A Role for Non-Covalent SUMO Interaction Motifs in Pc2/CBX4 E3 Activity
Figure 1
Pc2 contains two potential SIMs.
A) Human Pc2 is shown schematically, with the location of the CtBP-interaction motif (PIDLR in Pc2), the major sumoylation site in Pc2 (VKPE) and the two consensus SIMs. CD: chromodomain, His: poly-histidine stretch, C-box: carboxyl-terminal homology box. B) Alignments of the conserved regions surrounding SIM1 and SIM2 are shown, together with the SIM consensus sequences. Amino acid number are from human Pc2, sequences are from human, mouse, chicken, Xenopus laevis and tropicalis, zebra fish and Tetraodon nigroviridis. The PIDLR, adjacent to SIM2, is indicated by a line above the sequence. Identity (black) and similarity (gray) in at least 5/7 sequences is shown. C) An alignment of known SIMs (protein names to the left) is shown. The SIM in each is boxed, and acidic residues within 10 amino-acids of the SIM are shaded. D) His6-YFP fusion proteins encoding amino acids 250–560 of Pc2 (either wild type, or lacking SIM1, SIM2 or both, as indicated) were incubated with GST fusions to SUMO1 or SUMO2 bound to glutathione agarose, and interacting proteins were identified by western blot. A portion of the input proteins was analyzed by western blot (below), with either a His6 antibody for the Pc2 fusions, or a GST antibody for the SUMO fusions. E) COS1 cells were transfected with the indicated expression constructs and lysates analyzed for CtBP and sumoylated CtBP. F) The interaction of wild type or SIM1 mutant Pc2 with CtBP was determined by co-immunoprecipitation from COS1 cells transfected as indicated. Proteins were precipitated on anti-Flag agarose and analyzed by T7 western blot. Expression in the lysates was monitored by direct western (lower panels). The sequence around SIM1 and of the two Pc2 SIM1 mutants are shown below. The positions of molecular weight markers are indicated.