Plk1-Mediated Phosphorylation of TSC1 Enhances the Efficacy of Rapamycin

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Author Manuscript Published OnlineFirst on March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-3046 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Plk1-mediated phosphorylation of TSC1 enhances the efficacy of rapamycin

Zhiguo Li1, Yifan Kong1, Longzhen Song1, Qian Luo1, Jinghui Liu1, Chen Shao1, Xianzeng Hou1, and Xiaoqi Liu1,2,*

1Department of Biochemistry, Purdue University, West Lafayette, IN 47907 2Center for Cancer Research, Purdue University, West Lafayette, IN 47907

Running Title: Plk1 activates mTORC1

*To whom correspondence should be addressed: Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47907 Tel: 765-496-3764; Fax: 765-494- 7897; Email: [email protected].

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-3046 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

The AKT/TSC/mTOR axis is an important pathway controlling cell growth, survival and proliferation in response to extracellular cues. Recently, it was reported that AKT activity fluctuates across the cell cycle. However, it remains unclear whether downstream targets of AKT are also regulated by the cell cycle. Here we report that mTORC1 activity inversely correlates with AKT activity during the cell cycle. Mechanistically, Plk1 phosphorylation of TSC1 at S467 and S578 interfered with TSC1/TSC2 binding, destabilized TSC1, promoted dissociation of the

TSC complex from the lysosome, and eventually led to mTORC1 activation. Tumors derived from cancer cells expressing the TSC1-S467E/S578E mutant exhibited greater sensitivity to rapamycin than those expressing WT TSC1. Collectively, our data support a model in which

Plk1, instead of AKT, regulates the TSC/mTORC1 pathway during mitosis, eventually regulating the efficacy of rapamycin.

Introduction

The phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)

pathway is highly conserved and its activation is tightly controlled via a multistep process (1).

Upon stimulation with growth factors, PI3K is activated by receptor tyrosine kinases (RTKs) to

convert phosphatidylinositol 3,4-bisphosphate (PIP2) to phosphoinositide 3,4,5-trisphosphate

(PIP3). Phosphoinositide-dependent kinase 1 (PDK1) and AKT bind to PIP3, allowing PDK1 to

access and phosphorylate T308 in AKT and thereby activate AKT (2,3). AKT can subsequently

phosphorylate and inactive TSC2 by inducing its release from the lysosome (4-6). The lysosomal, small Ras-like GTPase, Rheb, which is regulated by the TSC complex, activates mTORC1 (7). mTORC1 substrates include the eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and ribosomal protein S6 kinase 1 (S6K1), which, in turn, phosphorylates the ribosomal protein S6 to promote protein synthesis (8). In addition, the PI3K/AKT/mTOR

pathway is important for the regulation of cell cycle progression (9-11). Consistent with these observations, it was reported that AKT activity is fluctuated across the cell cycle (12). Further, it

was shown that TSC1 is threonine-phosphorylated during nocodazole-induced G2/M arrest (13).

A significant number of studies have pointed to failure in various critical mitotic events as a

cause of aneuploidy in tumors (14-16). The regulation of proper mitotic progression is

predominantly controlled by several conserved serine/threonine kinases, such as Cdk1, Plk1,

and aurora kinases (17). It has been documented that Plk1 is involved in almost every step of

mitosis (18). Thus, it is not surprising that Plk1 is overexpressed in many cancer types (19-22).

More importantly, recent studies have also linked Plk1 with other cancer-associated pathways,

such as DNA damage response (23-28), p53 and the PI3K/AKT/mTOR pathway (29,30). For

example, a crosstalk between Plk1 and the p53 tumor suppressor has been described (31,32). In

another study, Plk1 elevation was shown to cause PTEN inactivation (33). In line with this

observation, Plk1-associated activity was demonstrated to contribute to the low-dose arsenic-

mediated metabolic shift via activation of the PI3K/AKT/mTOR pathway (34). Furthermore, it

was reported that the phosphorylated form of TSC1 interacts with Plk1, and that the interaction

between Plk1 and the TSC1/TSC2 complex regulates local mTOR activity (35).

Here we show that the activity of mTORC1 is correlated with Plk1 activity and inversely

correlated with AKT activity during cell cycle. Mechanistically, Plk1 directly phosphorylates

TSC1 at S467 and S578. We show that Plk1 phosphorylation of TSC1 leads to inactivation of the

TSC1 complex, thus activation of mTORC1 in mitosis, and that cells expressing the hyper- phosphorylated form of TSC1 have apparent mitotic defects, but with a higher sensitivity to rapamycin. Together, these observations and others’ previous findings support a new working

model in which AKT activates the TSC/mTORC1 axis in response to growth factors in

interphase, whereas Plk1, instead of AKT, regulates the TSC/mTORC1 pathway during mitosis.

Materials and Methods

Cell culture, Transfections, Constructs, and RNAi

The cell lines were obtained from ATCC. The cell lines were authenticated by ATCC and

tested for absence of mycoplasma contamination (MycoAlert, Lonza). The cells used in the

experiments were within 10 passages from thawing. HeLa and HEK293T cells were cultured in

DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100

units/ml streptomycin at 37˚C in 8% CO2. PC3 cells were cultured in F-12K medium supplemented with 10% FBS. After cells were transfected with plasmids with Liopfectamine

(Invitrogen) for 48 h, cells were harvested for IB or IF. myc-TSC1 and HA-TSC2 expression plasmids were obtained from Addgene. Various TSC1 mutants were created with the

QuikChange site-directed mutagenesis kit (Stratagene). The identities of all plasmids were confirmed by sequencing.

Cell synchronization by mitotic shake-off and double thymidine block (DTB)

To arrest cells at mitosis, cells growing in 100 mm dishes were treated with 100 ng/ml

nocodazole for 24 h. After floating cells were collected into 50 ml tubes containing 10 ml of pre-

cold phosphate-buffered saline (PBS), additional mitotic cells were collected by shaking off

dishes for 10 min on ice. The procedure was repeated one more time. Cells were spun down at

2000 rpm for 2 min, re-suspended in pre-cold 20 ml of PBS and kept on ice for 30 min. The

procedure was repeated 2 more times to completely remove nocodazole. After cells were

checked microscopically to ensure they are in good condition, cells were re-seeded at 80%

confluent to ensure cells are ready for experiments 24 h later. To arrest cells at G1/S boundary,

cells were treated with 2 mM of thymidine for 16 h, released for 8 h, and treated with 2 mM of

thymidine for 16 h again. After washing with PBS for three times, cells were released into fresh

medium for different times and harvested.

Antibodies

The phospho-specific antibodies against TSC1-S578 were generated by Proteintech. In brief, a peptide containing phospho-S578 was synthesized and used to immunize rabbits. After the antibodies were affinity purified, a series of control experiments were performed to confirm its specificity. The antibodies against mTOR (2983), p-mTOR-S2448 (2971), TSC1 (6935), TSC2

(4308), p-TSC2-T1462 (3611), AKT (9272), p-AKT-S473 (4060), p-AKT-T308 (4056), p-S6K-

T389 (9205), S6 ribosomal protein (2217), p-S6-S240/244 (2215), p-S6-S235/236 (4858), p-4E-

BP-1-S65 (9451), phospho-Hisone H3 (9701), Histone H3 (9717) and PTEN (9188) used in this study were purchased from Cell Signaling, whereas the antibodies against Plk1 (05-844) were from Millipore. We obtained antibodies against LAMP2 (sc-18822) and Ubiquitin (sc-8017) from Santa Cruz Biotechnology. Antibodies against Rheb (MAB3426) and Myc (M5546) were purchased from Nous Biologicals and Sigma, respectively.

Mouse xenograft model

All the animal experiments described in this study were approved by the Purdue University

Animal Care and Use Committee (PACUC). HeLa cells were transfected with different myc-

TSC1 constructs (WT, 2A or 2E) and treated with 2 mg/ml G418 for 1 week to select transfection-positive cells. Then, 1x106-selected cells were mixed with an equal volume of

Matrigel (Corning) and inoculated into the right flank of a nude mouse (Envigo, 5 mice per group, 4 groups, 20 mice total). One week later, mice were treated with 10 mg/kg rapamycin once a week and tumor volumes were measured twice a week using the following formula: V=L

Χ W2/2 (V, mm3; L, mm; W, mm).

Statistical analysis

All data are presented as means ± standard deviation (SD). Statistical calculations were performed with Microsoft Excel analysis tools. While a two-tailed, unpaired student's t test was

used to assess the difference between the effects of treatment in cell lines, one-way analysis of variance was used to determine statistically significant differences from the means in the animal study. P values of <0.05 were considered statistically significant. *p<0.05, **p<0.01.

Results

Phosphorylation of S6 is fluctuated during the cell cycle and inversely correlated with p-

AKT.

To elucidate the potential role of the PI3K/AKT/mTOR pathway during mitosis, we treated

HeLa cells with nocodazole to induce cell cycle arrest at prometaphase. While phosphorylation

of AKT was minimal at prometaphase, it gradually increases as cells exit from mitosis and enter

the interphase, confirming the work of others (12) (Fig. 1A). Unexpectedly, phosphorylation of

S6 (at both S235/S236 and S240/S244) was detected to be very high at prometaphase, but

quickly decreased upon mitotic exit (Figs. 1A, S1A). Of note, the finding is not cell type

specific, as the similar phenomena was observed in HEK 293T cells as well (Fig. S1B).

Therefore, our finding suggested an alternative activation of the PI3K/AKT/mTOR pathway in

response to growth factors, in which, the levels of p-S6 and p-AKT are strongly correlated (Fig.

1B). To further validate our finding, we also synchronized HeLa cells with the double thymidine

block protocol to arrest cells at the G1/S boundary. Consistent with the results based on the

release from prometaphase, the p-AKT signal was high in G1/S phase, but gradually decreased

as cells enter G2/M phase. In contrast, the p-S6 level was minimal in G1/S phase, but was

significantly increased as cells enter G2 and peaked in mitosis, matching the Plk1 level during

cell cycle progression (Figs. 1C, S1C). To confirm these novel findings, we conducted immunofluorescence (IF) studies to assess phosphorylation of S6 at various stages of mitosis in

HeLa cells. Consistent with the results of the IB analysis, the level of p-S6 becomes most abundant at mitosis (Figs. 1D, 1E).

PI3K/AKT-independent activation of mTORC1 in mitosis

We asked whether mitosis-associated S6 phosphorylation was under control of the

PI3K/AKT pathway. Accordingly, asynchronously growing and mitosis-arrested HeLa cells

were serum-starved for 12 h and then subjected to stimulation with insulin for 10 min. Pre-

incubation of serum-starved cells with LY294002 for 6 h completely prevented insulin-induced

mTORC1 kinase activity, as measured by reduced phosphorylation of S6K and S6 (Fig. S1D).

However, neither treatment of cells with starvation nor treatment of cells with insulin had any

effect on S6K, S6 or 4E-BP1 phosphorylation in mitotic cells (Figs. 2A, S1E). More

interestingly, treatment of mitotic cells with LY294002 failed to block S6K, S6 or 4E-BP1

phosphorylation, indicating that S6K, S6 or 4E-BP1 phosphorylation is independent of

PI3K/AKT pathway during mitosis (Figs. 2A, S1E).

To avoid the possibility of side effect of Nocodazole, we conducted IF studies in randomly growing HeLa cells. LY294002 pretreatment markedly reduced p-S6 signal in interphase cells

(Fig. 2B) but not in mitotic cells (Fig. 2B). Interestingly, mitosis-specific phosphorylation of S6 was inhibited by both mTORC1 and p70S6K inhibitors, indicating that mTORC1/p70S6K

pathway is responsible for mitotic-specific S6 phosphorylation (Fig. 2B). To substantiate this

further, HeLa cells were synchronized at the G2/M boundary with RO-3306, and then allowed

them to go through mitosis upon release, with most cells exiting mitosis at 4 h post-release.

Rapamycin, but not LY294002, pretreatment markedly reduced p-S6 signal (Figs. 2C, 2D).

Finally, we also asked whether amino acid starvation affects the observation we made. As

indicated, amino acid-starved cells still responded to nocodazole treatment by increased

phosphorylation levels of S6 at S235/6. In addition, mobility shift of 4E-BP1, an indicator of its

phosphorylation, was also clearly detected upon nocodazole treatment (Fig. S1F).

TSC1 is hyper-phosphorylated during mitosis

To further characterize the role of the PI3K/AKT/mTOR signaling during mitosis, we re-

examined changes in the activities of AKT and its downstream signaling proteins. Consistent

with the data in Fig. 2, hyper-phosphorylation of S6 and S6K was observed during mitosis in an

insulin-stimulation independent manner (Fig. 3A). Interestingly, under normal growth condition,

electrophoretic migration of TSC1 was predominantly detected as a single band (Fig. 3A),

whereas a second more slowly migrating band was observed in mitotic cell extracts (Fig. 3A).

This observation is consistent with a previous study (13). To test whether TSC1 is indeed hyper-

phosphorylated in mitosis, we arrested cells at G1, S, G2 and M phase. As indicated in Fig. 3B, the mitosis-enriched cells expressed a TSC1 form that migrated apparently slower than TSC1 from interphase cells. Since the change in electrophoretic mobility was completely reversed by treatment of extracts with -phosphatase (Fig. 3C), we concluded that TSC1 is hyper-

phosphorylated during mitosis. To identify kinases that are responsible for TSC1

phosphorylation in mitosis, we compared the TSC1 mobility upon inhibition of several mitotic

kinases. Addition of RO-3306, impaired the mobility shift of TSC1, suggesting that TSC1 might

be phosphorylated by Cdk1 in mitosis (Fig. 3D). In contrast, treatment with BI2536 (a specific

inhibitor of Plk1), VX-680 (a pan-Aurora inhibitor), or SB202190 (an inhibitor of p38 MAP

kinase) did not change the mobility of mitotic TSC1 significantly (Figs. 3E, S2A). Consistent

with the previous study (13), our data suggest that the majority of TSC1 phosphorylation during

mitosis likely depends on Cdk1. Considering the fact that Cdk1-mediated substrate

phosphorylation tends to create a docking site for the C-terminal polo-box domain of Plk1, we

tested the combination effect of RO-3306 plus BI2536. As indicted, Plk1 inhibition alone

slightly reduced mobility shifts of TSC1 and 4E-BP1, but a combination of RO-3306 and BI2536

completely blocked these events (Figs. 3F, 3G). In support, Plk1 overexpression led to increased

levels of p-S6K, p-4E-BP1, p-S6, but not p-AKT and p-TSC2 (Figs. 3H, S2B). Finally, hyper- phosphorylation of TSC1 during mitosis is independent of AKT kinase activity (Fig. S2C).

Phosphorylation of TSC1 by Plk1 in vitro and in vivo during mitosis

It has been documented that Cdk1 tends to function as a priming kinase for subsequent

phosphorylation by Plk1 for mitotic proteins (36). Cdk1 phosphorylates TSC1 at three possible

sites T417, S584 and T1047 (13). Further, TSC1 interacts with Plk1 in a T310 phosphorylation-

dependent manner (35). Therefore, we hypothesized that TSC1 might be a novel PIk1 substrate.

Previously, we also identified TSC1 as a potential Plk1 substrate (37). Co-immunoprecipitation

assays showed that endogenous TSC1 co-immunoprecipitated with endogenous Plk1 in

nocodazole-treated cells (Fig. 4A). Next, we asked whether Plk1 regulates mTORC1 kinase

activity. As indicated, depletion of Plk1 reduced the phosphorylation levels of S6K and S6 (Figs.

4B, S2D). These observations confirmed the previously published results (35). To directly test

whether TSC1 is a Plk1 substrate, in vitro kinase assays were conducted and showed that TSC1

is a direct Plk1 substrate (Fig. 4C). To determine if TSC1 phosphorylation by Plk1 was

modulated by Cdk1, we performed the sequential kinase assay. Pre-incubation with Cdk1 clearly

enhanced the subsequent Plk1-mediated phosphorylation of TSC1 (Fig. 4D). To directly map

potential phosphorylation sites of TSC1, various recombinant glutathione S-transferase (GST)-

fused TSC1 regions were generated. In vitro kinase assays showed that two regions of TSC1,

amino acids (aa) 301-474 and aa 475-660, contain the Plk1 phosphorylation sites (Fig. 4E).

Then, virtually every single serine and threonine residues of aa 301-474 and aa 475-660 was mutated into alanine to identify S467 and S578 as two Plk1 phosphorylation sites (Figs. 4F, 4G).

To further characterize the phosphorylation sites we mapped, we also generated a polyclonal

antibody that specifically recognizes the phosphorylated form of TSC1 at S578 (Fig. 4H). In

vivo, the pS578 antibody detected signals from cells expressing WT TSC1, but not from cells

expressing the S578A variant (Fig. 4I). More importantly, the pS578 antibody was able to detect

phosphorylation signals on TSC1 from HeLa cells, but the epitope was significantly reduced in

cells depleted of Plk1 (Fig. 4J). To determine if Cdk1 serves as a priming kinase for Plk1 in

cells, we depleted Cdk1 using RNAi and analyzed the TSC1-S578 phosphorylation. As indicated

in Fig. 4K, the pS578 level was significantly reduced in Cdk1-depleted cells. Finally,

overexpression of Plk1-WT form, but not the kinase-deficient mutant (K82M), led to elevation

of the pS578-TSC1 epitope (Fig. 4L). Alignment of TSC1 sequences indicates that the two

phosphorylation sites are highly conserved across different species (Fig. 4M).

Phosphorylation by Plk1 suppresses TSC1 function during mitosis

TSC1 and TSC2 are two tumor suppressor genes mutated in tumor syndrome TSC (35),

whereas increased Plk1 expression is detected in many types of cancer (19). We hypothesized

that Plk1 phosphorylation of TSC1 during mitosis might have an inhibitory effect on the

TSC1/TSC2 complex. Compared to cells expressing WT-TSC1, cells expressing the phospho-

mimetic TSC1-2E mutant clearly had an increased level of p-S6 upon insulin stimulation (Fig.

5A). Next, we co-transfected HeLa cells with different forms of myc-TSC1 with HA-TSC2 and treated with nocodazole. As indicated in Fig. 5B, while the p-S6 signals were decreased during

mitosis in both WT- and 2A-TSC1-expressing cells, expression of TSC1-2E failed to do so. To

further substantiate this, we synchronized HeLa cells in prometaphase by the mitotic shake-off

protocol and then allowed cells to exit mitosis upon release. As expected, compared with cells

expressing TSC1-2A, cells expressing TSC1-2E apparently showed a higher p-S6 level during

mitotic exit (Figs. 5C, S3A). We hypothesized that phosphorylation of TSC1 might also play a role in regulating its interaction with TSC2. As expected, WT TSC1 and TSC2 formed a stable

complex. However, we found that compared with TSC1-WT and -2A mutant, the amount of

TSC1-2E mutant bound to TSC2 was apparently reduced (Fig. 5D). To address if phospho-

mimetic mutant of TSC1 is less stable than TSC1-WT or -2A mutant, we transfected HeLa cells with different forms of myc-TSC1 constructs, followed by treatment with cycloheximide. As indicated in Fig. 5E, 12 h cycloheximide treatment did not affect the levels of TSC1-WT and -2A mutant, but significantly reduced the level of TSC1-2E mutant. To ensure our finding is not cell type specific, we repeated the experiment in PC3 cells and obtained the similar result (Fig. S3B).

A similar observation was made when HEK 293T cells were co-transfected with Myc-TSC1

(WT, 2A or 2E mutant) with HA-TSC2 (Fig. 5F). To confirm these findings, we further showed that the level of TSC1-2E mutant was markedly increased upon treatment with the proteasome inhibitor MG132 (Fig. 5G). Finally, a much higher level of polyubiquitination was detected in

TSC1-2E mutant than that of TSC1-WT and -2A mutant (Fig. 5H).

Phosphorylation of TSC1 by Plk1 affects mitotic progression and cell growth

Since phosphorylation of TSC1 is cell cycle regulated, we asked whether Plk1-dependent phosphorylation of TSC1 affects cell cycle progression. Interestingly, we found that overexpression of WT TSC1 affects mitotic progression (Fig. 6A). Next, we compared mitotic exit of cells expressing different forms of TSC1 at the Plk1 phosphorylation sites and found that cells expressing different forms of TSC1 (2A or 2E) go through mitosis with different kinetics.

As indicated, the level of p-H3 was already fairly low at 2 h post-release in cells expressing

TSC1-2A, but the level of p-H3 remained high even at 4 h post-release in cells expressing TSC1-

2E (Fig. 6B). To rule out the possibility that the mitotic delay was caused by nocodazole, we performed time-lapse live cell imaging of HeLa cells stably expressing GFP-H2B. Consistent with the nocodazole release experiments, live cell imaging analysis revealed that cells expressing

TSC1-2E showed a prolonged prometaphase associated with a significantly extended average time (63.2 ± 2.4 min) from nuclear envelope breakdown to the onset of anaphase compared with those of control cells (41.8 ± 1.5 min) (Fig. 6C). Experiments were then conducted to determine

if the mitotic defect observed in TSC1-2E-expressing cells was sensitive to mTOR inhibitor

rapamycin. Cells expressing TSC1-2E mutant showed slower mitotic exit (Fig. 6B), rapamycin

treatment reduced pS6 level (Fig. S3C), but did not reverse the TSC1-2E expression-induced

mitotic delay (Fig. 6D).

The mTOR signaling plays a role in coordinating cell growth and proliferation signals with

protein synthesis in a raptor-dependent manner during mitosis (38). To test whether the

Plk1/TSC1/mTORC1 axis also mediates cell growth, we exogenously expressed different forms

of myc-TSC1 constructs in HeLa cells. Under normal growth conditions, expression of different

TSC1 constructs all led to a decrease in the rate of cell growth. Unexpectedly, the growth rate of

TSC1-2E-expressing cells, which showed the constitutively active mTOR pathway (Fig. S3D),

was similar to those of cells expressing TSC1-WT and -2A (Fig. 6E). Interestingly, at a lower

dose of rapamycin, 15 nM, we observed that the overall growth rate was appreciably lower in

TSC1-2E-expressing cells than cells expressing TSC1-WT and -2A (Fig. 6F and 6G). Finally,

cells expressing different forms of TSC1 constructs were used to generate xenograft tumors.

Consistent to cell culture-based data in Fig. 6, tumors expressing TSC1-2E mutant were more

sensitive to rapamycin treatment than tumors derived from cells expressing TSC1-WT or -2A

mutant (Figs. 7A, 7B, S4A, S4B). Further IHC analysis indicates that tumors expressing the

TSC1-2E mutant had the lowest proliferation rate, indicated by Ki67 staining (Fig. S4C).

Interesting, both tumors expressing TSC-2A and -2E mutants had high levels of cleaved caspase

3 signals (Fig. S4D). Thus, the small tumor size of group expressing TSC1-2E is due to both

reduced proliferation and increased apoptosis, whereas the small tumor size of group expressing

TSC1-2A is mainly due to increased cell death. We also measured the baseline of apoptosis in an

in vitro experiment in the presence and absence of rapamycin in cells expressing different forms

of TSC1 (WT, 2A, 2E). Consistent with the general concept that rapamycin usually does not

induce apoptosis, we failed to detect apparent apoptosis in cultured cells (Fig. S4E). Apparently,

the tumors expressing TSC1-2E respond differently to rapamycin, and more experimentation is

needed to clarify the issue.

Plk1 phosphorylation of TSC1 leads to decreased lysosomal localization of the TSC

complex, activation of mTORC1, and increased sensitivity to rapamycin.

We then asked whether cells expressing different forms of TSC1 have different mTORC1

activities. As indicated, HeLa cells expressing TSC1-2E mutant indeed had a higher

phosphorylation level of S6K than cells expressing the 2A mutant (Fig. 7C). To ensure that our

finding is not HeLa cell specific, we repeated the experiment in PC3 cells and obtained similar

results (Figs. 7D, S3D). It was shown that AKT-mediated phosphorylation of TSC2 results in

dissociation of the TSC complex from the lysosome in response to insulin stimulation (6). As

expected, treatment of cells with AKT inhibitor MK2206 prevented the dissociation of the TSC

complex from the lysosome (Fig. 7E). Interestingly, treatment of cells with Plk1 inhibitor

BI2536 also reduced cytosolic localization, but increased lysosomal localization, of the TSC

complex (Fig. 7E). To further dissect how Plk1 phosphorylation of TSC1 affects the mTORC1

activation at the lysosome, we followed co-localization of different members of the mTOR

pathway upon expression of different forms of TSC1 (WT vs 2A). mTOR was co-localized with

its positive regulator Rheb at lysosome in cells expressing TSC1-WT, but expression of TSC1-

2A mutant apparently reduced the co-localization of Rheb with mTORC1 at lysosome (Figs. 7F,

7G). In striking contrast, co-localization of TSC2 with LAMP2, was increased in cells expressing

TSC1-2A mutant in comparison to cells expressing TSC1-WT (Figs. 7H, 7I). Altogether, we conclude that Plk1 phosphorylation of TSC1 leads to increased dissociation of the TSC complex from the lysosome.

Discussion

PI3K, AKT and mTOR are three hubs in the PI3K pathway to regulate cell growth and

proliferation (39). Despite the fact that all the inhibitors demonstrated very promising preclinical

activities, none of them is currently able to cure a single cancer patient, largely due to the

development of drug resistance. Unfortunately, the underlying molecular mechanism of

resistance to these inhibitors remains largely unknown (40,41). In case of mechanisms

responsible for resistance to mTOR inhibitors, one potential mechanism is due to the fact that

activation of oncogenic mTOR pathway can be achieved in diverse ways (41,42). Consistent

with this idea, our data clearly showed that the PI3K/AKT pathway, the well-established

upstream activating pathway of mTORC1, is downregulated during mitosis, whereas mTORC1

is clearly active. To probe the mechanism, we found that mitotic TSC1 is highly phosphorylated.

While it is well documented that AKT phosphorylation of TSC2 leads to inactivation of the

TSC1/2 complex, kinase(s) response for and functional significance of TSC1 phosphorylation

have not been described. Our observations confirmed the previous study that TSC1 is

phosphorylated by Cdk1 and the phosphorylated TSC1 interacts with Plk1 (13,35). Furthermore,

we indicate that Plk1 cooperates with Cdk1 to generate the mitotic-specific hyper-

phosphorylated TSC1, providing a novel mechanism for the regulation of mTORC1 activity

during mitosis. In this model, the TSC complex functions as a downstream target of Plk1. We showed that Plk1-mediated phosphorylation of TSC1 causes disruption of TSC complex formation and destabilization of TSC1 protein, eventually resulting in activation of mTORC1 in mitosis. Interestingly, it was recently shown that Plk1 protein levels are increased in TSC1-/- and

TSC2-/- cells (43). When combined with what we described here, these data implies that a

feedback mechanism might exist between Plk1 and TSC1.

It was shown that TSC1 physically and functionally interacts with Plk1 to regulate

centrosome biology and mitotic progression (35). We hypothesized that Plk1 phosphorylation of

TSC1 in M phase also regulates mitotic progression. Supporting this notion, we found that

expression of phospho-mimetic TSC1 mutant impairs normal mitotic progression. We then tested

the hypothesis that impaired mitotic progression observed in TSC1-2E-expressing cells might be

mTOR dependent. Unexpectedly, pre-treatment of cells expressing TSC1-2E with rapamycin had

no impact on mitotic progression. These results indicate that Plk1 phosphorylation of TSC1

affects its function through at least two mechanisms: First, phosphorylation of TSC1 impairs

normal mitotic progression; Second, phosphorylation of TSC1 activates the mTORC1 pathway.

Indeed, cells expressing TSC1-2E showed a slower growth in the presence of rapamycin,

supporting the notion that Plk1 phosphorylation of TSC1 plays a central role in the coordination

of cell cycle progression and cell growth. Plk1, a serine/threonine protein kinase that is normally

expressed in mitosis, is frequently overexpressed in multiple types of human tumors regardless

of the cell cycle stage. However, the causal relationship between overexpression of Plk1 and

tumorigenesis has not been fully investigated. In this study, we provide evidence that Plk1- mediated phosphorylation of TSC1 leads to aberrant mitosis and hyper-activation of mTOR pathway, both of which are hallmarks of cancer.

A previous study reported that shRNA-mediated Plk1 depletion decreased the

phosphorylation of mTOR substrates (44). It was also reported that Plk1 is a physical mTORC1

interactor and involved in autophagy pathway (45). However, the underlying mechanism how

Plk1 controls the mTORC1 pathway is still unclear. Here, we report a novel signaling pathway

where Plk1 regulates mTOR independently of AKT in mitosis. The data support the signaling

findings in both cancer and normal cell lines used. We acknowledge that the effects of the TSC1

mutants in proliferation and tumor growth appear puzzling. We are also surprised that the

phosphorylation mimic of the TSC1 phosphorylation site delays mitosis but has no effect on cell

proliferation when compared to WT TSC1 or alanine mutant. This could be due to the different

culture conditions. For the mitotic exit experiments, we used shake-off protocol to synchronize

all cells in prometaphase. The delayed mitosis exit observed in the phosphorylation mimic

mutant could be due to the defected recovery from the nocodazole arrest. The delayed mitotic

exit could also be the potential mechanism of unexpected observation – rapamycin-induced

apoptosis in TSC1-2E cells. Rapamycin almost never induces apoptosis, both in vitro and in

vivo. It is very surprising that TSC1-2A and TSC1-2E tumors treated with rapamycin have

increased apoptosis. Thus, it is possible that rapamycin-induced apoptosis under this condition is

due to defects in mitosis or due to aberrant activation of pro-survival pathways. The exact

mechanism deserves further experimentation.

In summary, we propose that mTOR activity is controlled by two different pathways during

cell cycle. In interphase, the PI3K/AKT pathway plays a key role to activate the mTOR pathway

by AKT-mediated phosphorylation of TSC2 in response to intracellular signaling. However, in

mitosis, Plk1 is the major kinase to activate the mTOR pathway by targeting TSC1. Instead of

activation of the mTOR pathway, Plk1 phosphorylation of TSC1 also leads to mitotic defects in an mTOR-independent manner.

Acknowledgments

Grant Support: This work was supported by NIH grants R01 CA157429 (X. Liu), R01 CA192894 (X. Liu), R01 CA196835 (X. Liu), R01 CA196634 (X. Liu), and P30 CA023168 (Purdue Center for Cancer Research).

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Figure Legends Figure 1. Phosphorylation of S6 is regulated during the cell cycle and inversely correlated with p-AKT.

(A) HeLa cells were subjected to mitotic shake-off protocol, released for the indicated periods, and harvested for immunoblotting (IB). (B) HeLa cells at 60% confluency were serum starved for 24 h, incubated with indicated inhibitors (25 µM of LY294002 for 1 h, 100 nM of rapamycin for 2 h, or 10 µM BI-D1870 for 1 h), stimulated with 100 nM of insulin for 10 min, and harvested. (C) HeLa cells were synchronized by the double thymidine block (DTB) protocol and released for the indicated periods. (D-E) After HeLa cells were seeded on coverslips at 40 % confluency for 24 h, cells were fixed in 4% paraformaldehyde for 15 min, extracted with 1%

Triton X-100 at room temperature for 15 min, and subjected to IF staining. White arrows indicate cells at mitosis. The data represent means (±SD) of three independent experiments from counting 500 cells each. Scale bars are 5 μm (1D) and 10 µm (1E).

Figure 2. mTORC1 activation was independent of AKT activity in mitosis.

(A) HeLa cells were cultured in 6-well plates, left reaching 80% confluence and treated with 100 ng/ml of nocodazole for 12 h to arrest at mitosis. Cells were collected, reseeded onto plates, and cultured for 24 h in medium without FBS. Upon incubation with or without 25 M of LY294002 for 6 h, cells were stimulated with insulin (100 nM) for 10 min before harvest. (B) Exponentially growing HeLa cells were incubated with three inhibitors (25 µM of LY294002 for 1 h, 100 nM of rapamycin for 2 h, or 10 µM BI-D1870 for 1 h), fixed in 4% paraformaldehyde for 15 min, and stained with p-S6 antibodies. Red and white arrows indicate interphase and mitotic cells, respectively. Quantification results are indicated at the right. The data represent means (±SD) of three independent experiments from counting 500 cells each. Scale bar, 5 μm. (C) HeLa cells at

50% confluency were treated with 10 M of RO-3306 for 18 h to arrest at the G2/M boundary,

and released into fresh medium in the presence or absence of 25 M of LY294002. (D) HeLa

cells were treated with 10 M of RO-3306 for 18 h and released in the presence or absence of

100 nM of rapamycin.

Figure 3. TSC1 is hyper-phosphorylated during mitosis.

(A) Immunoblot analysis of the PI3K/AKT/mTORC1 pathway-associated proteins obtained from

asynchronous and mitotic HeLa cells. HeLa cells at 80% confluency were treated with 100 ng/ml of nocodazole for 12 h, deprived of serum for 24 h as in Fig. 2B, stimulated with 100 nM of insulin for 10 min, and harvested for IB. The arrow indicates the phosphorylated form of TSC1.

(B) Mitotic TSC1 showed a reduced electrophoretic mobility. HeLa cells were treated with 300

M of mimosine, 500 M of hydroxyurea, 10 M of RO-3306 or 100 ng/ml of nocodazole to

arrest cells in G1, S, G2 or M phase, and harvested for IB. (C) The reduced electrophoretic

mobility of mitotic TSC1 is due to hyper-phosphorylation. Extracts from randomly growing

cells or cells arrested in mitosis by 100 ng/ml of nocodazole for 24 h or 100 nM of docetaxel for

24 h were incubated with -phosphatase (-PPase) for 30 min at 30oC, followed by IB. (D) HeLa cells were pretreated with 100 ng/ml of nocodazole for 24 h, incubated with 10 M of RO-3306

for different times, and harvested for IB. (E) HeLa cells were treated with 100 ng/ml of nocodazole for 24 h, followed by incubation with 10 M of RO-3306, 100 nM of BI2536, 10 M

of VX-680, or 10 M of SB202190 for 6 h. (F and G) HeLa cells were treated with 100 ng/ml of

nocodazole ± 10 M of RO-3306 ± 100 nM of BI2536 for 12 h. (H) Overexpression of Plk1 led

to mTORC1 activation. PC3 cells were infected with adenovirus expressing Plk1 for 2 days and

harvested for IB with various antibodies including pS235/6-S6, pS240/4-S6, and p-S6K.

Figure 4. Plk1 phosphorylates TSC1 at S467 and S578.

(A) Plk1 interacts with phosphorylated TSC1. 293T cells were treated with 100 ng/ml of

nocodazole for 24 h to arrest cells in mitosis and harvested for anti-Plk1 immunoprecipitation

(IP), followed by IB. (B) Depletion of Plk1 led to reduced S6 and S6K phosphorylation. PC3

cells were transfected with pBS/U6-Plk1 to deplete Plk1 and harvested for IB with indicated

antibodies. (C) Plk1 phosphorylates TSC1 in vitro. 293T cells were transfected with Myc-TSC1

and subjected to IP with Myc antibodies. After the IP pellet was incubated with recombinant

Plk1 in the presence of [γ-32P]ATP, and detected by autoradiography. (D) Priming

phosphorylation by Cdk1 enhances TSC1 phosphorylation by Plk1. 293T cells were transfected

with Myc-TSC1 and subjected to IP with Myc antibodies. The IP pellet was incubated with Cdk1

in the presence of unlabeled ATP, followed by incubation with Plk1 in the presence of [γ-32P]

ATP. (E-H) Plk1 phosphorylates TSC1 at S467 and S578 in vitro. Recombinant Plk1 was

incubated with various purified GST-TSC1 fragments (E) or mutants (F, G) as in D. While Plk1-

T210D is constitutively active, Plk1-K82M is a kinase inactive mutant. (H) The pS578-TSC1

antibody is specific. Plk1 was incubated with GST-TSC1 constructs, followed by anti-pS578-

TSC1 IB. (I) TSC1-S578 is phosphorylated in vivo. 293T cells were transfected with TSC1

constructs and subjected to anti-pS578-TSC1 IB. (J-L) Plk1 is responsible for phosphorylation of

TSC1 at S578 in vivo. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 (J) or pKD-

Cdk1 to deplete Cdk1 (K). (L) 293T cells were transfected with Plk1-WT or kinase-deficient

mutant for 2 days and harvested. (M) Alignment of TSC1 protein sequences containing the two

sites from different species.

Figure 5. Mutation of Plk1 phosphorylation sites alters the TSC activity.

(A-C) Substitution of TSC1 phosphorylation sites by acidic residues decreases the TSC1/TSC2 complex activity. (A) HeLa cells were seeded in 6-well dishes for 24 h and co-transfected with

Myc-TSC1 constructs with HA-TSC2. After 24 h of incubation, cells were treated with 100 nM

of insulin for 10 min, and harvested. (B) HeLa cells were co-transfected with Myc-TSC1 constructs with HA-TSC2, treated with 100 ng/ml of nocodazole for 12 h, and harvested.

nocodazole, and released for indicated times. (D) Acidic residue substitutions disrupt the

formation of the TSC1/TSC2 complex. 293T cells were co-transfected with Myc-TSC1 and HA-

TSC2 and harvested for anti-Myc IP, followed by anti-HA IB. (E-G) The phosphomimetic

mutant of TSC1 is unstable. (E) HeLa cells were transfected with Myc-TSC1 constructs, treated

with 10 μg/ml cycloheximide for different times, and harvested. (F) HEK293T cells were co-

transfected with Myc-TSC1 constructs with HA-TSC2, treated with 10 μg/ml cycloheximide for different times, and harvested. (G) PC3 cells were transfected with TSC1 constructs and treated with or without 10 µM MG132 for 12 h. (H) PC3 cells were co-transfected with Myc-TSC1 constructs with His-ubiquitin, treated with 10 µM MG132 for 12 h, and harvested for anti-Myc

IP, followed by anti-ubiquitin IB.

Figure 6. Phosphorylation of TSC1 by Plk1 affects mitotic progression and cell growth.

(A-C) TSC1-2E expression slows down mitotic exit. (A) HeLa cells were co-transfected with

Myc-TSC1 and HA-TSC2 constructs for 24 h, treated with 100 ng/ml of nocodazole, and

harvested by mitotic shake-off protocol. The collected mitotic cells were re-seeded for indicated

times for IB. (B) HeLa cells were co-transfected with Myc-TSC1 and HA-TSC2 constructs for

48 h, treated with 100 ng/ml of nocodazole for 24 h, and harvested by mitotic shake-off protocol.

The collected cells were then released for indicated times. (C) HeLa cells stably expressing GFP-

H2B were subjected to TSC1 transfection, followed by time lapse microscope analysis. Mitosis

duration was quantified (3 experiments with approximately 300 cells for per experiment). Scale

bars, 10 µm. (D) TSC1-2E expression slows down mitotic exit independent of mTORC1

pathway. HeLa cells were co-transfected with Myc-TSC1-2E and HA-TSC2 constructs, treated

with 10 M of RO-3306 for 18 h to arrested cells in late G2 phase and released in the presence

or absence of 100 nM of rapamycin for indicated times. (E-G) Cells expressing TSC1-2E are

more sensitive to rapamycin. (E) HeLa cells transfected with different myc-TSC1 constructs

were seeded in the medium. (F) HeLa cells transfected with the indicated TSC1 plasmids were

seeded in the medium containing 15 nM rapamycin. (G) Cells expressing TSC1-2E mutant are

hyper-sensitive to rapamycin. HeLa cells expressing myc-TSC1 constructs and HA-TSC2

constructs were seeded in plates for 5 days, treated with or without rapamycin.

Figure 7. Plk1 phosphorylation of TSC1 leads to decreased lysosomal localization of the

TSC complex, activation of mTORC1, and increased sensitivity to rapamycin.

(A and B) HeLa cells stably expressing myc-TSC1 constructs were obtained by G418 selection.

Tumors were generated into 20 nude mice (5 mice per group) by inoculating 1 x 106 cells/mouse.

At 7 days post-inoculation, mice were treated with 10 mg/kg rapamycin once a week and tumor

sizes were followed. (A) Tumor growth curves. (B) Representative images of tumors. (C and D)

HeLa (C) or PC3 (D) cells were transfected with Myc-TSC1 constructs for 1 day and harvested for IB. (E) PC3 cells were treated with 2 M of MK2206 for 30 min or 200 nM of BI2536 for 12

h and harvested for fractionation into heavy membrane (lysosome) and light membrane (cytosol)

fractions, followed by IB. (F) PC3 cells were co-transfected with TSC1 constructs, and subjected

to IF staining. Representative images taken (top panel, scale bar, 10 µm; bottom panel, scale bar,

50 µm). (G) Quantification of E to show the mean and SEM of Pearson’s coefficient that was

determined by Image J software. (H) PC3 cells transfected as in F were subjected to co-staining.

Representative images taken (top panel, scale bar, 10 µm; bottom panel, scale bar, 50 µm). (I)

Quantification of F to show the mean and SEM of Pearson’s coefficient that was determined by

Image J software. For quantification, at least three experiments were repeated, and at least three fields were chosen each time. *, P<0.05.

Plk1-mediated phosphorylation of TSC1 enhances the efficacy of rapamycin

Zhiguo Li, Yifan Kong, Longzhen Song, et al.

Cancer Res Published OnlineFirst March 20, 2018.

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