Phosphorylation-dependent β-TrCP interaction and ubiquitination of PAPC.

<p>(<b>A</b>) GST pull-down assay between PAPC-GST and β-TrCP. β-TrCP was pulled-down by PAPC, whereas PAPC without the substrate recognition domain (WD40 repeat; β-TrCPΔWD40) did not display an interaction with PAPC. Western blot bands of Myc-β-TrCP have smears in the upper regions. These are due to poly-ubiquitination of β-TrCP. (<b>B</b>) C-terminal domains of wild-type (Ct) and phosphorylation-mimic (CtSD) PAPC were able to pull-down β-TrCP. Non-phosphorylated form of PAPC C-terminal domain (CtSA) did not interact with β-TrCP. (<b>C</b>) The ubiquitination assay showed that PAPC is poly-ubiquitinated in <i>Xenopus</i> embryos but that this was abolished by expressing dominant-negative β-TrCP (β-TrCPΔF). (<b>D</b>) Non-phosphorylated form of PAPC (PAPC-SA13) and PAPC with lysine-to-arginine mutations in the cytoplasmic region (PAPC-KR) exhibited markedly reduced poly-ubiquitination. (<b>E</b>) The amino acid sequences of the PAPC DSR domain and the β-catenin destruction motif (DM) are weakly similar. The localizations of GFP-tagged PAPCΔDSR and GFP-tagged PAPC-β-catDM (PAPC whose DSR domain was replaced by β-catenin DM) were shown. The graph shows the relative signal intensity in the cytoplasm (c) and on the plasma membrane (m).