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Regulation of EGFR endocytosis by CBL during mitosis 1

Ping Wee and Zhixiang Wang* 2

Department of Medical Genetics and Signal Transduction Research Group, Faculty of Medicine 3

and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7 4

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Running Head: EGFR endocytosis during mitosis 6

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Keywords: EGFR, mitosis, endocytosis, CBL, ubiquitination 8

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ABSTRACT 10

Endocytosis plays important roles in regulating EGFR signaling. We previously found 11

that EGFR endocytosis during mitosis is mediated differently than during interphase. While the 12

regulation of EGFR endocytosis in interphase is well understood, little is known regarding the 13

regulation of EGFR endocytosis during mitosis. Here, we studied the mechanisms regulating 14

mitotic EGFR endocytosis. We found that contrary to interphase cells, mitotic EGFR endocytosis 15

is more reliant on the activation of the E3 ligase CBL. At high EGF doses, inhibition of inhibited 16

EGFR endocytosis of mitotic cells, but not of interphase cells. Moreover, the endocytosis of 17

mutant EGFR Y1045F-YFP was strongly delayed. The endocytosis of truncated EGFR Δ1044-18

YFP that does not bind to CBL was completely inhibited. EGF induced stronger ubiquitination 19

of mitotic EGFR than interphase EGFR and mitotic EGFR is trafficked to lysosome for 20

degradation. Furthermore, during mitosis low doses of EGF also stimulate EGFR endocytosis by 21

NCE. Contrary to interphase, CBL and the CBL-binding regions of EGFR were required for 22

mitotic EGFR endocytosis at low doses. This may be due to the mitotic ubiquitination of the 23

EGFR even at low EGF doses. In conclusion, mitotic EGFR endocytosis solely proceed through 24

CBL-mediated NCE. 25

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INTRODUCTION 33

The epidermal growth factor (EGF) receptor (EGFR), like other receptor tyrosine kinases 34

(RTKs), regulates key events in cell growth, differentiation, survival and migration [1-3]. 35

Aberrant signaling from EGFR has been implicated in many diseases [2, 4]. EGFR is historically 36

the prototypical RTK. It was the first of this large family of transmembrane receptors to be 37

cloned, and the first for which a clear connection between aberrant receptor function and cancer 38

could be drawn [2]. The binding of EGF to EGFR at the cell surface induces dimerization of 39

EGFR, which results in the activation of EGFR tyrosine kinase and EGFR trans-40

autophosphorylation [5, 6]. EGFR activation stimulates various signaling pathways that regulate 41

multiple cell functions [1, 3]. EGF stimulates cell proliferation by driving the cell cycle that is 42

comprised of four phases: G1, S, G2 and M [7, 8]. Binding of EGF also stimulates the rapid 43

internalization of EGFR [9]. EGFR endocytosis and EGFR-mediated cell signaling are mutually 44

regulated [10, 11]. 45

In spite of significant advances in our understanding of EGFR signaling and trafficking, 46

some critical knowledge is still lacking. Our current knowledge of EGFR signaling and EGFR 47

endocytosis comes mostly from the studies of cells in G1 phase of the cell cycle. Very little is 48

known regarding EGFR-mediated signaling and endocytosis in mitosis. 49

Mitosis represents a period where the needs and requirements of the cell differ vastly 50

from interphase cells. EGFR signaling has been shown to be regulated differently between 51

interphase and mitotic cells. We and others previously found that the EGFR of mitotic cells can 52

still be activated during mitosis, but that the signal transduction pathways are regulated 53

differently compared to interphase cells [12, 13]. We also previously found that EGFR 54

endocytosis of mitotic cells is regulated differently, in that EGFR is endocytosed at a slower rate 55

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[14]. At the time, we did not fully decipher the molecular mechanisms behind the differential 56

kinetics. Therefore, in this report, we further studied this phenomenon. 57

Endocytosis of the EGFR can lead to two distinct fates for the receptor: recycling back to 58

the plasma membrane or lysosomal degradation. As such, the route taken directly influences the 59

total number of receptors available for a subsequent signal transduction response. EGFR 60

recycling has been shown to be mediated by clathrin-mediated endocytosis (CME), whereas non-61

clathrin mediated endocytosis (NCE) targets receptors for lysosomal degradation. 62

CME is a mechanism of internalization that is dependent on the recruitment of clathrin to 63

the receptor. While this notion has been disputed by some studies [15-17], most data support the 64

theory that CME is inhibited in mitosis [18-27]. The mechanisms underlying CME inhibition are 65

still unknown, however, several mechanisms have been proposed and partially tested. These 66

mechanisms include “moonlighting” hypothesis [25, 26], the phosphorylation of endocytic 67

proteins [28, 29], and the unavailability of actin for CME [30]. In agreement with this, we 68

previously found that mitotic EGFR endocytosis was clathrin-independent as siRNA depletion of 69

clathrin heavy chain did not affect mitotic EGFR endocytosis [14]. We therefore hypothesized 70

that mitotic EGFR proceeded exclusively through NCE. 71

NCE has been described as having potential tumor suppressive characteristics {Caldieri, 72

2017}. NCE has also been described as initiating more slowly than CME [9, 31-34], which fits 73

with our observed delay in mitotic EGFR endocytosis [14]. Molecularly, EGFR NCE has been 74

described as only activated by physiologically high doses of EGF [32, 35, 36], which is likely a 75

mechanism evolved to compensate when the CME pathway is saturated and to prevent excessive 76

EGFR signaling [37]. EGFR NCE has been shown to be mediated by ubiquitination of the 77

receptor, and this ubiquitination has been shown to be limited by the activity of the E3 ligase c-78

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CBL [32, 35, 36]. Therefore, c-CBL (henceforth CBL) provides a critical negative regulatory 79

control of the EGFR, as it targets the EGFR for endocytosis and degradation. The activation of 80

CBL depends on its binding to the activated EGFR, either by direct interaction with pY1045, or 81

by indirect interaction through the adaptor GRB2, which binds to pY1068 or pY1086 [35, 36, 38, 82

39]. 83

In this report, we find that EGF-stimulated EGFR endocytosis proceeds exclusively by 84

NCE during mitosis. We find that contrary to interphase cells, mitotic EGFR endocytosis is more 85

reliant on the activation of CBL. At high EGF doses, inhibition of CBL by siRNA or mutation 86

inhibited EGFR endocytosis of mitotic cells, but not of interphase cells. Moreover, the 87

endocytosis of truncated EGFR Δ1044-YFP, which does not bind to CBL, was completely 88

inhibited. EGF induced stronger ubiquitination of mitotic EGFR than interphase EGFR and 89

mitotic EGFR is only trafficked to lysosomes for degradation. Furthermore, we found that during 90

mitosis, low doses of EGF also stimulate EGFR endocytosis by NCE. Contrary to interphase, 91

CBL and the CBL-binding regions of EGFR were required for mitotic EGFR endocytosis at low 92

doses. This was due to the mitotic ubiquitination of the EGFR even at low EGF doses. 93

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MATERIALS AND METHODS 95

Antibodies and chemicals 96

Antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), including: mouse anti-EGFR 97

(sc-373746), anti-pY99 (sc-7020), anti-CBL (sc-170), anti-Ubiquitin (sc-8017), anti-Cyclin B1 98

(sc-245), and anti-β-Tubulin (sc-5274), rabbit anti-GRB2 (sc-8034) and anti-SHC (sc-967), and 99

goat anti pY1068 (sc-16804) and pY1086 (sc-16804). LAMP-2 (AF6228) antibody was from 100

R&D Biosystems (Minneapolis, MN). The horseradish peroxidase (HRP)-conjugated secondary 101

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antibodies were from Bio-Rad (Hercules, CA) and the fluorescence-conjugated secondary 102

antibodies were from Jackson ImmunoResearch (West Grove, PA). Goat anti-mouse 103

immunoglobulin G (IgG) conjugated with agarose were from Sigma (St. Louis, MO). EGF was 104

from Upstate Biotechnology. 105

Plasmid construction 106

The EGFR-YFP, EGFR-Y1045F-YFP, EGFR-Δ991-YFP, and EGFR-Δ1044-YFP constructs 107

were described previously [40]. The c-CBL-YFP and 70z-CBL-YFP constructs were generous 108

gifts from the Sorkin Lab. 109

Cell Culture, transfection, and treatment 110

HeLa, 293T, and MCF-7 cells were growth at 37°C in Dulbecco’s modified Eagle’s medium 111

containing 10% fetal bovine serum and antibiotic/antimycotic solution maintained at 5% CO2 112

atmosphere. For transfection, MCF-7 cells in 24-well plates were transfected using 113

LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) as per the manufacturer’s protocol, 114

and 293T cells in 24-well plates were transfected using calcium phosphate precipitation with 115

BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer. MCF-7 and 293T cells were 116

chosen due to their low levels of endogenous EGFR. Small interfering RNA-mediated silencing 117

transfections were done using CBL siRNA (sc-29241; Santa Cruz Biotechnology, Santa Cruz, 118

Calif) in HeLa cells as per the manufacturer’s protocol. 119

Mitotic cells were collected by gentle mitotic shake-off as previously described [13]. Briefly, 120

cells were arrested in prometaphase by treating cells with nocodazole (200 ng/mL) in serum-free 121

media for 16h. The nocodazole-arrested cells were treated with EGF (2 ng/mL or 50 ng/mL) for 122

5, 30, and 45 min, or not treated with EGF (0 min). The EGF-containing media was then 123

removed and serum-free media was added. Cells were placed on ice and dislodged by gently 124

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tapping the plates for 5 min. The mitotic cell-containing media was centrifuged at 1000 rpm for 5 125

min. The obtained mitotic cells were then lysed with cold Mammalian Protein Extraction 126

Reagent (M-Per) (Thermo Fisher Scientific Inc, Rockford, IL USA) buffer in the presence of 127

phosphatase and protease inhibitors including 100 mm NaF, 5 mM MgCl2, 0.5 mM Na3VO4, 128

0.02% NaN3, 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 10 μg/ml aprotinin, and 1 μM 129

pepstatin A. To collect lysates for interphase cells, cells were serum-starved for 16h. Cells were 130

then treated with EGFR for 5, 30, and 45 min. To ensure consistency with the mitotic treatment, 131

the cells were also tapped on ice for 5 min to remove mitotic cells and then left on ice for 5 min. 132

The remaining interphase cells were collected by scraping on ice in cold M-Per in the presence 133

of phosphatase and protease inhibitors. For both interphase and mitotic cells, after lysing, the 134

samples were centrifuged at 21,000 ×g and the supernatant was collected for immunoblotting. 135

Immunoprecipitation and immunoblotting 136

Immunoprecipitation experiments were carried out as described previously [40]. Interphase or 137

mitotic cells were lysed with immunoprecipitation buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 138

1% NP40, 0.1% sodium deoxycholate, 100 mM NaF, 0.5 mM Na3VO4, 0.02% NaN3, 0.1 mM 139

4-(2-aminoethyl)-benzenesulfonyl fluoride, 10 μg/mL aprotinin, and 1 μM pepstatin A) for 15 140

min at 4°C. Cell lysates were then centrifuged at 21,000 ×g. The supernatant, containing 1 mg of 141

total protein, were incubated with 0.8 μg of mouse monoclonal anti-EGFR antibody A-10 (Santa 142

Cruz) for 2 h at 4°C with gentle mixing by inversion. Goat anti-mouse IgG conjugated with 143

agarose was added to each fraction and incubated for 2 h at 4°C with gentle mixing by inversion. 144

Next, the agarose beads were centrifuged, washed three times with immunoprecipitation buffer, 145

and 2× loading buffer was added. The samples were boiled for 5 min at 95°C and loaded for 146

SDS-PAGE for subsequent immunoblotting. 147

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Immunoblotting was performed as previously described [41]. Briefly, protein samples were 148

separated by SDS-PAGE and were transferred to nitrocellulose. The membranes were blocked 149

for non-specific binding, and incubated with primary antibody overnight. The membranes were 150

then probed with HRP-conjugated secondary antibody followed by detection with enhanced 151

chemiluminescence solution (Pierce Chemical, Rockford, IL) and light detection on Fuji Super 152

RX Film (Tokyo, Japan). 153

Indirect immunofluorescence 154

Indirect immunofluorescence was performed as previously described [14]. Cells were grown on 155

glass coverslips and serum-starved for 16 h. After treatment without or with nocodazole (200 156

ng/mL for 16h) and without or with EGF for various indicated times, the cells were fixed with 157

ice cold methanol for 10 min. The cells were then permeabilized with 0.2% Triton X-100 for 10 158

min on ice. Next, cells were blocked with 1% BSA for 1 h on ice. Cells were then incubated with 159

primary antibody overnight at 4°C. Primary antibody anti-CBL was used at 1:50, and anti-160

pEGFR-Y1086 and anti-EGFR were used at 1:200. Cells were then washed three times with 161

PBS, and incubated with rhodamine- or FITC-labeled secondary antibody for 1 h at 4°C. Cells 162

were then washed three times PBS, followed by nuclear staining with DAPI (4´6-diamidino-2-163

phenylindole) (300 nM). Finally, cells were washed three times and mounted. Images were taken 164

with DeltaVision deconvolution microscopy (GE Healthcare Life Sciences, Buckinghamshire). 165

Quantification of EGFR internalization was performed using ImageJ as previously described 166

[14]. Briefly, the cells were visualized by differential interface contrast (DIC). For each image, a 167

large polygon (VL) was drawn along the outer edge of the cell membrane to represent the entire 168

area of the cell. In addition, a small polygon (VS) was drawn along the inner edge of the cell 169

membrane to represent the cell interior. The VL and VS values were calculated for either stains of 170

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EGFR, pEGFR, or for YFP (for EGFR-YFP mutants), and membrane EGFR percentage was 171

obtained by the following equation: 172

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RESULTS 175

CBL interaction with EGFR during mitosis 176

EGFR expression at the plasma membrane does not change from interphase to mitosis 177

[13, 14, 42]. Previously, we found that similar to interphase, stimulation of nocodazole-arrested 178

mitotic HeLa cells with high doses of EGF (50 ng/mL) induced the phosphorylation of the EGFR 179

at all major tyrosine residues, including Y992, Y1045, Y1068, Y1086, and Y1173 [13]. 180

Moreover, this also phosphorylated CBL to similar levels [13]. 181

To confirm mitotic CBL activation by EGF stimulation, we observed CBL localization in 182

mitotic HeLa cells by immunofluorescence microscopy. Immunofluorescence co-staining using 183

anti-EGFR and anti-CBL antibodies revealed that CBL co-localizes with EGFR upon 5 min of 184

50 ng/mL EGF treatment in both interphase and mitotic cells (Figure 1A). Furthermore, 185

immunoprecipitation of EGFR using a monoclonal anti-EGFR antibody of both interphase and 186

mitotic cell lysates showed that mitotic cells stimulated with EGF for 5 mins had higher IPs of 187

CBL with EGFR than interphase cells (Figure 1B). Interestingly, CBL IP with EGFR decreased 188

after 30 min EGF in mitotic cells, whereas it increased for interphase cells, and continued 189

increasing at 45 mins EGF. Most surprisingly however, was that ubiquitination of the EGFR was 190

enhanced at all time points studied during mitosis compared to interphase (Figure 1B). Since 191

CBL also binds EGFR indirectly through the EGFR adaptor GRB2, we also immunoblotted 192

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EGFR immunoprecipitates for GRB2 and SHC. The results showed that during mitosis, GRB2 193

and SHC also bind to EGFR following EGF stimulation (Figure 1B). 194

In summary, double indirect immunofluorescence revealed that both EGFR and CBL co-195

localize after EGF stimulation during mitosis. Co-IP experiments also showed that EGF 196

stimulated the interaction between EGFR and CBL. In addition, EGFR is more strongly 197

ubiquitinated by EGF stimulation during mitosis. 198

Effects of altering CBL activity during mitosis 199

CME has been shown to be inhibited during mitosis [27, 29, 30]. Therefore, we sought to 200

discover whether altering CBL activity, the major mediator of NCE, would inhibit EGFR 201

endocytosis. We first silenced CBL in HeLa cells by siRNA transfection and found that 202

transfected mitotic cells had much less EGFR endocytosis following EGF (50 ng/mL) 203

stimulation, as observed by immunofluorescence (IF) staining of activated EGFR (Figure 2A). 204

In comparison, transfected interphase cells were little affected. Similarly, in MCF-7 cells 205

transfected with EGFR-YFP knockdown CBL by siRNA also inhibited mitotic endocytosis of 206

EGFR (Figure 2B). To further verify the role of CBL, we used the dominant-negative 70Z-CBL-207

YFP mutant, which has a deletion of 17 amino acids that disrupts the RING finger structure 208

making it unable to interact properly with ubiquitin-conjugating enzymes (E2 ligases) [43, 44]. 209

The 70Z-CBL-YFP protein can still bind to the cytoplasmic tail of activated EGFR [43, 45-47]. 210

Transfection with 70Z-CBL-YFP significantly inhibited EGF-induced EGFR endocytosis during 211

mitosis, but not in interphase (Figure 3A). Quantification of the data from Figure 3A showed 212

that mitotic cells transfected with 70Z-CBL-YFP retained EGFR at the plasma membrane when 213

compared with non-transfected cells, even after 60 minutes of EGF treatment (50 ng/mL) 214

(Figure 3B). Taken together, downregulating CBL activity decreased EGFR endocytosis in 215

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mitosis, but not in interphase. Therefore, CBL activity appears more important during mitotic 216

EGFR endocytosis than during interphase. 217

We next sought to see whether CBL overexpression could increase the rate of 218

endocytosis during mitosis. CBL overexpression in HeLa cells did not appear to induce 219

endocytosis at earlier time points, nor increase the rate of EGFR internalization (Figure 3C). 220

This is similar to interphase cells, where it was previously reported that overexpression of CBL 221

did not increase the rate of EGFR internalization [46, 48]. 222

Role of EGFR C-terminal domains for mitotic endocytosis 223

We previously showed that mitotic EGFR endocytosis requires EGFR kinase activity 224

[14]. Treatment with the EGFR-tyrosine kinase antagonist AG1418 inhibited mitotic EGFR 225

endocytosis, and washing away AG1478 restored endocytosis [14]. In contrast, interphase EGFR 226

could still undergo endocytosis in the presence of AG1478 [14]. To further explore the role of 227

EGFR kinase activity in the activation of CBL, we blocked EGFR activation with AG1478 for 1 228

h prior to EGF (50 ng/mL) treatment. As before, this treatment prevented EGFR endocytosis 229

during mitosis as visualized by IF [14] (data not shown). We next examined CBL 230

phosphorylation by immunoblotting with antibody to p-CBL and showed that treatment with 231

AG1478 inhibited EGF-induced CBL tyrosine phosphorylation in mitotic cells. (Figure 4A). 232

Therefore, EGFR kinase activity is required for CBL activation. 233

We next sought to investigate which EGFR domains were important for mitotic 234

endocytosis. We made use of previously constructed YFP-tagged EGFR mutants including 235

EGFR with Y1045F substitution (Y1045F-YFP, no direct CBL binding), EGFR truncated at 236

1045 (Δ1044-YFP, no CBL binding), EGFR truncated at 992 (Δ991-YFP, no internalization), 237

and WT (EGFR-YFP) (Figure 5F). We transfected these constructs into MCF-7 or HEK 293T 238

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cells, since they express low amounts of endogenous EGFR, then observed the effects of EGF 239

treatment on their plasma membrane localization using indirect immunofluorescence (Figure 240

4B-E & Figure 5A-E). 241

In non-EGF treated MCF-7 cells, all mutants exhibited high plasma membrane 242

localization and low cytoplasmic localization during both interphase and mitosis (Figure 5A-E). 243

For the cells in the interphase, treatment of EGF (50 ng/mL) for 30, 45, or 60 mins significantly 244

increased the internalization of EGFR-YFP, Y1045F-YFP, and Δ1044-YFP, but not Δ991-YFP 245

which is endocytosis deficient due to the lack of internalization motifs. The internalization levels 246

of EGFR-YFP, Y1045F-YFP, and Δ1044-YFP during interphase at all three time points were all 247

similar. In contrast, these mutants responded to EGF treatment differently from each other when 248

cells were in mitosis. For the cells in mitosis, EGF stimulated strong endocytosis of EGFR-YFP, 249

approximately two-thirds of EGFR-YFP was internalized following 30 min EGF treatment, with 250

more EGFR-YFP becoming internalized at 45 and 60 min. The internalization of both Y1045F-251

YFP and Δ1044-YFP however were impaired in mitosis. In mitosis, no endocytosis of Y1045F-252

YFP mutants was observed following addition of EGF for 30 min, and only very low level of 253

EGFR endocytosis at 45 min. Interestingly, a high proportion of them eventually became 254

endocytosed at 60 min. However, no EGF-induced endocytosis of Δ1044-YFP mutants was 255

observed even at 60 min following EGF addition (Figure 5). Similar results were observed when 256

the experiments were repeated in 293T cells (Figure 4B-E). 257

Taken together, this data shows that the CBL-binding domains of the EGFR are more 258

important for mitotic EGFR endocytosis than interphase. These results also suggest that GRB2 259

cooperation for indirect CBL-binding to EGFR dramatically increases mitotic EGFR 260

endocytosis. 261

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Endocytic trafficking of EGFR in mitosis 262

The endocytic pathway that the EGFR takes has been shown to influence the fate of the 263

EGFR. CME has been shown to lead to EGFR recycling, whereas NCE targets receptors for 264

lysosomal degradation [35, 36]. Since we observed that EGFR endocytosis during mitosis 265

proceeds exclusively in CBL-mediated NCE, we hypothesized that EGFR endocytosis during 266

mitosis should lead exclusively to lysosomal trafficking. To test this, we examined the co-267

localization of endocytic route markers with EGFR by fluorescence microscopy. The EGFR of 268

both mitotic and interphase cells showed strong co-localization with EEA-1 and RAB5 after 30 269

min EGF treatment, indicating that the EGFR is trafficked to early endosomes (Figure 6). EEA-270

1 and RAB5 did not co-localize with any EGFR at the plasma membrane of either mitotic or 271

interphase cells. Staining with the late endosomal markers LAMP-2 showed that EGFR and 272

LAMP-2 colocalized after EGF stimulation for 60 min. These data indicated that EGFR was 273

targeted to lysosomes through NCE during mitosis. 274

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Low EGF doses activate mitotic EGFR NCE 276

The above experiments were all performed using high concentrations of EGF (50 277

ng/mL). During interphase, low doses of EGF only activates CME, whereas high doses activate 278

both CME and NCE [32, 35, 36]. If this finding is also applied to mitosis, low doses of EGF 279

should not induce EGFR endocytosis in mitosis as CME is inhibited in mitosis and only high 280

doses of EGF activates NCE. However, we previously observed that low doses TR-EGF (2 281

ng/mL) could still lead to their internalization in mitotic HeLa and CHO cells in a similar pattern 282

as high dose of EGF [14]. We therefore decided to determine if EGFR endocytosis at both high 283

and low dose of EGF are regulated by CBL-mediated NCE. 284

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We hypothesized that similar to high dose, low dose EGF is still able to stimulate EGFR 285

endocytose through NCE in mitosis. To test this hypothesis, we first repeated experiments 286

described in Figure 3-5 with low dose of EGF (2 ng/ml), and we indeed obtained similar results 287

(Figure 6-7). As shown in Figure 6, in MCF-7 cells transfected with EGFR-YFP, EGF at low 288

dose (2 ng/mL) induced EGFR endocytosis in both interphase and mitotic cells (Figure 7) . The 289

endocytosis of Y1045F-YFP was only observed at 60 min following EGF addition in mitosis, 290

and Δ1044-YFP was deficient in endocytosis during mitosis (Figure 7). These data suggest that 291

at low dose EGF, mitotic EGFR endocytosis require its interaction with CBL. To further 292

determine the involvement of CBL in low dose EGF-stimulated EGFR endocytosis in mitosis, 293

we transfected Hela cells with either wild type CBL-YFP or non-functional 70z-YFP (Figure 8). 294

As in high dose EGF conditions, expression of 70z-YFP inhibited EGFR endocytosis in response 295

to low dose EGF. However, expression of CBL-YFP did not affect EGFR endocytosis in mitosis 296

in response to low dose EGF. Together these data suggest that EGFR endocytosis induced by 297

low dose EGF is also mediated by CBL 298

We then examined why low dose EGF is able to stimulate EGFR endocytosis through 299

CBL-mediated NCE in mitosis, but not in interphase. We examined whether this is due to CBL-300

mediated ubiquitination of EGFR. To this end, we treated Hela cells with EGF at 2 ng/mL or 50 301

ng/mL for 45 min and examined the ubiquitination of EGFR in both interphase and mitosis 302

(Figure 9A). Co-immunoprecipitation of EGFR and immunoblotting for ubiquitin revealed that 303

as before, high dose EGF (50 ng/mL) induced higher EGFR ubiquitination in mitosis more than 304

in interphase. Low dose EGF (2 ng/mL) did not induce the ubiquitination of EGFR in interphase, 305

as previously reported [32, 35, 36]. However, low dose EGF stimulation caused significant 306

EGFR ubiquitination in mitotic cells. Moreover, the binding of CBL to EGFR followed the same 307

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pattern as ubiquitination, with CBL again binding to EGFR at low doses during mitosis, but not 308

during interphase. 309

We also performed similar experiments with different times of EGF stimulation, at 5, 30, 310

and 45 mins (Figure 9B). The phosphotyrosine-specific antibody pY99 was used to confirm 311

EGFR phosphorylation. Blotting for ubiquitin revealed that during mitosis ubiquitination of 312

EGFR occurred at 5 mins, and is sustained through to 45 mins in response to low dose EGF 313

stimulation. In contrast, in interphase EGFR ubiquitination only occurred briefly at 5 min, 314

ubiquitination was very weak at later time points. In addition, CBL and SHC are pulled-down 315

with EGFR during both interphase and mitotic. Therefore, it appears that low dose EGF 316

stimulation differentially ubiquitinates mitotic EGFR, and not interphase EGFR. 317

Since mitotic EGFR is strongly ubiquitinated at low doses of EGF, and ubiquitination has 318

been associated with EGFR degradation, we hypothesized that low EGF doses could lead to 319

EGFR degradation during mitosis. Total cell lysates of interphase and mitotic cells treated with 320

low doses of and total EGFR levels were assayed by Western blot. Whereas interphase EGFR 321

levels remain constant throughout 45 mins of low dose EGF treatment, we found that mitotic 322

EGFR levels drop drastically with time (Figure 9C). Taken together, these results suggest that, 323

unlike interphase, low doses of EGF activate CBL-mediated EGFR degradation in mitotic cells. 324

Interestingly, by Western blotting, the CBL band appears smaller in mitotic samples than 325

interphase samples. 326

DISCUSSION 327

Our results showed that EGF-induced EGFR endocytosis during mitosis proceeds 328

exclusively by CBL-dependent NCE (Figure 10). NCE plays a major role in the regulation of 329

EGFR fate by targeting it to lysosomes for degradation. Our research has uncovered a temporal 330

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period by which to exclusively target EGFR for degradation. This bypasses the receptor 331

recycling pathway that is undesirable if the goal is EGFR attenuation, or if it is to deliver and 332

keep a pharmacological agent into a cell [49]. Targeting mitotic cells is feasible for EGFR-333

overexpressing cancer cells, as these cells intrinsically undergo more cell proliferation. In 334

addition, the population of mitotic cells can be increased by treatment with anti-mitotic drugs, 335

such as the commonly used taxanes and vinca alkaloids. Therefore, mitotic cells of EGFR-336

overexpressing cells can be targeted more directly. Moreover, the FDA-approved EGFR 337

antibody cetuximab has been shown to initiate receptor endocytosis [50]. Whether mitotic 338

EGFR treated with EGFR antibodies are also internalized by NCE remains to be investigated. 339

However, if it does, nano-conjugation of EGFR antibodies to pharmacological agents may 340

provide a targeted approach to treating these cancers. 341

The study of EGFR NCE thus far has relied on the inhibition of clathrin, as well as the 342

use of high doses of EGF to activate NCE. Our results indicate that mitotic EGFR endocytosis is 343

exclusively through NCE. Thus, mitotic cells offer an alternate system for studying the NCE of 344

the EGFR regardless of EGF dosage. In general, NCE is much more complicated and very little 345

understood. It is no surprise that very little is known regarding the regulation of mitotic NCE of 346

EGFR. Our findings that mitotic EGFR endocytosis is mediated by CBL through NCE at both 347

high and low dose of EGF advanced our understanding on both EGFR endocytosis and NCE in 348

general. 349

The theory that EGFR ubiquitination is absolutely necessary for endocytosis has been a 350

subject of controversy [51-54]. Our research supports the notion that ubiquitination by CBL is 351

important for NCE [32, 36]. Furthermore, our research provides strong support for the 352

requirement of CBL and GRB2 binding to the EGFR in order to cause its ubiquitination [35, 36]. 353

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Our results argue that GRB2-mediated CBL binding is more important than direct CBL-binding 354

during mitosis, as the internalization of Δ1044 mutant had significantly inhibited, whereas the 355

internalization of Y1045F mutant had only slightly inhibited. Overexpression of CBL did not 356

accelerate nor enhance mitotic EGFR endocytosis in response to EGF. This has also been 357

recently demonstrated by in vivo experiments [55]. NCE has been reported to initiate more 358

slowly than CME [9, 32-34, 43, 56], and it therefore appears that CBL overexpression is not the 359

limiting factor to the speed of EGFR NCE. Other important mediators of NCE, for example 360

EPS15, EPS15R, and EPSIN [32], or endoplasmic reticulum (ER)-resident protein reticulon 3 361

(RTN3) and CD147 [57] may warrant investigation. More importantly, the exact mechanism by 362

which the ubiquitination of the EGFR induces internalization is still unknown, and studies to 363

elucidate the precise molecular mechanism would be extremely impactful. 364

The inactivation of CBL has been shown to display pro-oncogenic features [58-62]. 365

Moreover, common pro-oncogenic EGFR mutations L858R and L858R/T790M have impaired 366

CBL-binding, slower endocytosis, and impaired degradation [63]. EGFRvIII, the most common 367

variant in gliomas, also has a reduced interaction with CBL and thus impaired ubiquitination 368

owing to hypophosphorylation of pY1045 [64, 65]. Here, we showed that CBL activity during 369

mitosis is even more important, and its activity is enhanced compared to interphase cells. 370

Evolutionarily, since mitotic cells do not have active CME [27, 29], the activation of NCE may 371

have been even more critical during mitosis to suppress EGFR overactivation. A loss of CBL 372

activity, whether by inactivating mutations to CBL or EGFR CBL-binding, would therefore have 373

a more pronounced effect during mitosis, as the EGFR would continue to signal excessively. The 374

functional role of mitotic EGFR activation is still not well known. It is unclear how abnormally 375

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sustained EGFR signaling during mitosis affects cellular processes, however, it does appear to 376

help mitotic cancer cells resist nocodazole-mediated cell death [13]. 377

We showed that the EGFR is more strongly ubiquitinated during mitosis at both low and 378

high doses of EGF, suggesting that CBL activity is enhanced during mitosis. How can CBL be 379

better primed to induce endocytosis, even at low concentrations of EGF during mitosis? It has 380

been shown that CBL also acts as an adaptor in the CME pathway. Since CME is no longer 381

active during mitosis, a possible explanation may be due to increased CBL protein availability, 382

as the cellular pool of CBL no longer needs to divide its time between CME and NCE. Another 383

explanation may be that CBL is modified during mitosis to be better primed for its E3 ligase 384

activity. Interestingly, probing with the CBL antibody revealed that the mitotic CBL band 385

appears smaller compared to interphase (Figure 8), although the exact significance is unknown. 386

Another alternative possibility may revolve around the DUBs (deubiquitinating enzymes) that 387

deubiquitinate EGFR. Fifteen DUBs have been reported to impact EGFR fate, although some 388

may be deubiquitinating non-EGFR component, such as EPS15 [66]. These DUBs could be shut 389

off during mitosis, causing ubiquitination to persist longer than during the interphase. 390

Mitosis represents a phase of tremendous transition to the cell. A significant change is the 391

global phosphorylation of mitotic proteins by mitotic kinases. For example, studies have shown 392

the mitotic phosphorylation of over 1000-6027 proteins, including 14,000-50,000 393

phosphorylation events depending on the study [67-69]. Interestingly, many phospho-sites 394

overlap between EGF-stimulated cells and mitotic cells [69]. Indeed, various components of the 395

EGFR signaling and endocytic pathways appear to play different roles in mitosis, a phenomenon 396

known as moonlighting [28, 70]. This includes important members of EGFR CME, such as 397

clathrin, dynamin, and AP-2 [28, 71-74]. Since these proteins and many others are moonlighting 398

19

in mitosis-related processes, their availability to participate in EGFR endocytosis during mitosis 399

may be compromised. This may also affect EGFR signaling. It has been shown that EGF-400

induced AKT activation requires EGFR residence in clathrin coated pits, but not internalization 401

[75, 76]. We previously showed that only AKT2, and not AKT1, becomes activated following 402

EGF stimulation during mitosis [13]. Since CME is shut down during mitosis, it can be 403

speculated that the differential activation of AKT during mitosis is a consequence of the inability 404

of clathrin to be involved in mitotic EGFR endocytosis. Therefore, the changes imparted by 405

global mitotic phosphorylation and mitotic cell rounding cannot be discounted to EGFR 406

signaling, and likely of other signaling receptors as well. 407

In our study, we made use of the microtubule depolymerizer nocodazole to arrest cells in 408

mitosis. So far, nocodazole is still the most widely used drug for arresting cells in mitosis [69, 409

77-79]. We decided to use nocodazole in our research in order to obtain synchrony between our 410

Western blots, co-IPs, and immunofluorescence experiments, as it has been shown that the sub-411

stage of mitosis can influence the kinetics of endocytosis [14]. Previous research has showed that 412

factors such as serum starvation, nocodazole, and other mitotic inhibitors could inhibit CME 413

[80]. However, it should be noted that the researchers were evaluating transferrin receptors, 414

which is endocytosed by constitutive endocytosis rather than the ligand-induced mechanism used 415

by EGFR. Furthermore, our previous study that showed that clathrin downregulation by siRNA 416

had no effect on mitotic EGFR endocytosis was performed without the use of nocodazole [14]. 417

We have also previously shown that 16 h nocodazole treatment does not lead to significant cell 418

apoptosis [13]. 419

The EGFR uses various signaling pathways to achieve numerous pro-oncogenic cellular 420

outcomes. Endocytosis downregulates EGFR signaling from the cell surface, but initiates 421

20

intracellular signaling from endosomes [81]. In this way, endocytosis controls EGFR signaling, 422

spatially and temporally, making it an indispensable part of receptor signaling. Therefore, the 423

interplay between EGFR signaling and endocytosis critically determines cellular outcome. 424

In conclusion, our research further showed that mitotic EGFR endocytosis proceed through 425

CBL-mediated NCE, which supports the notion that mitotic endocytosis is not completely 426

inhibited, but proceeds through NCE. The unique property of mitotic EGFR endocytosis offers 427

an important opportunity for developing cancer therapy that targets both EGFR and mitosis. 428

429

430

431

21

ACKNOWLEDGEMENTS 432

We thank Dr. A. Sorkin (Department of Cell Biology, University of Pittsburgh School of 433

Medicine, Pittsburgh, PA, USA 15261) for generously sending us the CBL constructs including 434

c-CBL-YFP and 70z-CBL-YFP. This work was supported in part by grants from the Canadian 435

Institutes of Health Research (CIHR). Ping Wee is supported in part by a scholarship from the 436

Natural Sciences and Engineering Research Council. 437

22

FIGURES 438

Figure 1. CBL is activated by EGF-stimulation during mitosis. A) Direct 439

immunofluorescence images of HeLa cells stained with CBL (green), EGFR pY1086 (red), and 440

DAPI (blue). Cells were treated with EGF (50 ng/mL) for the indicated times. B) Co-441

immunoprecipitation of EGFR from asynchronous (interphase) or nocodazole-arrested (mitosis) 442

HeLa cells. EGF (50 ng/mL) was used to treat cells for the indicated times. Immunoblotting was 443

performed with the specified antibodies. Mitotic EGFR is more strongly ubiquitinated than 444

interphase. Total cell lysates (input) are also shown. Results are representative of at least two 445

biological replicates. Size bar: 20µm. 446

447

Figure 2. siRNA downregulation CBL inhibits mitotic endocytosis of EGFR. Indirect 448

immunofluorescence to observe EGFR endocytosis in cells treated with CBL siRNA or with 449

scramble siRNA in: A) HeLa cells stained for CBL (green) and EGFR pY1086 (red) and treated 450

with EGF (50 ng/mL) for 15 mins; and B) MCF-7 cells transfected with EGFR-YFP and treated 451

with EGF (50 ng/mL) for 45 mins. MCF-7 cells were treated with nocodazole (200 ng/mL) for 452

16 h. Size bar: 20µm. 453

454

Figure 3. The effects of downregulation and overexpression of CBL on mitotic endocytosis 455

of EGFR. Indirect immunofluorescence to observe EGFR endocytosis in HeLa cells transfected 456

with dominant-negative CBL (70z-YFP) or wild type c-CBL-YFP. Following the transfection of 457

CBL, the cells were treated with nocodazole (200 ng/mL) for 16 h. The cells were then treated 458

with EGF (50 ng/mL) for the indicated times and were stained for pY1086 (red), and DAPI 459

(blue). The transfected cells were green. (A) EGFR endocytosis in cells transfected with 70z-460

23

YFP. (B) Quantification of EGFR retained in the plasma membrane in mitotic cells from 461

experiments described in (A). Each data is the average of at least 10 mitotic cells. Control are 462

mitotic cells treated with EGF (50 ng/mL) for 2 mins and with fully plasma membrane-localized 463

EGFR. (C) The endocytosis of EGFR in the cells transfected with wild type c-CBL-YFP. The * 464

represents interphase cell, # represents mitotic cell, and #’ represents transfected cell. Size bar: 465

20µm. 466

467

Figure 4. The role of EGFR kinase activation in mitotic EGFR endocytosis. A) The effects 468

of AG1478 on EGFR and CBL phosphorylation. Asynchronous (I-phase) and nocodazole-469

arrested (M-phase) HeLa cells were pre-treated with AG1478 1 hour prior to EGF treatment, 470

then treated with EGF (50 ng/mL) for the indicated times. To study the role of EGFR C-terminal 471

domains, 293T cells were transfected with with B) EGFR-YFP (positive control), C) EGFR-472

Y1045F-YFP (no direct CBL binding), D) EGFR-Δ1044-YFP (no CBL binding), and E) EGFR-473

Δ991-YFP (negative control). Cells were treated with EGF (50 ng/mL) for the specified times 474

and observed by indirect immunofluorescence. Cell cycle phase of cells were determined by 475

DNA morphology (not shown). Cells were treated with nocodazole (200 ng/mL) for 16 h. Size 476

bar: 20µm. 477

478

Figure 5. Necessity of EGFR’s CBL-binding domains for EGFR endocytosis. MCF-7 cells 479

were transfected with A) EGFR-YFP (positive control), B) EGFR-Y1045F-YFP (no direct CBL 480

binding), C) EGFR-Δ1044-YFP (no CBL binding), and D) EGFR-Δ991-YFP (negative control). 481

Cells were treated with nocodazole (200 ng/mL) for 16 h and with EGF (50 ng/mL) for the 482

specified times and observed by indirect immunofluorescence. Cell cycle phase of cells were 483

24

determined by DNA morphology (not shown). E) Quantification of plasma membrane retainment 484

of YFP for A)-C) for at least 10 cells (see Materials and Methods). F) Illustration of EGFR 485

mutants used and their ability to bind CBL. Size bar: 20µm. 486

487

Figure 6. Mitotic EGFR is sorted to early endosomes and lysosomes. HeLa cells were treated 488

with nocodazole (200 ng/mL) and EGF (50 ng/mL) for 30 mins and EGFR co-localization with 489

early endosome markers and lysosomal markers were observed by indirect immunofluorescence. 490

Cells were stained with EGFR (green), DAPI (blue) and either: A) RAB5 (red), B) EEA-1 (red), 491

or C) LAMP-2 (red). Size bar: 20µm. 492

493

Figure 7. Effects of low EGF dose on mitotic EGFR endocytosis. Cells were treated with EGF 494

(2 ng/mL) to only activate CME for the specified times and observed by indirect 495

immunofluorescence. Cell cycle phase of cells were determined by DNA morphology (not 496

shown). MCF-7 cells were transfected with A) EGFR-YFP (positive control), B) EGFR-497

Y1045F-YFP (no direct CBL binding), C) EGFR-Δ1044-YFP (no CBL binding), and D) EGFR-498

Δ991-YFP (negative control). Cells were treated with nocodazole (200 ng/mL) for 16 h. Size 499

bar: 20µm. 500

501

Figure 8. Effects of low EGF dose on endocytosis in cells with CBL alterations. HeLa cells 502

were transfected with E) Dominant-negative CBL (70z-YFP) and F) CBL-YFP and treated with 503

low dose EGF (2 ng/mL) for the indicated times. Cells were stained with EGFR pY1086 (red) 504

and DAPI (blue). The * represents interphase cell, # represents mitotic cell, and #’ represents 505

transfected cell. Cells were treated with nocodazole (200 ng/mL) for 16 h. Size bar: 20µm. 506

25

507

Figure 9. Ubiquitination and CBL-binding of EGFR during low EGF doses. Co-508

immunoprecipitation of EGFR from asynchronous (interphase) or nocodazole-arrested (mitosis) 509

HeLa cells. A) Cells were treated with EGF for 45 mins using low and high dose EGF (2 and 50 510

ng/mL). B) Cells were treated with low dose EGF (2 ng/mL) for 0, 5, 30, or 45 mins. High dose 511

EGF treatments for 45 minutes are included for reference. C) Immunoblotting of total cell lysate 512

(TCL) with the specified antibodies. Results are representative of at least two biological 513

replicates. 514

515

Figure 6. Model of EGFR endocytosis during mitosis. In interphase cells, low EGF doses (>2 516

ng/mL) only activates clathrin-mediated endocytosis (CME), leading to receptor recycling. High 517

EGF doses (>20 ng/mL) also activates CME, but can also activate non-clathrin mediated 518

endocytosis (NCE) due to the dose-dependent activation of CBL and EGFR ubiquitination. NCE 519

leads to lysosomal degradation of EGFR. In mitotic cells, CME is shut off. Therefore, EGFR 520

endocytosis must proceed by NCE. Both low and high concentrations of EGF activate NCE 521

during mitosis, and this may be because contrary to interphase cells, low EGF concentrations can 522

activate CBL and EGFR ubiquitination. Therefore, mitotic EGFR endocytosis leads exclusively 523

to lysosomal degradation. 524

525

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