemical evidence for a function of Par-1 in the Hpo pathway.
Although the conserved function of Hpo has been well studied, the regulatory mechanism of its kinase activity is still largely obscure. Currently, the regulatory mechanism of Hpo kinase activity was believed to mainly be dependent on autophosphorylation by altering the phosphorylation status of the Thr195 site [37,53,57]. However, whether the uncharacterized phosphorylation events of Hpo, which have been identified in several recent proteome-wide phosphorylation studies [39,40], contributed to the regulation of Hpo activity is still unknown. By studying the mechanism underlying Par-1 function in Hpo signaling, we demonstrated that Par-1 induced Hpo phosphorylation at Ser30 and this lead to the regulation of Hpo kinase activity.
Although we have extensively studied how Par-1 regulates the Hpo pathway in this study, several unresolved questions remain. The interaction between Par-1 and Hpo/Sav may be tightly regulated because full-length Par-1 only weakly interacted with Hpo/Sav, unlike the interaction with the N-terminal fragment of Par-1 ( Figure 5A-5B). However, the triggering signal for Par-1 to interact with Hpo/Sav is still unknown. It has been reported that Par-1 was activated by Tao-1 and LKB1 [49,50]. In this study, we established that Par-1 antagonized Tao-1 in Hpo signaling, and interestingly, in Drosophila, the antagonistic relationship between Par-1 and Tao-1 in microtubule regulation has been previously reported [57][58][59]. Thus, it is unlikely that Tao-1 functions as the trigger. We then investigated whether LKB1 functioned as an activator of Par-1 in Hpo signaling by expressing the LKB1 transgene in different organs. Unlike Par-1, ectopic LKB1 expression limited both wing and eye growth (unpublished data), indicating that LKB1 was also not the trigger.
We have shown that Par-1 and Tao-1 exhibited opposing effects on Hpo signaling ( Figure S5F). Given that Tao-1 and Par-1 were partners that regulated microtubule dynamics via the phosphorylation of Tau [48], Tau may have a function in Hpo signaling. To investigate this hypothesis, we employed genetic and biochemical studies and found that Tau RNAi failed to suppress the expression of Hpo pathway-responsive genes ( Figure S8A-S8B). In addition, Tau did not trigger Hpo phosphorylation and Sav dissociation in vitro ( Figure S8C-S8D), indicating that Par-1 regulated Hpo signaling independent of Tau. Interestingly, it has been previously suggested that Par-1 did not regulate Tau activity in Drosophila [46], indicating an evolutionary difference between Par-1 and Tau-1 function.
We have provided evidence that Par-1 regulated Hpo signaling via the phosphorylation of Hpo or the destruction of the Hpo/Sav complex. Because Par-1 is a well-known polarity regulator and polarity components, such as Crumb and Lgl, have been shown to be involved in the Hpo signaling pathway [35], it is possible that Par-1 may regulate Hpo signaling via a polarity complex, or its activity might be regulated via a polarity complex. Indeed, the localization of Crumb and Patj were affected by Par-1 expression (unpublished data). Thus, further studies on polarity complexes and Hpo signaling will help elucidate this problem.

Cloning, Mutants, Transgenes, and Drosophila Genetics
Par-1 fragments were amplified from the BDGP DGC Clone (number RE47050) using the PCR. The sequence of full-length Par-1 used in this study was the same as Par-1-N1S (accession number NP_001014542). which has been previously described [43]. The Par-1 kinase-dead mutant was generated by converting the conserved Ser412 and Thr408 into alanine at the activation loop of the Par-1 catalytic domain. All of the primers used in this study are available upon request.
The EP library for the screen was a gift from Jianming Chen (Third Institute of Oceanography, State Oceanic Administration, China). Par-1 w3 is a null allele from the St Johnston lab, and FRT/FLP-mediated mitotic recombination was used to generate mutant clones, as previously described [20]. The genotypes used to generate the clones were the following: FRTG13 Par-1 w3 /eyflp; FRTG13 ubi-GFP. Par-1 RNAi fly was purchased from the Bloomington Drosophila Stock Center (stock number 32410). To generate the Par-1 RNAi-2 transgenic fly, an artificial microRNA method was adopted, which was reported to efficiently silence gene expression [60]. Briefly, we designed two hairpin oligos that were targeted to the 1227-1247 base pair and the 1551-1571 base pair regions of Par-1-N1S (accession number NM_001014542). Next, these two hairpin oligos were ligated to create a tandem hairpin RNA. The procedure for this complete construction can be found in the supplementary information of Wang et al.'s study [61]. The following transgenes were used in this study: bantam-lacZ (a gift from Wei Du, The University of Chicago), UAS-ex-RNAi (V22994, VDRC), UAS-fat-RNAi (V9396, VDRC), and UAS-Tau-RNAi (V25024, VDRC). Other stocks included: bantam sensor mic32-GFP, ex-lacZ, diap1-GFP3.5, hh-Gal4, GMR-Gal4, MS1096, act . CD2 . Gal4, eyless-Gal4, diap1-lacZ, UAS-Yki, UAS-Yki-RNAi, UAS-Sd-RNAi, hpo BF33 , wts latsX1 , and Sav SH13 , which have all been previously described [20,53]. The generation of the transgenes at the attP locus has been previously described [20]. Unless otherwise indicated, all of the flies were cultured at 25uC.
Cell Culture, Transfection, Immunoprecipitation, Western Blot Analysis, Immunostaining, and the Luciferase Reporter Assay S2 cells were maintained in Drosophila Schneider's Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. The cells were incubated at 25uC in a humidified air atmosphere.
Plasmid transfection was performed using Lipofectamine (Invitrogen), according to the manufacturer's instructions. For all of the transfection experiments, a ubiquitin-Gal4 construct was cotransfected with the pUAST expression vectors.
For the luciferase reporter assay, the 36Sd2-Luc reporter has been previously described [20]. The luciferase assay was performed using the Dual Luciferase Assay System (Promega).

Phosphorylation Mobility Shift Assay
For the phosphorylation mobility shift assays, Phos-Tag AAL-107 (FMS Laboratory) was introduced to enlarge the mobility shift. The operating procedure was performed according to the manufacturer's instructions. For all of the mobility shift assays, the protein samples were processed using an SDS-PAGE gel under a low voltage. According to the molecular weight of the protein, a 6% or 8% resolving gel was used for the Wts and the Sav and Hpo mobility shift assays, respectively.

In Vitro Kinase Assay
Immunoprecipitated cell lysates or purified protein were directly incubated in 20-40 ml of kinase assay buffer (250 mM HEPES, pH 7.4, 0.2 mM EDTA, 1% glycerol, 150 mM NaCl, and 10 mM MgCl 2 ). The reaction were initiated by the addition of an ATP mixture (2 ml 1 mM ATP, 0.2 ml [c-32 P] ATP [10 mCi/ml]) and then incubated at 30uC for 30 min. The reactions were terminated by the addition of an SDS sample buffer. Next, the samples were boiled for 5 min at 100uC followed by SDS-PAGE and autoradiography.

Mapping Phosphorylation Sites by Mass Spectrometry
S2 cells were transfected with Flag-Hpo or cotransfected with Flag-Hpo and Par-1. 48 h after transfection, the cells were harvested and then lysed. SDS-PAGE and Colloidal Blue staining (Invitrogen, LC6025) were then performed on the protein samples. The Hpo protein was cut from the gel and sent to the Protein Center, SIBCB for mass spectrometric analysis. A detailed procedure of the mass spectrometric analysis may be obtained from the Protein Center, SIBCB. The candidate sites were identified by the increased phosphorylation abundance in the cotransfected Flag-Hpo and Par-1 cells versus Flag-Hpo transfected cells.

Statistical Analysis
All of the data in this study were expressed as the mean 6 standard error of the mean (SEM) and were analyzed using Student's t test by R 2.9.0. The results were considered statistically significant if p,0.05. (indicated by arrows). Note that the upregulation of diap1-LacZ induced by ectopic Par-1 was completely suppressed by Yki RNAi. (C-F9) Par-1 is functionally dependent on Sd in the Hpo pathway. Adult wings expressing UAS-2*Myc Par-1; UAS-Sd RNAi (C9) showed a similar phenotype as that of Sd RNAi (C). Furthermore, wild-type wing discs (D-D9), wing discs expressing UAS-2*Myc-Par-1 (E-E9) or UAS-Myc-Par-1; UAS-Sd RNAi (F-F9) in the Pcompartment were immunostained to demonstrate the expression of diap-GFP3.5. The P-compartment was marked by the loss of Ci or Myc tag (red) and is indicated by arrows. Note that coexpression of Sd RNAi reversed the upregulation of diap1-GFP 3.5 induced by Par-1. (G-G90) Drosophila wings of the indicated genotypes are shown. Note that the enlarged wing size induced by ex RNAi was reversed by Par-1 RNAi. (H-H90) Drosophila wings of the indicated genotypes are shown. Note that Par-1 RNAi reduced wing size even in the fat-RNAi condition. (TIF) Figure S4 (A) A schematic representation of the Par-1 full-length structure or its truncated forms. (B) Immunoprecipitation between Par-1-N, Wts and Mats. S2 cells were transfected with the indicated constructs followed by co-immunoprecipitation. Note that Wts and Mats were unable to interact with the N-terminal of Par-1. (TIF) Figure S5 (A) Par-1 regulates Hpo phosphorylation in vitro. S2 cells were transfected with the indicated constructs. Cell lysates were subjected to the phosphorylation mobility shift assay. Note the phosphorylation shift of Hpo in the presence of Par-1 but not with Par-1 KD. (B-C) Hpo(S30A) mutants blocked the Par-1induced Hpo phosphorylation shift but Hpo (T615A), Hpo(S66A) and Hpo(Y365E) did not. (D-E) Par-1 induces Ser30 phosphorylation of Hpo in S2 cells. S2 cells were transfected with the indicated constructs. The cell lysates were subjected to a Western blot analysis. Note that the phospho Hpo(Ser30) antibody could only detect Hpo but not Hpo(S30A) phosphorylation induced by Par-1. (F) Par-1 and Tao-1 antagonization regulates Hpo phosphorylation. S2 cells were transfected with the indicated plasmids, and the cell lysates were subjected to a direct Western blot analysis or a phosphorylation mobility shift assay. Note that Tao-1 partially inhibited the Par-1-induced Hpo phosphorylation mobility shift, while Par-1 inhibited Tao-1-induced Hpo Thr195 phosphorylation. (G-I) Hpo(S30A) showed enhanced activity compared with wild-type Hpo in vivo. Adult wings of wild-type (G), wings expressing Hpo (G9), and wings expressing Hpo(S30A) (G0) under the control of tub-Gal80 ts ; Ci-Gal4. Note that the Hpo Ser30 mutant induced smaller wings (H) and a higher mortality rate (I) compared to wild-type Hpo. The relative wing size (H) was quantified using an unpaired t-test. The results represented the mean 6 SEM. **p,0.01, (n.6) for each genotype. The percentage of lethal flies (I) was calculated by dividing the number of lethal pupas by the total number of pupas. To induce Hpo expression, fly progeny were transferred to a 29uC incubator at different developmental stages. (TIF) Figure S6 (A) Par-1 destabilizes Hpo-induced Sav accumulation independent of Par-1 phosphorylation at the Hpo Ser30 site. S2 cells were transfected with the indicated constructs followed by a Western blot analysis. Note that both wild-type Hpo and Hpo(S30A) could stabilize Sav. In addition, stabilized Sav can be destabilized by Par-1 overexpression. (B) par-1 functions upstream of sav in the Hpo pathway. Clones were generated using the MARCM system. The following genotypes were used: eyflp, Ubi-Gal4, UAS-GFP; FRT82B Sav SH13 /FRT82B Gal80 (left panel), and eyflp, ubiGal4, UAS-GFP; Par-1-RNAi; FRT82B Sav SH13 / FRT82B Gal80 (right panel). (C) Quantification of the relative clone size. The relative clone size was calculated as the GFP area divided by the entire disc area. All of these data were expressed as the mean 6 SEM. **p,0.01. **p,0.001. n.5, for each group. (TIF) Figure S7 (A) MARK1 significantly enhances the transcriptional activity of Yap. HEK293T cells were transfected with the indicated plasmids, and the cell lysates were directly subjected to a dual luciferase reporter assay. Note that MARK1 synergized with Yap to promote the transcriptional activity of TEAD. (B) MARK1 induces a MST2 phosphorylation mobility shift. HEK293T cells were transfected with the indicated plasmids and cell lysates were directly subjected to a phosphorylation mobility shift assay. Note that MST2 showed a shift band by MARK1 coexpression. (TIF) Figure S8 (A-B9) Wing discs expressing Tau RNAi (A-B9) with hh-Gal4 were immunostained with anti-Ci (red) and anti-DIAP1(green A-A9) to demonstrate the expression level of DIAP1 (A-A9) and mic32-GFP (B-B9). Note that the expression of Tau RNAi failed to affect Hpo pathway-responsive gene expression. The arrows indicate the P-compartment. (C-D) S2 cells were transfected with the indicated constructs followed by Western blot analyses. Note that Tau affects neither the phosphorylation status of Sav (C) nor the mobility shift of Hpo (D). (TIF)