FIGURE 6.

Pin1 catalytic and binding activities are required for enhancing LSF transactivation by facilitating dephosphorylation of Ser-291 and Ser-309. A, NIH 3T3 cells were infected with a parental control retrovirus (−), or retrovirus-overexpressing FLAG-tagged wild-type (WT) Pin1, the Y23A (WW domain) mutant of Pin1, or the R68A/R69A (PPiase domain) mutant of Pin1. These cells were cotransfected with pCMV or pCMV-LSF, the luciferase reporter plasmid pGL3B-WT4E1b and phRL-TK, as described in the legend to Fig. 4A. Activation by LSF of the reporter construct in the presence of each Pin1 mutant is normalized to the degree of activation by LSF in cells infected with the control parental retrovirus. The results are a compilation of two independent experiments, error bars represent S.E., and asterisks represent p < 0.05 by pair-wise t test. B, immunoblot analysis of extracts prepared in passive lysis buffer from a representative experiment described in A. The extracts were resolved by SDS-PAGE on a 12.5% gel and immunoblotted with anti-LSF-314, anti-S291p, anti-S309p, and anti-Pin1 (Calbiochem).