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. 2017 Oct 13;18(12):2119–2130. doi: 10.15252/embr.201744034

Figure 3. 14‐3‐3 outcompetes ITSN for binding to phosphorylated DENND2B.

Figure 3

  1. Purified GST‐14‐3‐3 WT or K50E was incubated with lysates of HEK‐293T cells expressing GFP‐DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.
  2. HEK‐293T cells expressing Flag‐DENND2B were treated with DMSO or 250 nM OA, and cell lysates were incubated with GST‐14‐3‐3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.
  3. Quantification of (B) where bound DENND2B is normalized to the SM. Mean ± SD. Welch's t‐test **P = 0.006. n = 8 for both treatment groups from five independent experiments.
  4. HEK‐293T cells expressing Flag‐DENND2B were treated with 250 nM OA alone or with 50 μm PKD inhibitor CID755673, and cell lysates were incubated with GST‐14‐3‐3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.
  5. Quantification of (D) where bound DENND2B is normalized to the SM. Mean ± SD. Welch's t‐test *P = 0.040. n = 5 for both treatment groups from three independent experiments.
  6. GST‐SH3A was incubated with lysates of HEK‐293T cells expressing GFP‐DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.
  7. Lysates of HEK‐293T cells expressing Flag‐DENND2B were pre‐incubated with increasing concentrations of purified 14‐3‐3 WT and subsequently incubated with GST‐SH3A (top panel) or GST‐Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.
  8. Regulation of the ITSN‐DENND2B interaction. In the absence of phosphorylation, DENND2B binds the SH3A (A) domain of ITSN. Phosphorylation of DENND2B by PKD occurs on Ser30 residue within the first proline‐rich domain (PRD). Phosphorylation of Ser30 recruits 14‐3‐3 which outcompetes ITSN binding.

Source data are available online for this figure.