Figure 2.

Altered iron regulation and protein degradation of the S138E phosphomimetic mutant of IRP1. Cells expressing IRP1^S138A, IRP1^S138E or IRP1^S138E/3C>3S were grown without or with 100 μM hemin or 100 μM desferal for 24 h. (A) RNA-binding activity of IRP1^S138A, IRP1^S138E or IRP1^S138E/3C>3S determined by quantitative EMSA. Cells were incubated with no addition (Control), 100 μM hemin or 100 μM desferal for 24 h. Results are expressed as percent of control value for each clone and are mean±s.e.m. (n=3). An asterisk indicates that desferal value is significantly different from control (P<0.05), while two asterisks indicate a hemin effect relative to control or desferal. (B) Representative immunoblot of IRP1^S138A, IRP1^S138E, and IRP1^S138E/3C>3S in lysates from cells incubated as in panel A. Treatments are indicated by Control (C); hemin (H); and desferal (D). (C) The half-life of IRP1^S138E was 4.1±0.3 h in hemin, but greater than 18 h in desferal (n=3) (details in Materials and methods). (D) The half-life of IRP1^S138E/3C>3S was 5.1±1.1 h in the presence of hemin, and was greater than 18 h in the presence of desferal (n=3) (details in Materials and methods). Representative decay curves are shown.