Regulation of EGFR endocytosis by CBL during mitosis · 11 Endocytosis plays important roles in...
Transcript of Regulation of EGFR endocytosis by CBL during mitosis · 11 Endocytosis plays important roles in...
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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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742
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