1. Volume 22, Issue 5, Pages 967-978 (May 2012)
ERK1/2 Regulate Exocytosis through Direct Phosphorylation of the Exocyst Component Exo70 
Jinqi Ren, Wei Guo 
Developmental Cell 
Volume 22, Issue 5, Pages (May 2012)
DOI: /j.devcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Developmental Cell 2012 22, 967-978DOI: (10.1016/j.devcel.2012.03.005)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 ERK1/2 Phosphorylate Exo70 at Serine 250 in Response to EGF
(A) Exo70 contains a conserved ERK1/2 phosphorylation motif “PYSP” (underlined). The asterisk indicates the potential phosphorylation site of rat Exo70 at Serine 250 (S250).
(B) Phosphorylation of endogenous Exo70. Exo70 was immunoprecipitated from EGF treated (5 min) HEK293 cells and then immunoblotted with the anti-Exo70 antibody or anti-ERK1/2 substrate antibody that specifically recognizes the phosphorylation motif in ERK1/2 phosphorylated proteins. The cell lysates were also probed for ERK1/2 and phospho-ERK1/2 (p-ERK1/2) with antibodies against ERK1/2 and phopsph-ERK1/2, respectively (bottom).
(C) ERK1/2 phosphorylate Exo70 at Serine 250. HEK293 cells were transfected with Flag-tagged wild-type Exo70, the Exo70(S250A) mutant, and the Flag vector control (Flag). Flag-tagged Exo70 proteins were immunoprecipitated from the cell lysates using the anti-Flag antibody. The immunoprecipitated Exo70 were analyzed with the anti-ERK1/2 phospho-substrate antibody or the anti-Flag antibody. The anti-ERK1/2 phospho-substrate antibody recognizes the wild-type Exo70, but not the Exo70(S250A) mutant (top). The bottom panel shows the total amounts of immunoprecipitated Flag-Exo70 and Flag-Exo70(S250A).
(D) EGF stimulated the phosphorylation of Exo70. Cells transfected with Flag-tagged Exo70 were stimulated with EGF for 5 or 10 min. Exo70 was immunoprecipitated and analyzed as described above.
(E) Cells were incubated with ERK inhibitor U0126 for 30 min prior to EGF treatment (5 min). Exo70-Flag proteins were immunoprecipitated from cell lysates followed by western blotting as above. U0126 blocked EGF-induced phosphorylation of Exo70 (top). U0126 also blocked ERK1/2 phosphorylation as demonstrated by western blot analysis of the amounts of ERK1/2 and phospho-ERK1/2 in the cell lysates (bottom), indicating the effectiveness of the drug treatment.
(F) U0126 inhibits phosphorylation of Exo70 in a dose-dependent manner. Exo70-Flag transfected HEK293 cells were preincubated with the indicated amounts of U0126 for 30 min and subsequently stimulated with EGF. Phosphorylation of Exo70 and ERK1/2 was analyzed as above.
(G) ERK2 phosphorylates Exo70 in vitro. Purified full-length Exo70 and Exo70 fragment containing amino acids 191–651 (Exo70-C) were incubated with constitutively activated recombinant ERK2 and [32P] γ-ATP in an in vitro kinase assay. The samples were analyzed by SDS-PAGE and autoradiography. The phosphorylation signal was detected in Exo70 and Exo70-C, but not GST.
(H) ERK2 phosphorylates Exo70 at Serine 250 in vitro. Recombinant wild-type Exo70 or S250A mutant was incubated with constitutively-activated ERK2 (ERK2-CA) or kinase-dead mutant ERK2 (ERK2-KD). Samples were subjected to SDS-PAGE and autoradiography. The top panel shows the phosphorylation of Exo70 and ERK2. The bottom panel is Coomassie blue-stained gel showing the amounts of the purified wild-type and S250A mutant Exo70 together with the recombinant ERK2 used in the kinase assay.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2 Phosphorylation of Exo70 by ERK1/2 Promotes VSV-G Exocytosis
(A) ERK1/2 promotes post-Golgi VSV-G exocytosis. HeLa cells were transfected with ts045-VSV-G-GFP and maintained at 40°C for 16 hr. The temperature was shifted to 20°C for 2 hr to allow the exit of ts045-VSV-G-GFP from the ER but arrested at the TGN. The cells, with or without U0126 pretreatment for 30 min, were then shifted to 32°C for 30 or 60 min for ts045-VSV-G vesicles to exit Golgi and transport to the plasma membrane. The cells were processed for immunofluorescence staining with a monoclonal antibody (8G5) against the extracellular domain of VSV-G. The distribution of total (green) and plasma membrane-incorporated ts045-VSV-G-GFP (red, stained by 8G5) were observed by fluorescence microscopy. Scale bar represents 5 μm.
(B) Quantification of VSV-G surface incorporation. Fluorescence signals of the cells were quantified by ImageJ software. VSV-G cell surface incorporation is presented as the ratio of cell surface antibody staining fluorescence to total VSV-G-GFP fluorescence of the cell. Three independent experiments were performed. At least 50 cells were quantified for each group in each experiment. The data were statistically analyzed by student's t test. ∗∗p < 0.05; ∗∗∗p < A.U., arbitrary units.
(C) Western blot showing the levels of knockdown of endogenous Exo70 and the expression of Flag-tagged rat Exo70 variants detected by the anti-Exo70 antibody and anti-Flag antibody. The endogenous Exo70 and Flag-tagged Exo70 are indicated on the blot as they have similar molecular weights. Actin was used as loading control.
(D) Exo70 phosphorylation at Serine 250 is important for VSV-G exocytosis. HeLa cells were treated with Exo70 siRNA, and then cotransfected with ts045-VSV-G-GFP with Flag-tagged rat Exo70, Flag-Exo70(S250A), or Flag-Exo70(S250D). VSV-G transport was then examined in cells expressing these Flag-tagged Exo70 variants. VSV-G incorporation to the plasma membrane was reduced in cells expressing Exo70(S250A), but increased in cells expressing Exo70(S250D). Scale bar represents 5 μm.
(E) Quantification of VSV-G surface incorporation in cells expressing wild-type, S250A, and S250D mutant Exo70. VSV-G cell surface incorporation was quantified as described above. Comparing to cells expressing wild-type Exo70, cell surface incorporation of VSV-G was reduced in cells expressing Exo70(S250A), but increased in cells expressing Exo70(S250D) at both 30 min and 60 min after VSV-G release from the TGN. n = 50; ∗p < A.U., arbitrary units.
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 3
Exo70 Phosphorylation Promotes Its Interactions with Other Exocyst Components in Cells
(A) HEK293 cells expressing Flag-tagged Exo70 or mutant Exo70(S250A) were serum-starved and then treated with EGF or U0126 as indicated. Exo70-Flag was immunoprecipitated from the cell lysates with the anti-Flag monoclonal antibody. The immunoprecipitates were analyzed by western blotting with antibodies against Exo84 and Sec8 (top). The cell lysates (5% of the inputs) were also analyzed for Exo70-Flag, Sec8, Exo84, ERK1/2, and phospho-ERK1/2 (p-ERK1/2).
(B and C) Quantification of the relative binding of Sec8 and Exo84 with Flag-tagged Exo70 and Exo70(S250A) in the presence of EGF and U0126. The levels of binding were normalized to the basal level (-EGF/-U0126). ∗p < 0.05 comparing to the basal level; n = 3. A.U., arbitrary units.
(D) HEK293 cells coexpressing Flag-tagged Sec8 and Myc-Exo70 or Myc-Exo70(S250A) were treated with EGF or U0126 as described above and used for immunoprecipitation with the anti-Flag antibody. The amounts of Exo70 and Exo84 coimmunoprecipitated with Flag-Sec8 were detected by western blotting. The amount of Exo84 and Myc-Exo70 that coimmunoprecipitated with Sec8 increased with EGF stimulation, and U0126 blocked the stimulatory effect. The cell lysates (5% of the inputs) were also analyzed for Exo70-Myc, Sec8-Flag, Exo84, ERK1/2, and phospho-ERK1/2 (p-ERK1/2).
(E and F) Quantification of the binding of Sec8 and Exo84 with Flag-tagged Exo70 and Exo70(S250A) in the presence of EGF and U0126 as shown in (D).
(G) HeLa cells were treated with EGF and U0126 as described above. Immunoprecipitation was performed using a monoclonal antibody against the endogenous Exo70. The amount of Sec8 that coimmunoprecipitated with Exo70 increased with EGF stimulation. U0126 blocked the stimulatory effect.
(H) Quantification of the binding of Sec8 with the endogenous Exo70 in the presence of EGF and U0126 as shown in (G).
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 4
Exo70 Phosphorylation Enhances Its Binding to Sec8 and Exo84 In Vitro
(A) Flag-tagged Exo70 and Exo70(S250A) were immuno-purified from the EGF-treated HEK293 cells. Sec8 and Exo84 were in vitro translated in the presence of [35S]-methionine, and used for binding to the Exo70 variants. Wild-type Exo70 and Exo70(S250D) had stronger interactions with Sec8 and Exo84 than Exo70(S250A). As a control, anti-Flag immunoglobulin beads incubated with lysates of the untransfected cells did not bind to Sec8 or Exo84.
(B and C) Quantification of the binding of Sec8 and Exo84 with Flag-tagged Exo70 in the presence of EGF or U0126 as shown in (A). The levels of binding were normalized to the basal level. ∗p < 0.05 comparing to the basal level; n = 3.
(D) Flag-tagged Exo70 purified from cells treated with EGF has stronger interaction with in vitro translated Sec8 and Exo84 than those purified from untreated cells or cells preincubated with U0126. As a control, anti-Flag immunoglobulin beads incubated with lysates of the untransfected cells did not bind to Sec8 or Exo84.
(E and F) Quantification of the binding of in vitro translated Sec8 and Exo84 shown in (D).
(G) GST-tagged wild-type and mutant Exo70 were incubated with in vitro translated Sec8 and Exo84. The phospho-mimic Exo70(S250D) mutant has a stronger binding to Sec8 and Exo84.
(H and I) Quantification of the binding of in vitro translated Sec8 and Exo84 with recombinant GST-tagged Exo70, Exo70(S250A), and Exo70(S250D) as shown in (G).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 5 Exo70 Phosphorylation Promotes Exocyst Complex Assembly
(A) HeLa cells were treated with EGF for 5 min, or preincubated with U0126 for 30 min before EGF treatment. Cell extracts were fractionated on a Superdex /300 GL gel filtration column and analyzed by SDS-PAGE and western blotting with the anti-Sec8 and anti-Exo70 antibodies. The molecular weight standards for the fractions are marked at the top.
(B) Western blot showing the levels of Exo70 in cells stably expressing Flag-tagged rat Exo70 and Exo70(S250A) as detected by the anti-Exo70 antibody and anti-Flag antibody. The endogenous Exo70 and Flag-tagged Exo70 are indicated to the right. Actin was used as loading control.
(C) The Exo70 knockdown cells expressing Flag-tagged rat Exo70 or Exo70(S250A) were treated with EGF for 5 min, and the lysates were prepared for gel-filtration chromatography. The fractions were collected and analyzed by western blotting using the anti-Flag antibody.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

8. Figure 6
ERK1/2 Phosphorylation of Exo70 at S250 Is Important for Invadopodia Formation and MMP Secretion
(A) ERK1/2 signaling is required for invadopodia formation. MDA-MB-231 cells treated with DMSO or 20 μM U0126 were cultured on Alexa-568-gelatin coated coverslips. The cells were fixed and stained with Alexa-488-phalloidin (green). The right panel indicates the areas of Alexa-568-gelatin digestion generated by invadopodia (black holes) from these cells. Scale bars represent 10 μm.
(B) Quantification of areas of digestion shows that the invadopodia activity was significantly reduced in cells treated with U0126. A.U., arbitrary units. Error bars represent SD. ∗p < 0.01.
(C) Effect of U0126 on MMP secretion in MDA-MB-231 cells. MDA-MB-231 cells treated with DMSO or 20 μM U0126 were incubated with serum-free medium for 16 hr. The conditioned media were subjected to in gel zymography.
(D) Quantification of secreted MMP levels in cells treated with DMSO or 20 μM U0126 were performed using ImageJ software (n = 3). Error bars represent SD. ∗p < 0.01.
(E) MDA-MB-231 parental cells or cells stably expressing rat Exo70-Flag or Exo70(S250A)-Flag mutant were transfected with siRNA against luciferase or endogenous human Exo70. The cells were cultured on Alexa-568-labeled gelatin matrix for invadopodia formation. The cells were then stained with Alexa-594-phalloidin (blue) together with either the anti-Exo70 antibody (parental cell line, top two panels) or anti-Flag antibody (Exo70-Flag stable cell lines, bottom two panels) (green). Scale bars represent 10 μm.
(F) Quantification of areas of degradation per cell. Three independent experiments (over 50 cells each) for each treatment were carried out. Error bars represent SD. ∗p < 0.05.
(G) In-gel zymography analysis of MMP-2 and MMP-9 secreted from the cells. The positions of MMP-2 and MMP-9 on SDS-PAGE are labeled to the right.
(H) Quantification of secreted MMP levels were performed by Image J (n = 3). Error bars represent SD. ∗p < 0.05.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions