FIGURE 1.
Pin1 binds the Ser/Thr-Pro motifs of p27Kip1 through its WW and PPIase domain. A, schematic illustration of the potential Pin1-binding sites in p27Kip1, which includes the S10P (Ser-10-Pro), S178P (Ser-178-Pro), and T187P (Thr-187-Pro) motifs. NLS, nuclear localization signal. B, immunoblotting analysis following the use of GST-Pin1, GST-Pin1-W34A (W34A mutant), and GST-Pin1-R2A (R68A/R69A mutants) to pull down FLAG-p27Kip1. GST beads were used as a control. GST and GST-Pin1 proteins were stained as loading controls. C, immunoblotting (IB) analyses following the use of GST-Pin1 or GST beads to pull down either FLAG-p27Kip1 or -p27Kip1 mutants, in the absence (left panel) or presence (right panel) of CIP. OA, okadaic acid; CIP, calf intestinal phosphatase. D, co-immunoprecipitation (IP) assay using FLAG-M2 beads. GFP-Pin1-S16A was co-immunoprecipitated with FLAG-p27Kip1, but not a FLAG-p27Kip1-3A mutant. E, GFP-Pin1-S16A was co-immunoprecipitated with either the FLAG-p27Kip1-WT or the FLAG-p27Kip1-S10A mutant but not with the FLAG-p27Kip1-T187A mutant.