NOTCH1 intracellular domain negatively regulates PAK1 signaling pathway through direct interaction

Biochimica et Biophysica Acta 1863 (2016) 179–188

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NOTCH1 intracellular domain negatively regulates PAK1 signaling pathway through direct interaction Ji-Hye Yoon a, Jung-Soon Mo a, Eun-Jung Ann a, Ji-Seon Ahn a, Eun-Hye Jo a, Hye-Jin Lee a, Se-Hoon Hong a, Mi-Yeon Kim a, Eung-Gook Kim b, Keesook Lee a, Hee-Sae Park a,⁎ a b

Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju, Republic of Korea Department of Biochemistry and Medical Research Center, College of Medicine, Chungbuk National University, Cheongju, Republic of Korea

a r t i c l e

i n f o

Article history: Received 21 May 2015 Received in revised form 1 November 2015 Accepted 3 November 2015 Available online 10 November 2015 Keywords: NOTCH1-IC PAK1 ILK1 Localization Protein–protein interaction Migration

a b s t r a c t p21-Activated kinase 1 (PAK1) is a serine/threonine protein kinase implicated in cytoskeletal remodeling and cell motility. Recent studies have shown that it also promotes cell proliferation, regulates apoptosis, and increases cell transformation and invasion. In this study, we showed that NOTCH1 intracellular domain (NOTCH1-IC) negatively regulated PAK1 signaling pathway. We found a novel interaction between NOTCH1-IC and PAK1. Overexpression of NOTCH1-IC decreased PAK1-induced integrin-linked kinase 1 (ILK1) phosphorylation, whereas inhibition of NOTCH1 signaling increased PAK1-induced ILK1 phosphorylation. Notably, ILK1 phosphorylation was higher in PS1,2−/− cells than in PS1,2+/+ cells. As expected, overexpression of NOTCH1-IC decreased ILK1-induced phosphorylation of glycogen synthase kinase 3 beta (GSK-3beta). Furthermore, NOTCH1-IC disrupted the interaction of PAK1 with ILK1 and altered PAK1 localization by directly interacting with it. This inhibitory effect of NOTCH1IC on the PAK1 signaling pathway was mediated by the binding of NOTCH1-IC to PAK1 and by the alteration of PAK1 localization. Together, these results suggest that NOTCH1-IC is a new regulator of the PAK1 signaling pathway that directly interacts with PAK1 and regulates its shuttling between the nucleus and the cytoplasm. © 2015 Elsevier B.V. All rights reserved.

1. Introduction p21-Activated kinase 1 (PAK1) is a serine/threonine (Ser/Thr) protein kinase that is activated by external stimuli through various cell surface receptors, including G-protein-coupled receptors and receptor tyrosine kinases in a small GTPase-dependent or GTPase-independent manner [1–3]. PAK1 is a binding partner of Rho GTPases Cdc42 and Rac1 [4] and is involved in diverse cellular processes such as cytoskeletal remodeling and cell motility. Recent studies have shown that PAK1 also promotes cell proliferation, regulates apoptosis, and increases cell transformation and invasion [5]. PAK1 is significantly overexpressed in some cancers such as ovarian, breast, and bladder cancers [6]. Several functional studies have reported that PAK1 is highly associated with cell transformation and tumorigenesis, as evidenced by the development of premalignant lesions and tumor formation due to PAK1 overexpression and hyperactivation [7,8]. The kinase activity of PAK1 is required for Ras-induced transformation. Further, the activity of PAK1 is dependent on the phosphorylation of Ser/Thr residues [9,10]. To date, most studies have focused on the cytosolic functions of PAK1. However, one

Abbreviations: NOTCH1-IC, NOTCH1 intracellular domain; PAK1, p21-activated kinase 1; ILK1, integrin-linked kinase 1. ⁎ Corresponding author at: School of Biological Sciences and Technology, Chonnam National University, Yongbong-dong, Buk-ku, Gwangju 500-757, Republic of Korea. E-mail address: [email protected] (H.-S. Park).

http://dx.doi.org/10.1016/j.bbamcr.2015.11.001 0167-4889/© 2015 Elsevier B.V. All rights reserved.

study showed that PAK1 binds to and phosphorylates histone H3 and that endogenous PAK1 is localized in the nucleus of 18%–24% interphase cells [11]. Singh et al. identified the signaling sequences of PAK1 that are involved in its nuclear localization and showed that PAK1 regulates the expression of its targets in a positive as well as negative manner [12]. These findings highlight the opportunity to determine new functions of PAK1 in the nucleus, including its possible role in cell cycle regulation, mitosis, and cancer. PAK1 is involved in the regulation of cell cycle, and its overexpression in human breast cancer cells results in the abnormal accumulation of centrosomes and aberrant mitosis [7]. Recent studies have shown that functions of PAK1 may be regulated by its intracellular location. While the role of PAK1 in the cytoplasm is well established, its role in the nucleus is unknown. Studies indicate that PAK1 localizes to and performs several functions within the nucleus. However, the mechanisms regulating the localization of PAK1 to the nucleus are unknown. Integrin-linked kinase 1 (ILK1) decreases the stability of NOTCH1 intracellular domain (NOTCH1-IC) through Fbw7 ubiquitin ligasemediated degradation via ubiquitin–proteasome pathway [13]. ILK1 is a pivotal effector in various cellular processes such as cell migration, invasion, proliferation, differentiation, metabolism, and survival [14–17]. Studies have shown that ILK1 regulates cell motility and migration through the small GTPases Rac1 and Cdc42 [18]. PAK1 and ILK1 regulate some common physiological processes [18,19]. ILK1 is a substrate of PAK1 in cellular processes such as cytoskeletal remodeling, cell motility, proliferation, apoptosis, transformation, and invasion. Studies have

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reported that ILK1 undergoes phosphorylation-dependent shuttling between the nucleus and the cytoplasm [20]. Chun et al. suggested that ILK1 localizes to the nucleus through a putative nuclear localization signal [21]. Previously, we reported the colocalization of ILK1 and NOTCH1-IC in both the nucleus and the cytoplasm of HaCaT and melanoma cells [13]. ILK1 controls tumor growth and angiogenesis by inducing the production of vascular endothelial growth factor (VEGF) through downstream effectors Akt1 and glycogen synthase kinase 3 beta (GSK-3beta) [16]. In addition, ILK1 negatively regulates NOTCH1 signaling in a GSK-3beta-independent manner [13]. In the present study, we evaluated the crosstalk between NOTCH1IC and PAK1 signaling and observed a novel interaction between these two proteins. Further, we suggested that the interaction of NOTCH1-IC with PAK1 inhibited the kinase activity of ILK1. Notably, NOTCH1-IC disrupted the interaction of PAK1 with ILK1 and altered the localization of PAK1. This inhibitory effect of NOTCH1-IC on the PAK1 signaling pathway was mediated by the binding of NOTCH1-IC to PAK1 and by the inhibition of its localization to the cytoplasm. Together, these results suggest that NOTCH1-IC is a new regulator of PAK1 that directly interacts with PAK1 and regulates its shuttling between the nucleus and the cytoplasm. In addition, our study provided evidence that NOTCH1 signaling negatively regulated PAK1-mediated signaling. 2. Materials and methods 2.1. Cell culture and transfection Human embryonic kidney 293 (HEK293) cells and PS1,2+/+ and PS1,2−/− MEFs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2. All media were supplemented with 1% penicillin/ streptomycin. The cells were transiently transfected with recombinant vectors by using calcium phosphate method or Lipofectamine-2000 (Invitrogen), according to the manufacturer's protocol [13]. 2.2. Immunoblotting analysis For immunoblotting, the cells were harvested after 48 h of transfection and were lysed in RIPA buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol [DTT], and 2 μg/ml each of leupeptin and aprotinin). The cell lysates were centrifuged at 12,000 ×g and 4 °C for 20 min. The soluble fraction was boiled in Laemmli buffer and was resolved by performing SDS-PAGE. After the electrophoresis, the separated proteins were transferred onto PVDF membranes by using a semi-dry transfer cell system. The membranes were blocked using phosphatebuffered saline (PBS; pH 7.4) containing 0.1% Tween 20 and 5% non-fat milk.

(GSH)–agarose beads (Sigma), in accordance with the manufacturer's instructions. For GST-pull down assay, equal amount of GST or GST– NOTCH1-IC was incubated for 4 h at 4 °C with the lysates of HEK293 cells transfected with the different expression vectors. For in vitro binding assay, GST–NOTCH1-IC eluted from GSH–agarose bead was incubated for 4 h at 4 °C with bead-binding GST–PAK1. After incubation, the beads were washed 3 times with ice-cold PBS and were boiled with 20 μl Laemmli buffer. The precipitates were separated by performing SDS-PAGE, and the pulled down proteins were identified by immunoblotting with specific antibodies.

2.5. Immunocomplex kinase assay Cultured cells were lysed in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 25 mM glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM sodium fluoride, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS for 30 min at 4 °C. The cell lysates were centrifuged at 12,000 ×g and 4 °C for 20 min. The soluble fraction was incubated with appropriate antibodies against the indicated protein kinase for 3 h at 4 °C. The immunocomplexes formed were incubated with protein A–agarose beads for 1 h at 4 °C, after which they were pelleted by centrifugation. The immunopellets were rinsed 3 times with lysis buffer and 2 times with 20 mM HEPES (pH 7.4). Immunocomplex kinase assays were conducted by incubating the immunopellets with 2 μg of substrate protein in 20 μl of reaction buffer containing 0.2 mM sodium orthovanadate, 10 mM MgCl2, 2 μCi [32P]ATP, and 20 mM HEPES (pH 7.4) for 30 min at 30 °C. The phosphorylated substrate was quantified using FLA7000 phosphorimager (Fuji) [13].

2.6. Immunofluorescence assay The cells were fixed with 4% paraformaldehyde in phosphatebuffered saline (PBS), and then permeabilized with 0.1% Triton-X 100 in PBS. Cells were blocked in 1% BSA in PBS. Primary antibodies were used as indicated at a dilution of 1:100. After incubation with the primary antibodies, the cells were washed three times in PBS. Alexa Fluor®488 (Invitrogen) or Alexa Fluor®532 (Invitrogen) conjugated anti-mouse secondary antibody (1:100) was added, and then the DNA dye TO-PRO®-3 Iodide was used for visualize cell nuclei. The stained cells were evaluated for localization via confocal microscopy (Leica TCS SPE). Each image is a single Z section at the same cellular level. The final images were obtained and analyzed using confocal microscopy with LAS AF software (Leica). Scale bars represent 25 μm as indicated [13].

2.3. Coimmunoprecipitation assay

2.7. Subcellular fractionation

HEK293 cells were transfected with the indicated expression vectors for 48 h and were washed with ice-cold PBS. The cells were lysed in RIPA buffer, and the cell lysates were centrifuged at 12,000 ×g and 4 °C for 20 min. The supernatant obtained was incubated with indicated antibodies, followed by incubation with 20 μl protein A–agarose beads. The beads were washed 3 times with ice-cold PBS and were boiled in Laemmli buffer. The pellets were heated for 5 min at 95 °C and were resolved by performing SDS-PAGE. Immunoblotting was conducted using indicated antibodies.

After 48 h of transfection with the indicated expression vectors, the cultured HEK293 cells were rinsed with ice-cold PBS and were resuspended in ice-cold buffer A containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.05% NP-40, and 2 μg/ml each of leupeptin and aprotinin. After 15-min incubation on ice, the suspended cells were harvested by centrifuging at 800 × g and 4 °C for 10 min to separate the cytoplasm from the nucleus. The supernatant was used as the cytosolic fraction. The pellet was homogenized in buffer B containing 5 mM HEPES (pH 7.9), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 26% glycerol. The pellet was then lysed and sonicated on ice to release nuclear proteins. After 30-min incubation on ice, the homogenates were centrifuged at 14,000 ×g and 4 °C for 20 min. The resultant supernatant was used as the nuclear fraction. Proteins in the nuclear and cytosolic fractions were quantified using Bradford assay, and 20 μg of each fraction was analyzed by western blotting [13].

2.4. GST-pull down and in vitro binding assay Recombinant GST–NOTCH1-IC and GST–PAK1 were expressed in Escherichia coli strain BL21 by using a pGEX system as indicated [13]. The recombinant GST–NOTCH1-IC was purified using glutathione

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2.8. Wound healing migration assay HeLa cells were cultured in 6-well plates and were transfected with the indicated expression vectors. After 48 h, cell monolayers were wounded using a sterile 200 μl pipette tip and were washed with the culture medium. The wound-healing images were taken at 12 hour intervals using a microscope system. The cell movement velocity was determined by measuring the distance of sketched area at different time points, using Image J program. 2.9. NOTCH1 knockdown To generate scrambled short hairpin RNA (shRNA) and shRNA targeting NOTCH1, double-stranded oligonucleotides targeting the specific genes were cloned into BglII and HindIII digested pSUPER vector (Oligoengine). The shRNA targeting NOTCH1 was generated using the following target sequence: 5′ AAG TGT CTG AGG CCA GCA AGA 3′. Sequence specificity was determined by performing BLAST searches for uniqueness and was validated using a scrambled shRNA sequence (5′ AAC AGT CGC GTT TGC GAC TGG 3′) that did not match any known mammalian sequence in GenBank. HEK293 or HeLa cells were transfected with the scrambled shRNA or NOTCH1 shRNA by using Lipofectamine-2000, according to the manufacturer's instructions [13]. 2.10. RNA isolation and RT-PCR and Q-PCR Total RNA was isolated using Trisol reagent (Invitrogen, Camarillo, CA, USA). All samples were treated with RNase-free DNase I (Takara, Tokyo, Japan) at 37 °C for 30 min under the following conditions: 20 mg of RNA, 40 mM Tris–HCl, 8 mM MgCl2, 5 mM dithiothreitol, 0.4 U/ml of RNase inhibitor (Promega, Madison, WI, USA), and 10 units of DNase I in a volume of 50 ml. Then a phenol/chloroform extraction was performed and the RNA precipitated. The total RNA (2 μg) was used to synthesize cDNA with Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions; contaminating DNA was removed by treating the samples with RNasefree DNase. The primers used in PCR analysis for human VEGF were: forward, 5′-ctacctccaccatgccaagt-3′; and reverse, 5′-gcagtagctgcgctgataga3′. GAPDH was used as the control in PCR analysis. 2.11. Statistical analysis All graphs were produced using SigmaPlot software (SYSTAT). Statistical comparisons of means were performed using unpaired Student's 2-tailed t-test for 2 data sets. For all statistical tests, a p value less than 0.05 was considered significant. For all experiments with error bars, standard deviation (SD) was calculated to indicate the variation in experiments and their associated data. All values are represented as mean ± SD. 3. Results 3.1. NOTCH1-IC physically interacts with PAK1 in intact cells To investigate the role of NOTCH1-IC in PAK1 signaling, we examined whether these two proteins interacted physically in intact cells. HEK293 cells were cotransfected with vectors expressing GFP–PAK1 and Myc–NOTCH1-IC. The cells were lysed, and their lysates were used for co-immunoprecipitation (Co-IP) analysis by using anti-Myc antibody. Immunoblotting of the anti-Myc immunoprecipitates by using an anti-GFP antibody showed that GFP–PAK1 was physically associated with Myc–NOTCH1-IC within the cells (Fig. 1A). Next, we examined whether endogenous NOTCH1-IC and PAK1 interacted in intact cells. Immunoblotting of NOTCH1-IC immunoprecipitates with an anti-PAK1 antibody indicated that these two endogenous proteins were physically associated in intact cells (Fig. 1B). Furthermore we

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showed the direct interaction between PAK1 and NOTCH1-IC by using bacterially purified GST–PAK1 and GST–NOTCH1-IC in vitro (Fig. 1C and D). NOTCH1-IC has a CDC domain that includes a RAM domain, 7 ankyrin (ANK) repeats, a polyglutamine (OPA) domain, and a proline-, glutamate-, serine-, and threonine-rich sequence (PEST) domain. We examined whether any of these domains were involved in the interaction between NOTCH1-IC and PAK1. For this, we cotransfected HEK293 cells with vectors expressing GFP–PAK1 and 3 Flag–NOTCH1-IC deletion mutants Flag–NOTCH1-IC-RAM-ANK (CDC domain), Flag–NOTCH1-ICOPA, and Flag–NOTCH1-IC-PEST. The cells were then lysed, and the proteins were coimmunoprecipitated using specific antibodies. Our results showed that PAK1 could bind to NOTCH1-IC-PEST but not to NOTCH1IC-RAM-ANK and NOTCH1-IC-OPA (Fig. 1E), indicating that the PEST domain was critical for the interaction of NOTCH1-IC with PAK1. Moreover, we examined the interaction between NOTCH1-IC with PAK1 and its isoforms (PAK2-4). NOTCH1-IC showed a most strong interaction with PAK1 but a weaker interaction with PAK4. PAK2 and PAK3 slightly interacted with NOTCH1-IC. These results suggest that PAK1 is an important effector of NOTCH1 signaling among PAKs (Fig. 1F). 3.2. NOTCH1-IC decreases PAK1-mediated ILK1 phosphorylation To determine whether the interaction of NOTCH1-IC with PAK1 played a role in PAK1 signaling, we performed immunocomplex kinase assays. We observed that ILK1 phosphorylation by PAK1 was markedly inhibited in NOTCH1-IC-expressing cells (Fig. 2A). To determine whether NOTCH1-IC modulated ILK1 phosphorylation by PAK1, we treated HEK293 cells with EGF and examined the role of NOTCH1-IC in EGFinduced ILK1 phosphorylation by PAK1. We observed that EGFinduced ILK1 phosphorylation by endogenous PAK1 was remarkably inhibited by NOTCH1-IC (Fig. 2B). To investigate the significance of endogenous NOTCH1-IC in PAK1 signaling, we inhibited endogenous NOTCH1 signaling by using DAPT, a gamma-secretase inhibitor. DAPT efficiently blocked presenilin/gamma-secretase complex, consequently preventing the activation of the NOTCH1 signaling pathway. DAPTinduced NOTCH1 inhibition altered ILK1 phosphorylation by PAK1. ILK1 phosphorylation by PAK1 was upregulated in DAPT-treated cells (Fig. 2C). ILK1 phosphorylation by PAK1 was further evaluated by knocking down NOTCH1 by using a specific shRNA. Cells transfected with NOTCH1 shRNA showed lower NOTCH1-IC expression than cells transfected with control shRNA. As expected, ILK1 phosphorylation by PAK1 was upregulated in cells transfected with NOTCH1 shRNA (Fig. 2D). These data suggested that the level of NOTCH1-IC expression affected the PAK1 signaling pathway. We next examined the effects of genetic suppression of endogenous NOTCH1-IC on EGF-induced endogenous PAK1 activation in MEFs from PS1,2-knockout (PS1,2−/−) mice, which showed impaired gamma-secretase-mediated processing of endogenous NOTCH1-IC, compared with MEFs from wild-type (PS1,2+/+) mice. Treatment with EGF more intensively increased the level of ILK1 phosphorylation by PAK1 in PS1,2−/− MEFs compared with that in PS1,2+/+ MEFs (Fig. 2E). These results suggested that NOTCH1-IC regulates ILK1 phosphorylation induced by PAK1. To confirm whether NOTCH1-IC inhibits the ILK1 phosphorylation by reducing PAK1 activity, we tested that NOTCH1-IC affects the phosphorylation level of PAK1 at Thr423 by using a specific antibody in HeLa cells. Phosphorylation of Thr423 in the activation loop of PAK1 is important for PAK1 activation catalyzed by EGF at the plasma membrane in a PDK1 dependent manner [22]. But NOTCH1-IC did not significantly regulate the phosphorylation of PAK1 Thr423 induced by EGF (Fig. 2F). These results suggested that NOTCH1-IC regulates ILK1 phosphorylation by interacting with PAK1, but not by direct regulation of PAK1 activation. 3.3. NOTCH1-IC inhibits PAK1-induced ILK1 signal pathway Previous reports have shown that ILK1 phosphorylates GSK-3beta at Ser9 and that the phosphorylation of GSK3β inhibits its activity [23,24].

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Fig. 1. NOTCH1-IC binds to PAK1 in intact cells. (A) Co-IP assay in HEK293 cells transiently transfected with the vector expressing GFP–PAK1 or Myc–NOTCH1-IC. Expression of PAK1 or NOTCH1-IC was analyzed by immunoblotting with anti-GFP or anti-Myc monoclonal antibody, respectively. (B) Endogenous Co-IP assay using anti-NOTCH1-IC and anti-PAK1 antibodies. Cell lysates were immunoblotted with anti-PAK1 and anti-NOTCH1-IC antibodies as a control. (C) GST-pull down assay by using GST or GST–NOTCH1-IC immobilized on GSH–agarose beads in HEK293 cells transfected with the vector expressing GFP–PAK1 or an empty vector. The input represents 1% of the cell lysate before the pull down assay. (D) In vitro binding assay by using GST–NOTCH1-IC eluted from GSH- agarose beads and GST–PAK1 immobilized on GSH–agarose beads. Purified proteins were stained with Coomassie brilliant blue R250 dye as a control. (E) Co-IP assay of PAK1 and NOTCH1-IC domains in HEK293 cells transfected with vectors expressing Flag–NOTCH1-IC-RAM-ANK, Flag–NOTCH1-IC-OPA, Flag– NOTCH1-IC-PEST, and GFP–PAK1. The cell lysates were also immunoblotted using anti-Flag and anti-GFP antibodies. (F) Co-IP assay of NOTCH1-IC and PAK1 and its isoforms in HEK293 cells transfected with Flag-NOTCH1-IC and Myc-PAK1-4. The cell lysates were also immunoblotted using anti-Flag and anti-Myc antibodies. Results of 3 independent experiments are presented as mean ± SD.

To determine whether NOTCH1-IC modulated the kinase activity of ILK1, we examined the role of NOTCH1-IC in HEK293 cells transiently transfected with ILK1. GSK-3beta phosphorylation by ILK1 was markedly decreased in NOTCH1-IC-expressing cells (Fig. 3A). Moreover, expression of NOTCH1-IC remarkably decreased EGF-induced stimulation of GSK-3beta phosphorylation by ILK1 (Fig. 3B). These data showed that NOTCH1-IC overexpression blocked EGF-induced ILK1 activation. ILK1 activation was further evaluated by inhibiting NOTCH1 by using DAPT. We observed that DAPT-induced NOTCH1 inhibition upregulated ILK1 activation and increased GSK-3beta phosphorylation by ILK1 (Fig. 3C). The role of endogenous NOTCH1 in the ILK1 signaling pathway was further evaluated by knocking down NOTCH1 by using a specific shRNA (Fig. 3D). GSK-3beta phosphorylation by ILK1 was upregulated in cells transfected with NOTCH1 shRNA. These data suggested that the level of NOTCH1 expression affected the ILK1 signaling pathway. We next examined the effects of genetic suppression of endogenous NOTCH1-IC on EGF-induced endogenous ILK1 activation in PS1,2−/− and PS1,2+/+ MEFs. Treatment with EGF remarkably increased GSK-3beta phosphorylation by endogenous ILK1 in PS1,2−/− MEFs compared with that in PS1,2+/+ MEFs (Fig. 3E). We next examined whether ILK1 activity can be regulated by the expression of NOTCH1-IC. Previously it has been reported that ILK1 was activated by phosphorylation at Ser246 and Thr173, and that these residues were phosphorylated by PAK1 [20]. We used phosphorylation-specific antibody to examine the phosphorylation of ILK1 Thr173. PAK1 markedly increased the phosphorylation of ILK1 at Ser173. But PAK1-induced phosphorylation of ILK1 Thr173 was reduced by NOTCH1-IC expression (Fig. 3F). Previous studies have shown that GSK-3beta degrades multifunctional oncogenic β-catenin

through phosphorylation in cytoplasm and that the phosphorylation and accumulation of β-catenin are important for tumorigenesis and cancer progression [25,26]. Because a Rac1/PAK1 cascade also controls β-catenin Ser675 phosphorylation and full activation in colon cancer cells [27], we tested that β-catenin Ser675 phosphorylation is regulated by NOTCH1-IC in HeLa cells. Phosphorylation at Ser675 of β-catenin was stimulated by PAK1. But expression of NOTCH1-IC remarkably inhibited PAK1-induced β-catenin Ser675 phosphorylation (Fig. 3G). Recently, it has been showed that ILK1 is responsible for angiogenesis by upregulating VEGF expression [16,28]. In line with these previous reports, we examined the mRNA level of VEGF in HeLa cells expressing PAK1 and NOTCH1-IC. The mRNA level of VEGF was increased by PAK1, and this PAK1-mediated induction of VEGF suppressed by NOTCH1-IC (Fig. 3H). These results suggested that NOTCH1-IC regulates the ILK1 and its downstream signaling pathway in a PAK1 dependent manner. 3.4. NOTCH1-IC disrupts the interaction between PAK1 and ILK1 by interacting with PAK1 in intact cells To investigate whether the kinase activity of PAK1 influenced its interaction with NOTCH1-IC, we performed Co-IP analysis in HEK293 cells expressing PAK1 variants, i.e., a constitutively active (CA) T423E mutant and a kinase-deficient (KD) K299R mutant. Interestingly, the interaction between NOTCH1-IC and PAK1 decreased in cells expressing PAK1-CA but rather increased in cells expressing PAK1-KD, in comparison with the interaction in cells expressing PAK1-WT (Fig. 4A). To ascertain this result, we performed Co-IP analysis in cells treated with PAK1 inhibitor, IPA3. The interaction between PAK1 and NOTCH1-IC was

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Fig. 2. NOTCH1-IC decreases ILK1 phosphorylation by PAK1. (A) Immunocomplex kinase assay of PAK1 in HEK293 cells transiently transfected with the vector expressing GFP–PAK1 or Myc–NOTCH1-IC. (B) Immunocomplex kinase assay of PAK1 in HEK293 cells transiently transfected with the vector expressing Myc–NOTCH1-IC. The cells were serum-starved for 24 h then treated with 100 nM EGF for 20 min at 37 °C. (C) Immunocomplex kinase assay of PAK1 in HEK293 cells transiently transfected with the vector expressing GFP–PAK1. After 42 h, the cells were treated with 1 μM DAPT for 6 h at 37 °C. (D) Immunocomplex kinase assay of PAK1 in HEK293 cells transfected with NOTCH1 shRNA (pSUPER-shNOTCH1) or control shRNA (pSUPER-shCon) and GFP–PAK1. (E) Immunocomplex kinase assay of PAK1 in PS1,2+/+ and PS1,2−/− MEFs treated with 100 nM EGF for 20 min at 37 °C after serum starvation. (A–E) Immunocomplex PAK1 kinase assays were conducted using His–ILK1 as the substrate. (F) Western blot analysis by using antibody against PAK1 phosphorylated at Thr423 in HeLa cells expressing GFP–PAK1 and Myc–NOTCH1-IC and treated with 100 nM EGF for 20 min at 37 °C after serum starvation. (A–F) Cell lysates were also immunoblotted using the indicated antibodies. Results of 3 independent experiments are presented as mean ± SD.

elevated by PAK1 inhibition (Fig. 4B). This result showed that the inactivated PAK1 more strongly interacts with NOTCH1-IC. Basing on the fact that NOTCH1-IC inhibited ILK1 activation through PAK1 but it did not directly regulate PAK1 activation, we speculated that NOTCH1-IC is involved in the formation of PAK1 and ILK1 complex. CoIP analysis indicated that HA–ILK1 was associated with GFP–PAK1 in the transfected cells. However, the association between ILK1 and PAK1 was disrupted in the presence of NOTCH1-IC (Fig. 4C). A previous study has shown that ILK1 interacts with NOTCH1-IC and decreases its stability through Fbw7 ubiquitin ligase-mediated degradation via the ubiquitin–proteasome pathway [13]. To characterize the interaction between NOTCH1-IC and ILK1 or PAK1, Co-IP analysis was performed using HEK293 cells cotransfected with vectors expressing V5–ILK1, GFP–PAK1, and Myc–NOTCH1-IC. We observed that NOTCH1-IC interacted with ILK1 and PAK1 but did not form a trimeric complex (Fig. 4D). These results indicated that NOTCH1-IC interacts with PAK1 or ILK1 and disrupts the interaction between PAK1 and ILK1. 3.5. NOTCH1-IC alters the localization of PAK1 and inhibits PAK1-mediated cell migration It has been reported that ILK1 is a PAK1 substrate that undergoes phosphorylation-dependent shuttling between the nucleus and the cytoplasm [20]. Previously, we also reported that NOTCH1-IC enhances the nuclear accumulation of ILK1 [13]. To determine the significance of the PAK1 activation in the localization of ILK1, we performed immunofluorescence analysis by using HEK293 cells transfected with the vector expressing V5–ILK1, Myc–PAK1-CA, or Myc–PAK1-KD. As expected, we found that ILK1 and PAK1 were predominantly localized in the cytoplasm (80% stained cells). We then assessed the subcellular localization of ILK1 in HEK293 cells transfected with the vector

expressing PAK1-CA or PAK1-KD. In cells expressing PAK1-CA or PAK1-KD, ILK1 was localized in the cytoplasm (70% stained cells) or both in the cytoplasm and the nucleus (80% stained cells), respectively (Fig. 5A). Previous studies have established that PAK1 translocates into the nucleus upon EGF stimulation and that EGF-induced nuclear import induces activation of PAK1 [29]. In cells treated with EGF, PAK1 and ILK1 localized both in the cytoplasm and in the nucleus. Interestingly, PAK1 and ILK1 translocated into the nuclear under the treatment of IPA3 (Fig. 5B). These results implied that the nuclear-cytoplasmic shuttling of PAK1 contributed to both activation and inactivation of PAK1. Next, we determined whether NOTCH1-IC influenced the alteration of ILK1 localization by PAK1. In cells expressing PAK1, ILK1 was localized in the cytoplasm. However, in cells expressing NOTCH1-IC, ILK1 was localized in the nucleus and the cytoplasm similar to that observed in cells expressing PAK1-KD (Fig. 5C). These results suggested that NOTCH1-IC affected the alteration of ILK1 localization by PAK1. NOTCH1-IC is predominantly located in the nucleus while PAK1 is predominantly located within focal adhesions in the cytoplasm. Therefore, we conducted immunofluorescence analyses to determine whether PAK1 was colocalized with NOTCH1-IC. To identify the cellular localization of PAK1 and NOTCH1-IC, we transfected HEK293 cells with the vector expressing GFP–PAK1 or Myc–NOTCH1-IC. As expected, PAK1 and NOTCH1-IC were predominantly localized in the cytoplasm and the nucleus, respectively. We then assessed the subcellular localization of PAK1 in HEK293 cells expressing NOTCH1-IC and observed that PAK1 was localized in both the nucleus and the cytoplasm (Fig. 5D). To further investigate the nuclear accumulation of PAK1, we examined the nuclear and cytoplasmic localization of PAK1 in the presence or absence of NOTCH1-IC (Fig. 5E). EGF induced the nuclear accumulation of both of PAK1 and phosphorylated PAK1 at Thr423. But NOTCH1-IC did not affect the nuclear translocation of phosphorylated PAK1. We next

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Fig. 3. NOTCH1-IC inhibits ILK1 signal pathway induced by PAK1. (A) Immunocomplex kinase assay of ILK1 in HEK293 cells expressing HA–ILK1 or Myc–NOTCH1-IC. (B) Immunocomplex kinase assay of ILK1 in HEK293 cells transiently transfected with the vector expressing Myc–NOTCH1-IC. The cells were serum-starved for 24 h then treated with 100 nM EGF for 20 min at 37 °C. (C) Immunocomplex kinase assay of ILK1 in HEK293 cells transiently transfected with the vector expressing V5–ILK1. After 42 h, the cells were treated with 1 μM DAPT for 6 h at 37 °C. (D) Immunocomplex kinase assay of ILK1 in HEK293 cells transfected with NOTCH1 shRNA (pSUPER-shNOTCH1) or control shRNA (pSUPER-shCon) with V5–ILK1. (E) Immunocomplex kinase assay of ILK1 in PS1,2+/+ and PS1,2−/− MEFs treated with 100 nM EGF for 20 min at 37 °C after serum starvation. (A–E) Immunocomplex ILK1 kinase assays were conducted using GST–GSK-3beta as the substrate. (F) Western blot analysis by using antibody against ILK1 phosphorylated at Thr173 in HeLa cells expressing HA-ILK1, GFP-PAK1 and Myc-NOTCH1-IC. (G) Western blot analysis by using antibody against β-catenin phosphorylated at Ser675 in HeLa cells expressing GFP–PAK1 and Myc–NOTCH1-IC. (A–G) The cell lysates were also immunoblotted with the indicated antibodies. (H) Real-time q-PCR using primers detecting VEGF in HeLa cells expressing GFP–PAK1 and Myc–NOTCH1-IC. Results of 3 independent experiments are presented as mean ± SD. *P b 0.01.

examined whether localization of β-catenin can be modulated by NOTCH1-IC–PAK1 pathway. In the presence of EGF stimulation, nuclear localization of β-catenin was stimulated by PAK1. But, under the NOTCH1-IC expression, PAK1 translocated into nuclear and consequently nuclear accumulation of β-catenin was decreased (Fig. 5F). These data indicated that NOTCH1-IC altered the localization of PAK1 leading to the inhibition of its downstream pathway. To explore the potential role of NOTCH1-IC in PAK1-mediated cell migration, we measured the migration capacity of cancer cells (Fig. 5G). The difference in the migration distance between control cells and cells expressing NOTCH1-IC was compared at 0, 12, 24 and 36 h. HeLa cells expressing NOTCH1-IC showed reduced motility. Quantification of migration distances showed that HeLa cells expressing NOTCH1-IC showed decreased migration compared with cells transfected with pCS2 control vector. In contrast, HeLa cells expressing PAK1 showed increased migration compared with control cells. However, the migration capacity of cells coexpressing PAK1 and NOTCH1-IC was significantly

decreased. These results indicated that NOTCH1-IC reduced PAK1mediated cell migration.

4. Discussion Recently, several groups reported the tumor suppressive role of the NOTCH1 signaling pathway [30]. The tumor suppressive role of NOTCH1 was first reported by genetic experiments involving mouse skin [31] and human keratinocytes [32]. A recent study reported the tumor suppressive role of NOTCH1 by identifying loss-of-function mutations in myeloid cancer [33], squamous cell carcinomas of the lungs [34] and the head and neck [35,36], and human bladder cancer [37]. However, the mechanisms underlying the regulation of cancer by the NOTCH1 signaling pathway are still unclear. The results of the present study allow us to understand the molecular basis of the tumor suppressive role of NOTCH1-IC.

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Fig. 4. NOTCH1-IC inhibits the interaction between PAK1 and ILK1. (A) Co-IP assay in HEK293 cells expressing Myc–PAK1, Myc–PAK1-CA, Myc–PAK1-KD, and Flag–NOTCH1-IC. (B) Co-IP assay in HEK293 cells transfected with Myc–PAK1 and Flag–NOTCH1-IC and treated with 10 μM IPA3 for 4 h at 37 °C after serum starvation. (C) Co-IP assay in HEK293 cells expressing HA– ILK1, GFP–PAK1, and Myc–NOTCH1-IC. (D) Co-IP assay in HEK293 cells expressing V5–ILK1, GFP–PAK1, and Myc–NOTCH1-IC. (A–D) The cell lysates were also immunoblotted with the indicated antibodies. Results of 3 independent experiments are presented as mean ± SD.

We have previously reported that ILK1 controls NOTCH1 signaling by decreasing its stability through Fbw7 ubiquitin ligase-mediated degradation [13]. We have also reported the colocalization of ILK1 and NOTCH1IC in both the nucleus and the cytoplasm in HaCaT and melanoma cells [13]. We found that NOTCH1-IC facilitates the nuclear accumulation of ILK1 under certain conditions. Further ILK1 negatively regulates NOTCH1 signaling in a GSK-3beta-independent manner [13]. A previous study indicated that PAK1 has a role as an upstream kinase of ILK1 signaling. PAK1 phosphorylates ILK1 and regulates its subcellular localization in a phosphorylation-dependent manner [20]. PAK1-mediated ILK1 phosphorylation controls ILK1-regulated cellular processes. In this study, we provide evidence that NOTCH1-IC is a new regulator of PAK1 that directly interacts with PAK1 and regulates its shuttling between the nucleus and the cytoplasm. In addition, we found that NOTCH1-IC affected PAK1 nuclear localization consequently leading to nuclear translocation of ILK1 and that the nuclear localization of PAK1-CA was reduced. This is consistent with that PAK1-CA slightly bound to NOTCH1-IC compare with PAK1-WT, suggesting the interaction with NOTCH1-IC might link to the translocation of PAK1 into the nuclear. Previous data and our finding implicated that NOTCH1-IC might play an important role in the regulation of subcellular localization of PAK1 and ILK1. PAK1 is significantly overexpressed in some cancers such as ovarian, breast, and bladder cancers [6]. Several functional studies have reported that PAK1 is highly associated with cell transformation and tumorigenesis, as evidenced by the development of premalignant lesions and tumor formation due to PAK1 overexpression and hyperactivation [7,8]. Recent studies have shown that the functions of PAK1 may be regulated by its intracellular location. However, little is known about the regulator that contributes to the localization of PAK1. A previous study was the first to show the nuclear localization of PAK1 upon growth factor stimulation in MCF-7 breast cancer cells [12]. EGFstimulated nuclear localization of PAK1 highlights the potential novel

functions of PAK1 in the nucleus that might be important for growth factor signaling [38]. Several studies have suggested that PAK1 could play a crucial role in cancer progression [39–45]. Previous studies have reported that Snail protein interacts with PAK1 in the cytoplasm [46–49]. These studies have also mentioned that it is unclear whether PAK1 and Snail translocate into the nucleus together or individually and whether the interaction between these two proteins is responsible for EMT induction through the suppression of E-cadherin expression. It has been reported that ILK1 contributes to tumor progression by increasing the expression of VEGF via the activation of PKB/Akt and HIF1alpha, thereby stimulating angiogenesis [16]. In this study, PAK1 upregulated the mRNA level of VEGF. But, PAK1-induced VEGF level was reduced under overexpression of NOTCH1-IC. Overexpressed PAK1 contributes to beta-catenin signaling through S675 phosphorylation and accumulation in colon cancer cells [27]. In our experiments PAK1-induced phosphorylation and nuclear accumulation of beta-catenin were inhibited by NOTCH1-IC. Our data have demonstrated that NOTCH1 functions as tumor suppressor via inhibition of PAK1-ILK1 pathway. In conclusion, the present paper provides the first evidence of a novel interaction between NOTCH1-IC and PAK1. We also observed that NOTCH1-IC increased the nuclear localization of PAK1 and consequently inhibited ILK1 signaling pathway (Fig. 6). This observation places NOTCH1 in an important position in regulation of PAK1 pathway. Since NOTCH1 and PAK1 signaling play the crucial part in tumor formation and invasiveness, this study provide important new insights into how these pathways converge to regulate the cell migration that is critical for cancer invasion and metastasis. Transparency document The Transparency document associated with this article can be found, in online version.

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Fig. 5. NOTCH1-IC alters the localization of PAK1 and inhibits PAK-mediated cell migration. (A) Immunofluorescence staining of HEK293 cells transiently transfected with expression vectors encoding V5–ILK1, Myc–PAK1-CA, or Myc–PAK1-KD. (B) Immunofluorescence staining of HEK293 cells transfected with V5–ILK1 and GFP–PAK1 and treated with 10 μM IPA3 for 4 h at 37 °C after serum starvation. (C) Immunofluorescence staining of HEK293 cells expressing V5–ILK1, GFP–PAK1 and Myc–NOTCH1-IC. (D) Immunofluorescence staining of HEK293 cells expressing GFP–PAK1 and Myc–NOTCH1-IC. (A–D) The overlays are shown in the right panels, and the color yellow denotes colocalization. Quantification of the number of cells showing PAK1 or ILK1 in the nucleus is shown on the right side of the panel. *P b 0.001, **P b 0.01. (E) Cytosolic (C) and nuclear (N) fractionations of HEK293 cells expressing GFP–PAK1 and Myc–NOTCH1-IC. The cell lysates were also immunoblotted using anti-GFP, anti-Myc, anti-Lamin, and anti-β-actin antibodies as a control. Immunofluorescence staining of HEK293 cells expressing GFP–PAK1 and Myc–NOTCH1-IC. (F) Immunofluorescence staining of HEK293 cells expressing GFP–β-catenin, Myc–PAK1 and Flag–NOTCH1-IC. The overlays are shown in the right panels, and the color yellow denotes colocalization. Quantification of intensity of β-catenin is shown on the right side of the panel. *P = 0.002. (G) Photomicrographs from a wound-healing assay of HeLa cells transfected with the expressing NOTCH1-IC and PAK1. The wound closure was quantified at every 12 h post-wound by measuring the remaining unmigrated area using Image J. Results of 3 independent experiments are presented as mean ± SD. *P = 0.002. Results of 3 independent experiments are presented as mean ± SD.

Acknowledgments We thank R. Kopan (Washington University Medical School) for the NOTCH1 constructs, B. Maria Carla Parrini (Institut Curie) and Jianxin Gu (Fudan University) for the PAK1 constructs, S. Dedhar

(British Columbia Cancer Agency) for the ILK1 constructs, Eek-hoon Jho (The University of Seoul) for the β-catenin antibody, and Chi Dae Kim (Pusan National University) for the phospho-β-catenin Ser675 specific antibody. This research was supported by Basic Science Research Program through the National Research Foundation

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Fig. 6. NOTCH-IC regulates PAK1 signaling through altering its localization. PAK1 phosphorylates ILK1 in the cytoplasm, and regulates cytoskeletal remodeling, cell motility, proliferation, apoptosis, transformation, and invasion. The interaction of NOTCH1-IC with PAK1 inhibits the kinase activity of ILK1. NOTCH1-IC disrupts the interaction of PAK1 with ILK1 and alters the localization of PAK1. ILK1 interacts with NOTCH1-IC and translocates in the nucleus [13]. This inhibitory effect of NOTCH1-IC on the PAK1 signaling pathway is mediated by the binding of NOTCH1-IC to PAK1 and by the inhibition of its localization to the cytoplasm. Results of 3 independent experiments are presented as mean ± SD.

of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2014R1A4A1003642).

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