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. 2010 Jun 14;189(6):997–1011. doi: 10.1083/jcb.200912082

Figure 8.

Figure 8.

Sec16 is a target of the MAPK pathway. (A) Cells were transfected with GFP-Sec16 cDNA. After 8 h, medium was replaced by complete medium (Cont) or medium without serum (starved). Sec16 was immunoprecipitated with anti-GFP, subjected to SDS-PAGE, and immunoblotted with anti-Sec16 (top). The blot was stripped and reprobed with anti–MAPK substrate (bottom). The numbers above the blot indicate relative amounts determined by densitometric scanning. The intensity of the MAPK substrate band was normalized to the Sec16 band. To facilitate comparisons, the value of the control band was set to 1. Note that starvation reduces phosphorylation of Sec16 by 40% (means ± SD; three independent experiments; P < 0.05). (B) Cells were transfected with GFP-Sec16 and, after 8 h, serum starved. Sec16 was immunoprecipitated with anti-GFP. A parallel cell culture was treated with EGF. Active ERK was immunoprecipitated with agarose-coupled anti–phospho-ERK and incubated with the immunoprecipitated active ERK. Immunoprecipitated GFP-Sec16 and immunoprecipitated phospho-ERK were mixed, and the kinase reaction was performed for 30 min at 30°C (reaction). 50% of the reaction was separated by SDS-PAGE and immunoblotted with anti-Sec16 (top). The blot was stripped and reprobed with anti–MAPK substrate (bottom). (C) Cells were transfected with GFP-Sec16 cDNA (wild type [wt] or T415I). Immunoprecipitated Sec16 (anti-GFP) was coincubated with active ERK2 and ATP for 30 min at 30°C. 50% of the reaction was loaded, subjected to SDS-PAGE, and immunoblotted with anti-Sec16 (top). The blot was stripped and reprobed with anti–MAPK substrate antibody (bottom). The numbers above the blot indicate relative amounts determined by densitometric scanning. The intensity of the MAPK substrate band was normalized to the Sec16 band. To facilitate comparisons, the value of the control band was set to 1. Note that the mutation of T415I reduces the ability of ERK2 to phosphorylate Sec16 by at least 65% (means ± SD; three independent experiments). (D) Phosphorylation of purified GST-tagged Elk1 by immunoprecipitated phospho-ERK1/2 (IP p-ERK) as described in B or recombinant active ERK2 (rec. ERK2). The reaction was performed at 30°C for 30 min either with buffer (−) or with the kinase (+). (E) HeLa cells were transfected with a plasmid encoding GFP-Sec16 lacking the N-terminal 923 aa. Sec16 was immunoprecipitated with anti-GFP and incubated with active ERK2 and ATP for 30 min at 30°C. Samples were subjected to SDS-PAGE and immunoblotting with anti-Sec16 (top). The blot was stripped and reprobed with anti–MAPK substrate antibody (bottom). The arrow indicates the position where a band would be expected in case of corresponding phosphorylation. (F) HeLa cells expressing GFP-tagged truncation mutant of Sec16 lacking the N-terminal 923 aa (delta) were serum starved for 8 h before the FRAP experiment (delta-ut). Afterward, cells were treated with EGF for 10 min, and further FRAP curves were recorded from the same dish (delta+EGF). (G) Quantification of the mobile fractions from the curves in F (see Materials and methods; means ± SD; three independent experiments). (H) Cells were transfected with GFP-Sec16 cDNA (wild type [wt] or T415I). Sec16 was immunoprecipitated with anti-GFP, subjected to SDS-PAGE, and immunoblotted with anti-Sec16 (top). The blot was stripped and reprobed with anti–MAPK substrate (bottom). The numbers above the blot indicate relative amounts determined by densitometric scanning. The intensity of the MAPK substrate band was normalized to the Sec16 band. To facilitate comparisons, the value of the control band was set to 1. Note that the T415I mutation reduces the phosphorylation of Sec16 down to 30% (means ± SD; four independent experiments). (I) HeLa cells expressing GFP-tagged Sec16-T415I were serum starved for 8 h before the FRAP experiment (T415I-ut). Thereafter, the cells were treated with EGF for 10 min, and additional FRAP curves were recorded from the same dish (T415I+EGF). (J) Quantification of the mobile fractions from the curves in I (see Materials and methods; means ± SD; three independent experiments). (A–E and H) Black lines on the blots indicate the position of the 250-kD (A–C), 50-kD (D), 150-kD (E), or 250-kD (H) molecular mass marker.