Actin is SUMOylated in vivo. (A and B) COS-7 cells were cotransfected with His-tagged SUMO proteins and various myc-tagged actin constructs. His-tagged proteins were purified on a Ni2+ column and analyzed with a myc antibody. Myc-actin (A) and myc-NLS-actin (B) are modified in vivo predominantly by SUMO 2 and 3. Modification of actin by SUMO2 and SUMO3 is increased when the amount of nuclear actin is increased (compare A and B). Vector indicates cells that were only transfected with myc-actin or myc-NLS-actin. (C) SUMOylation of nuclear targeted actin: COS-7 cells were cotransfected with His-SUMO2 and either myc-actin, myc-NLS-actin, or myc-actin-NES-1. Myc-NLS-actin and myc-actin-NES-1 are targeted to the nucleus via the NLS or are retained in the nucleus because of a mutation in the NES. The two actin constructs that are targeted to the nucleus show strong SUMOylation. In contrast, myc-actin, which is predominantly cytoplasmic, shows weaker modification. Molecular mass markers are indicated in kD. Asterisks, SUMOylated actin.
