Identification of residues in CITED2 wild type or CITED2T166N phosphorylated by ERK2 (MAPK1).

<p>(A) GST-CITED2 wild type or mutant GST-CITED2T166N were incubated with Mg [³²P]ATP in the presence of ERK2 and subjected to SDS-PAGE. The phosphorylated CITED2 was excised from the gel, digested with trypsin and the peptide separated by chromatography on a Vydac C18 column. The column was developed with an acetonitrile gradient (broken line) and ³²P-radioactivity is shown (full line). The phosphopeptides A1 to A6 are indicated. (B) The major peaks (A1 to A6) of ³²P-radioactivity were analysed by MALDI-TOF, MALDI TOF-TOF, Edman degradation and phosphoamino acid analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046256#s4" target="_blank">Materials and Methods</a> and the data for A1–A3 is shown. The sequence inferred from this data is shown underneath each figure.