Interaction of the RPEL repeats of scapinin with cellular actin.

<p>(<i>A</i>) The primary structure of scapinin. The N-terminal region that is conserved in the scapinin/phactr family (NC), the proline-rich region (PR), the three tandem repeats of PREL motifs (RPEL repeats 1, 2, and 3), and the PP1-binding domains (PP1) are indicated. In addition to the N-terminal conserved region (NC), the RPEL-repeat and PP1-binding domains are highly conserved in the scapinin/phactr family. (<i>B</i>) Three RPEL motifs are compared. The RPEL motif (pfam PF02755) is named according to the conserved amino acid sequence, RPxxxEL. (<i>C</i>) Interactions between the RPEL repeats of scapinin and cellular actin. The RPEL repeats of scapinin were produced as fusion proteins with glutathione S-transferase (GST) in <i>E. Coli</i> as illustrated. In the RP>AA mutant, both the arginine and proline residues in the second motif of the RPEL repeats were substituted with alanine residues. Purified GST-RPEL constructs were separated by SDS-polyacrylamide gel electrophoresis and stained with coomassie brilliant blue (CBB). GST-RPEL constructs were covalently conjugated to CNBr-agarose beads (5 mg protein/ml bed volume) and subjected to a pull-down assay with HL-60 cell lysates (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004247#s2" target="_blank">Methods</a>’). Actin in the pull-down-samples was determined by Western blotting (WB) with anti-actin antibody. The GST-RPEL construct (350–465 aa) contained substantial amounts of cleaved products (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004247#s3" target="_blank">Results</a>’).