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Fig. 3. Binding of N3 to itself is regulated by phosphorylation. (A) Equal amounts of proteins from cell lysates of SW480 or Colo320 cells transfected with GFP-N3 or GFP alone were prepared and immunoprecipitated with a monoclonal anti-N-APC antibody. Total lysates (left panels) and proteins bound to the immunoprecipitated material (right panels) were probed with an anti-GFP antibody. Only the longer N-APC in SW480 cells bound to GFP-N3. (B) Endogenous N-APC was immunoprecipitated from SW480 cells and treated with -phosphatase (-ppase; +) or control buffer (–). Purified His-tagged C-APC, C3 or N3 were added and bound proteins were detected using a monoclonal anti-His antibody. Only the binding of N3 was significantly inhibited when N-APC was dephosphorylated. In addition, the precipitated material was probed with a monoclonal anti-N-APC antibody to confirm that equal amounts of N-APC were recovered in each case (upper panel) and also to illustrate that treatment with -phosphatase increased the electrophoretic mobility of the N-APC in these cells, confirming that the N-terminal third of APC is normally phosphorylated in cells. (C) Immunoprecipitated N-APC from SW480 cells was incubated with purified His-tagged C-APC, C3 or N3 in the presence (+) or absence (–) of GST-tagged 14-3-3 protein. Bound C-APC, C3 and N3 were detected with a monoclonal anti-His antibody, and the presence of 14-3-3 was confirmed with a polyclonal anti-GST antibody (lower panel). (D) Immunoprecipitated endogenous APC from SW480 or Colo320 cells was incubated with purified GST–14-3-3 in the presence (+) or absence (–) of -phosphatase. Only the N-APC from SW480 cells bound 14-3-3 proteins strongly and this binding required phosphorylation of N-APC, consistent with the idea that the N3 region (absent in the APC in Colo320 cells) is required for this interaction. (E) Immunoprecipitated endogenous APC from SW480 cells was treated with -phosphatase or control buffer before purified GST–14-3-3 was added to the beads. After washing, purified N3 was added to each sample. Bound proteins were detected with the indicated antibodies. The control sample did not include the cell lysate. Both phosphorylation and the binding of 14-3-3 enhance the ability of N3 to bind N-APC.

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