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ULK1 phosphorylates Exo70 to suppress breast cancer metastasis

Mao, Liyuan, Zhan, Yan-yan, Wu, Bin, Yu, Qiang, Xu, Liang, Hong, Xiaoting, Zhong, Linhai, Mi, Panying, Xiao, Li, Wang, Xinquan, Cao, Hanwei, Zhang, Wenqing, Chen, Binbin, Xiang, Jingzhou, Mei, Kunrong, Radhakrishnan, Ravi, Guo, Wei, Hu, Tianhui·2020·Nature Communications
DOI 10.1038/s41467-019-13923-7PMC PMC6949295
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Abstract

Increased expression of protein kinase ULK1 was reported to negatively correlate with breast cancer metastasis. Here we report that ULK1 suppresses the migration and invasion of human breast cancer cells. The suppressive effect is mediated through direct phosphorylation of Exo70, a key component of the exocyst complex. ULK1 phosphorylation inhibits Exo70 homo-oligomerization as well as its assembly to the exocyst complex, which are needed for cell protrusion formation and matrix metalloproteinases secretion during cell invasion. Reversely, upon growth factor stimulation, Exo70 is phosphorylated by ERK1/2, which in turn suppresses its phosphorylation by ULK1. Together, our study identifies Exo70 as a substrate of ULK1 that inhibits cancer metastasis, and demonstrates that two counteractive regulatory mechanisms are well orchestrated during tumor cell invasion.

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Fig. 1 ULK1 inhibited migration and invasion of breast cancer cells. a The levels of ULK1 expression in breast cancer cell lines of different metastasis ability were examined by western blotting. β-Actin was used as a loading control. b, c Effects of ULK1 and ULK1(M92A) on the migration and invasion of MDA-MB-231 cells. Transwell assay was carried out using cells transfected with empty vector, HA-ULK1, or HA-ULK1(M92A) plasmid. Representative images (scale bar: 100 μm) (b) and cell counts (c) were shown (n = 6 biologically independent samples). AU: arbitrary unit; I/M: ratio of invasion (I) to migration (M). d, e Overexpressed ULK1 but not ULK1(M92A) significantly inhibited the degradation of extracellular matrix by MDA-MB-231 cells. Representative images (d) and gelatin degradation levels from 45 cells (e) for each group were shown (n = 3 independent experiments). Scale bar: 10 μm. f–i ULK1 knockdown increased the invasion and migration ability of MCF-7 cells as examined by the transwell assay (f, g) (scale bar: 100 μm) and wound healing assay (h, i) (scale bar: 200 μm). Representative images (f, h), cell counts (g), and quantification of wound distance (i) were shown (n = 6 biologically independent samples for f, g, n = 5 biologically independent samples for h, i). Data represented the means ± SEM. P values were analyzed by unpaired two-tailed Student’s t test and Kruskal–Wallis test (e).
Fig. 1 ULK1 inhibited migration and invasion of breast cancer cells. a The levels of ULK1 expression in breast cancer cell lines of different metastasis ability were examined by western blotting. β-Actin was used as a loading control. b, c Effects of ULK1 and ULK1(M92A) on the migration and invasion of MDA-MB-231 cells. Transwell assay was carried out using cells transfected with empty vector, HA-ULK1, or HA-ULK1(M92A) plasmid. Representative images (scale bar: 100 μm) (b) and cell counts (c) were shown (n = 6 biologically independent samples). AU: arbitrary unit; I/M: ratio of invasion (I) to migration (M). d, e Overexpressed ULK1 but not ULK1(M92A) significantly inhibited the degradation of extracellular matrix by MDA-MB-231 cells. Representative images (d) and gelatin degradation levels from 45 cells (e) for each group were shown (n = 3 independent experiments). Scale bar: 10 μm. f–i ULK1 knockdown increased the invasion and migration ability of MCF-7 cells as examined by the transwell assay (f, g) (scale bar: 100 μm) and wound healing assay (h, i) (scale bar: 200 μm). Representative images (f, h), cell counts (g), and quantification of wound distance (i) were shown (n = 6 biologically independent samples for f, g, n = 5 biologically independent samples for h, i). Data represented the means ± SEM. P values were analyzed by unpaired two-tailed Student’s t test and Kruskal–Wallis test (e).CC BY · PubMed Central
Fig. 2 Exo70 interacts with ULK1. a Interaction of Exo70 and ULK1 in 293T cells was detected by co-immunoprecipitation assay. Whole-cell lysates (WCLs) and immunoprecipitated (IP) proteins were analyzed by western blotting. b The interaction of endogenous Exo70 with ULK1 in MCF-7 and MDA-MB-231 cells were examined by co-immunoprecipitation using anti-Exo70 or anti-IgG (negative control) antibody. c The interaction of Exo70 and ULK1 was determined by GST pull-down assay. Purified HA-ULK1 protein was incubated with recombinant GST-Exo70 protein conjugated to the Glutathione Sepharose. GST-Exo70 was examined by Coomassie Blue staining (lower panel) and the bounded HA-ULK1 was detected with anti-HA antibody (upper panel). GST was used as a negative control. d The co-localization of endogenous Exo70, ULK1, and cortactin was demonstrated with immunofluorescence in MCF-7 and MDA-MB-231 cells. Scale bar: 10 μm.
Fig. 2 Exo70 interacts with ULK1. a Interaction of Exo70 and ULK1 in 293T cells was detected by co-immunoprecipitation assay. Whole-cell lysates (WCLs) and immunoprecipitated (IP) proteins were analyzed by western blotting. b The interaction of endogenous Exo70 with ULK1 in MCF-7 and MDA-MB-231 cells were examined by co-immunoprecipitation using anti-Exo70 or anti-IgG (negative control) antibody. c The interaction of Exo70 and ULK1 was determined by GST pull-down assay. Purified HA-ULK1 protein was incubated with recombinant GST-Exo70 protein conjugated to the Glutathione Sepharose. GST-Exo70 was examined by Coomassie Blue staining (lower panel) and the bounded HA-ULK1 was detected with anti-HA antibody (upper panel). GST was used as a negative control. d The co-localization of endogenous Exo70, ULK1, and cortactin was demonstrated with immunofluorescence in MCF-7 and MDA-MB-231 cells. Scale bar: 10 μm.CC BY · PubMed Central
Fig. 3 Exo70 is phosphorylated by ULK1 on Ser47, Ser59, and Ser89. a The level of threonine/serine phosphorylation of endogenous Exo70 under starvation conditions or rapamycin treatment. MDA-MB-231 cells were placed in starvation medium (EBSS, HBSS, or DMEM medium sugarless) or treated with rapamycin (50 nM) for 2 h, and then subjected to immunoprecipitation using anti-Exo70 antibody. Whole-cell lysates (WCLs) and immunoprecipitated (IP) proteins were analyzed by western blotting. Threonine/serine phosphorylation on Exo70 was detected by anti-p-Ser/Thr antibody. b 293T cells were transfected with empty vector or HA-ULK1 plasmid, and immunoprecipitation assay was carried out with anti-Exo70 antibody. Overexpression of ULK1 (HA-ULK1) increased the threonine/serine phosphorylation of endogenous Exo70 protein. c In vitro phosphorylation of Exo70 by ULK1. Bacterially purified Exo70 was incubated with purified HA-ULK1 and HA-ULK1(M92A) in the in vitro phosphorylation assay as described in Materials and methods. d Potential ULK1-phosphorylated sites within the N terminus of Exo70 based on the consensus ULK1 phospho-substrate sequence. e Effects of Exo70 mutations on its phosphorylation induced by ULK1. HA-ULK1 was co-transfected with the indicated FLAG-Exo70 plasmids in 293T cells, and then subjected to immunoprecipitation with anti-Flag antibody. Phosphorylation of Exo70 at serine/threonine was examined with p-Ser/Thr antibody. f Ser89 was identified to be another potential ULK1-phosphorylated site using immunoprecipitation in combination with mass spectrometry (IP-MS). g In vitro phosphorylation of wild-type and mutant Exo70 by purified ULK1. Exo70 at serine/threonine was examined with p-Ser/Thr antibody.
Fig. 3 Exo70 is phosphorylated by ULK1 on Ser47, Ser59, and Ser89. a The level of threonine/serine phosphorylation of endogenous Exo70 under starvation conditions or rapamycin treatment. MDA-MB-231 cells were placed in starvation medium (EBSS, HBSS, or DMEM medium sugarless) or treated with rapamycin (50 nM) for 2 h, and then subjected to immunoprecipitation using anti-Exo70 antibody. Whole-cell lysates (WCLs) and immunoprecipitated (IP) proteins were analyzed by western blotting. Threonine/serine phosphorylation on Exo70 was detected by anti-p-Ser/Thr antibody. b 293T cells were transfected with empty vector or HA-ULK1 plasmid, and immunoprecipitation assay was carried out with anti-Exo70 antibody. Overexpression of ULK1 (HA-ULK1) increased the threonine/serine phosphorylation of endogenous Exo70 protein. c In vitro phosphorylation of Exo70 by ULK1. Bacterially purified Exo70 was incubated with purified HA-ULK1 and HA-ULK1(M92A) in the in vitro phosphorylation assay as described in Materials and methods. d Potential ULK1-phosphorylated sites within the N terminus of Exo70 based on the consensus ULK1 phospho-substrate sequence. e Effects of Exo70 mutations on its phosphorylation induced by ULK1. HA-ULK1 was co-transfected with the indicated FLAG-Exo70 plasmids in 293T cells, and then subjected to immunoprecipitation with anti-Flag antibody. Phosphorylation of Exo70 at serine/threonine was examined with p-Ser/Thr antibody. f Ser89 was identified to be another potential ULK1-phosphorylated site using immunoprecipitation in combination with mass spectrometry (IP-MS). g In vitro phosphorylation of wild-type and mutant Exo70 by purified ULK1. Exo70 at serine/threonine was examined with p-Ser/Thr antibody.CC BY · PubMed Central
Fig. 4 Phosphorylation of Exo70 on Ser47/59/89 inhibited breast cancer metastasis. a–d Exo70 phosphorylation on Ser47, Ser59, and Ser89 inhibited the migration and invasion of MDA-MB-231 cells as analyzed by transwell assay (a, b) (scale bar: 100 μm) and wound healing assay (c, d) (scale bar: 200 μm). Control (Scramble) and Exo70 shRNA knockdown cells were transfected with empty vector, Exo70-Flag or Exo70 mutant variants. Representative images (a, c), cell counts (b), and quantification of wound distance (d) were shown (n = 6 biologically independent samples for a, b, n = 3 biologically independent samples for c, d). AU: arbitrary unit. e, f Exo70 phosphorylation on Ser47, Ser59, and Ser89 suppressed the metastasis of MDA-MB-231 cells to the lung. MDA-MB-231 cells stably expressing empty vector, Exo70-Flag, Exo70(3D)-Flag, or Exo70(3A)-Flag were subjected to tail-vein injection into nude mice (vector n = 6, WT n = 6, 3D n = 8, 3A n = 5). Representative images (e) and quantification (f) of Luciferase expression within the lungs of nude mice in each group were shown. Data represented the mean ± SEM. The difference in significance was analyzed by one-way analysis of variance (ANOVA) (b, d) or unpaired two-tailed Student’s t test (f no adjustment was made for multiple comparisons). *P < 0.05; **P < 0.01; ***P < 0.001; and n.s., nonsignificant vs. the indicated control.
Fig. 4 Phosphorylation of Exo70 on Ser47/59/89 inhibited breast cancer metastasis. a–d Exo70 phosphorylation on Ser47, Ser59, and Ser89 inhibited the migration and invasion of MDA-MB-231 cells as analyzed by transwell assay (a, b) (scale bar: 100 μm) and wound healing assay (c, d) (scale bar: 200 μm). Control (Scramble) and Exo70 shRNA knockdown cells were transfected with empty vector, Exo70-Flag or Exo70 mutant variants. Representative images (a, c), cell counts (b), and quantification of wound distance (d) were shown (n = 6 biologically independent samples for a, b, n = 3 biologically independent samples for c, d). AU: arbitrary unit. e, f Exo70 phosphorylation on Ser47, Ser59, and Ser89 suppressed the metastasis of MDA-MB-231 cells to the lung. MDA-MB-231 cells stably expressing empty vector, Exo70-Flag, Exo70(3D)-Flag, or Exo70(3A)-Flag were subjected to tail-vein injection into nude mice (vector n = 6, WT n = 6, 3D n = 8, 3A n = 5). Representative images (e) and quantification (f) of Luciferase expression within the lungs of nude mice in each group were shown. Data represented the mean ± SEM. The difference in significance was analyzed by one-way analysis of variance (ANOVA) (b, d) or unpaired two-tailed Student’s t test (f no adjustment was made for multiple comparisons). *P < 0.05; **P < 0.01; ***P < 0.001; and n.s., nonsignificant vs. the indicated control.CC BY · PubMed Central
Fig. 5 ULK1 phosphorylation suppressed Exo70 oligomerization. a The 293T cells were co-transfected with HA-tagged and Flag-tagged wild-type and mutant Exo70 in the presence or absence of Myc-ULK1, and then subjected to co-immunoprecipitation assay. b Self-interaction of Exo70 proteins was inhibited by its phosphorylation on Ser47, Ser59, and Ser89. The 293T cells were co-transfected with HA- or Flag-tagged Exo70 or its mutant variants, and then subjected to co-immunoprecipitation assay. c Recombinant Exo70 protein was loaded onto a Superdex 200 10/300GL column and the elution was analyzed. The elution peak of Exo70-3D shifted to a lower-molecular weight position compared to Exo70-WT. d Purified recombinant Exo70 protein was loaded onto a Superdex 200 10/300GL column and the eluted fractions were analyzed by SDS-PAGE and Coomassie blue staining. e Exo70-WT and Exo70-3A induced much more protrusions than Exo70-3D. MDA-MB-231 cells were transfected with GFP-Exo70, GFP-Exo70(3D), or GFP-Exo70(3A), and then stained with Rhodamine-phalloidin to label F-action. Scale bar: 10 μm. Magnification of boxed area was shown in the lower panel. f Protrusions index from at least 31 cells for each group was shown, distinguished by whether the number of filopodia in each cell is greater than five. Data represented the mean ± SEM (n = 3, three independent experiments), and the P values were analyzed by unpaired two-tailed Student’s t test. g The 293T cells were transfected with Exo70-Flag or Exo70(3D)-Flag, together with HA-Sec6, HA-Sec8, or HA-Exo84, and then subjected to co-immunoprecipitation assay. Interaction of Exo70 with Sec6, Sec8, and Exo84 were inhibited by phosphorylation of Exo70 on Ser47, Ser59, and Ser89. h Effects of Exo70 mutations on MMP-9 secretion in MDA-MB-231 cells as analyzed by zymography. Cells were transfected with empty vector, HA-Exo70, or HA-Exo70(3D) plasmid. Cellular proteins were also prepared and subjected to western blotting, with β-actin as an internal control. i The levels of Exo70 phosphorylation on Ser89 and the expression levels of ULK1 in five pairs of primary breast tumor tissues and the matched adjacent normal tissues. β-Actin was used as an internal control. N, normal; T, tumor.
Fig. 5 ULK1 phosphorylation suppressed Exo70 oligomerization. a The 293T cells were co-transfected with HA-tagged and Flag-tagged wild-type and mutant Exo70 in the presence or absence of Myc-ULK1, and then subjected to co-immunoprecipitation assay. b Self-interaction of Exo70 proteins was inhibited by its phosphorylation on Ser47, Ser59, and Ser89. The 293T cells were co-transfected with HA- or Flag-tagged Exo70 or its mutant variants, and then subjected to co-immunoprecipitation assay. c Recombinant Exo70 protein was loaded onto a Superdex 200 10/300GL column and the elution was analyzed. The elution peak of Exo70-3D shifted to a lower-molecular weight position compared to Exo70-WT. d Purified recombinant Exo70 protein was loaded onto a Superdex 200 10/300GL column and the eluted fractions were analyzed by SDS-PAGE and Coomassie blue staining. e Exo70-WT and Exo70-3A induced much more protrusions than Exo70-3D. MDA-MB-231 cells were transfected with GFP-Exo70, GFP-Exo70(3D), or GFP-Exo70(3A), and then stained with Rhodamine-phalloidin to label F-action. Scale bar: 10 μm. Magnification of boxed area was shown in the lower panel. f Protrusions index from at least 31 cells for each group was shown, distinguished by whether the number of filopodia in each cell is greater than five. Data represented the mean ± SEM (n = 3, three independent experiments), and the P values were analyzed by unpaired two-tailed Student’s t test. g The 293T cells were transfected with Exo70-Flag or Exo70(3D)-Flag, together with HA-Sec6, HA-Sec8, or HA-Exo84, and then subjected to co-immunoprecipitation assay. Interaction of Exo70 with Sec6, Sec8, and Exo84 were inhibited by phosphorylation of Exo70 on Ser47, Ser59, and Ser89. h Effects of Exo70 mutations on MMP-9 secretion in MDA-MB-231 cells as analyzed by zymography. Cells were transfected with empty vector, HA-Exo70, or HA-Exo70(3D) plasmid. Cellular proteins were also prepared and subjected to western blotting, with β-actin as an internal control. i The levels of Exo70 phosphorylation on Ser89 and the expression levels of ULK1 in five pairs of primary breast tumor tissues and the matched adjacent normal tissues. β-Actin was used as an internal control. N, normal; T, tumor.CC BY · PubMed Central
Fig. 6 ULK1 phosphorylation of Exo70 was suppressed by ERK1/2 phosphorylation. a ULK1 phosphorylation of Exo70 on Ser89 was affected by EGF and PD98059 treatment. The 293T cells transfected with ULK1-Flag and/or HA-Exo70 as indicated were treated with EGF or PD98059. The cell lysates were then subjected to immunoprecipitation with anti-HA antibody. Phosphorylation of Exo70 at Ser89 was examined by the anti-pSer89 antibody and quantified by densitometry. Data represented the mean ± SEM (n = 3, three independent experiments), and the P values were analyzed by unpaired two-tailed Student’s t test. b ULK1 phosphorylation of Exo70 on Ser89 was lower than that of Exo70(S250D). The 293T cells were co-transfected with ULK1-Flag and HA-Exo70 or HA-Exo70(S250D), and then subjected to immunoprecipitation with anti-HA antibody. Phosphorylation of Exo70 at Ser89 was examined by the anti-pSer89 antibody. c The interaction of ULK1 with Exo70(S250D) was compared with that of ULK1 and wild-type Exo70 by co-immunoprecipitation assay. d Regulation of Exo70 by ULK1 and ERK1/2 under different growth condition. Upon energy stress, ULK1 was activated and phosphorylated Exo70 on Ser47, Ser59, and Ser89, subsequently inhibiting the self-oligomerization of Exo70 and its interaction with other exocyst subunits; thereafter, decreasing filopodial protrusions formation and MMP secretion, which led to the suppression of breast cancer cell migration and invasion. Under growth stimuli, activated ERK1/2 phosphorylated Exo70 on Ser250, which facilitates cell invasion via enhancing the interaction of Exo70 with other exocyst subunits and thus increasing invadopodia formation and MMP secretion (our published data18); additionally, Exo70 phosphorylation on Ser250 by ERK1/2 could also suppressed ULK1-induced phosphorylation of Exo70 to release metastasis inhibition.
Fig. 6 ULK1 phosphorylation of Exo70 was suppressed by ERK1/2 phosphorylation. a ULK1 phosphorylation of Exo70 on Ser89 was affected by EGF and PD98059 treatment. The 293T cells transfected with ULK1-Flag and/or HA-Exo70 as indicated were treated with EGF or PD98059. The cell lysates were then subjected to immunoprecipitation with anti-HA antibody. Phosphorylation of Exo70 at Ser89 was examined by the anti-pSer89 antibody and quantified by densitometry. Data represented the mean ± SEM (n = 3, three independent experiments), and the P values were analyzed by unpaired two-tailed Student’s t test. b ULK1 phosphorylation of Exo70 on Ser89 was lower than that of Exo70(S250D). The 293T cells were co-transfected with ULK1-Flag and HA-Exo70 or HA-Exo70(S250D), and then subjected to immunoprecipitation with anti-HA antibody. Phosphorylation of Exo70 at Ser89 was examined by the anti-pSer89 antibody. c The interaction of ULK1 with Exo70(S250D) was compared with that of ULK1 and wild-type Exo70 by co-immunoprecipitation assay. d Regulation of Exo70 by ULK1 and ERK1/2 under different growth condition. Upon energy stress, ULK1 was activated and phosphorylated Exo70 on Ser47, Ser59, and Ser89, subsequently inhibiting the self-oligomerization of Exo70 and its interaction with other exocyst subunits; thereafter, decreasing filopodial protrusions formation and MMP secretion, which led to the suppression of breast cancer cell migration and invasion. Under growth stimuli, activated ERK1/2 phosphorylated Exo70 on Ser250, which facilitates cell invasion via enhancing the interaction of Exo70 with other exocyst subunits and thus increasing invadopodia formation and MMP secretion (our published data18); additionally, Exo70 phosphorylation on Ser250 by ERK1/2 could also suppressed ULK1-induced phosphorylation of Exo70 to release metastasis inhibition.CC BY · PubMed Central

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Authors
Mao, L., Zhan, Y.-Y., Wu, B., Yu, Q., Xu, L., Hong, X., Zhong, L., Mi, P., Xiao, L., Wang, X., Cao, H., Zhang, W., Chen, B., Xiang, J., Mei, K., Radhakrishnan, R., Guo, W., & Hu, T.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-01-08
Volume
11
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-019-13923-7
PMC
PMC6949295
Cited by
58
References
2
Language
en
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Funders
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R01GM111128)
  • National Natural Science Foundation of China (81572589, U1405228, 81472568, 31770860)

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Mao, L., Zhan, Y.-Y., Wu, B., Yu, Q., Xu, L., Hong, X., Zhong, L., Mi, P., Xiao, L., Wang, X., Cao, H., Zhang, W., Chen, B., Xiang, J., Mei, K., Radhakrishnan, R., Guo, W., & Hu, T. (2020). ULK1 phosphorylates Exo70 to suppress breast cancer metastasis. Nature Communications. https://doi.org/10.1038/s41467-019-13923-7