Figure 4. Phosphorylation of p27 at T197 promotes interaction with 14-3-3.

A, A protein array composed of 14-3-3 isoforms or other protein domains indicated in protein-protein interactions were fused to GST and spotted in duplicate onto a glass chip. Protein arrays were then incubated with phosphorylated and non-phosphorylated peptides corresponding to S10, S118, S140 and T197/T198 phosphorylation sites of p27 (predicted to be 14-3-3 interaction sites) that were labeled with Cy3. Interaction between Cy3-labeled peptides and the protein array was then visualized. A key indicating the area on the chip corresponding to each 14-3-3 isoform is shown below. B, ELT-3 cells were transfected with Flag-p27-WT or Flag-p27-T197A and used to generate whole-cell lysates. Lysates were equalized for p27-WT or p27-T197A expression and affinity purified using GST-14-3-3 or GST alone. Lysates and affinity-purified lysates were immunoblotted using an anti-p27 antibody. C, ELT-3 cells were transfected with Flag-p27-WT, Flag-p27-T197A or Flag-p27-T197E and used to generate whole-cell lysates. Lysates were equalized for exogenous p27 expression and affinity purified using GST-14-3-3. Lysates and affinity-purified lysates were immunoblotted using an anti-p27 antibody.