Figure 3.

The threshold effect for EGFR ubiquitination occurs at the PM. (A) Top, HeLa cells were subjected to dynamin 2-KD and treated for 2 min with EGF at the indicated concentrations. IP and IB were as shown. (B) EGFR internalization kinetics in dynamin 2-KD cells was measured using ¹²⁵I-EGF at low (1 ng/ml) or high (30 ng/ml) EGF concentrations. Results are expressed as the internalization rate constant (Ke, left panel) or as % of Ke in control cells (right panel), and are the mean of triplicate points (s.e.m.<8%). Dynamin 2-KD (Dyn 2-KD) severely impaired EGFR internalization both at low and high EGF concentrations, reducing rates to background levels. Similar background levels have previously been observed by us in clathrin-KD+filipin-treated HeLa cells, in which both CME and NCE are inhibited (Sigismund et al, 2005, 2008). These results confirm that both CME and NCE of the EGFR are dynamin 2 dependent. Comparable results were obtained with two different silencing oligos for dynamin 2 (data not shown). (C) Lysates of HeLa cells, control and dynamin 2-KD, stimulated with EGF for 2 min at the indicated concentrations were subjected to ELISA, forward approach (Supplementary Figure 2A), using anti-Ub (FK2) and anti-pY as detecting antibodies. Results are shown as a percentage of the maximal tyrosine phosphorylation or ubiquitination (% of max, see Materials and methods). Graph error bars indicate s.d. calculated on at least three independent experiments. All P-values were calculated using two-way ANOVA analysis. As shown the Ub curves were not significantly different between control and KD; the same was true for the pY curves. Conversely, the Ub curves were significantly different from the pY curves.

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