Fig. 3. (A) Mapping of in vivo MARK3 phosphorylation sites. Kinase inactive (D196A) GST-MARK3 purified from HEK 293 cells on glutathione-Sepharose, was excised from a colloidal Coomassie-Blue-stained polyacrylamide gel, and digested with trypsin. One tenth of the digest was subjected to LC-MS with precursor ion scanning and the phosphorylated residues were identified by manual inspection of the acquired MSMS spectra (left panel) as described in Materials and Methods. The identified peaks are listed in the table, with phosphorylated residues in bold. Similar results were obtained in two subsequent experiments. In a further experiment, using wild type MARK3, the same sites were identified, except that T541 and S543 were assigned as the sites of phosphorylation in the ENSTIPDQRTPVASTHSISSAATPDR peptide (marked with an asterisk). (B) Location of phosphorylation sites in MARK3. Schematic view of MARK3, with each phosphopeptide represented by a box, in which phosphorylated residues have been listed. Phosphorylation sites are represented by vertical lines in the MARK3 structure. Phosphorylation of S619, reportedly phosphorylated in xPAR-1 (Kusakabe and Nishida, 2004), was not detected in our analysis but is included in the figure for reference. (C) Conservation of MARK3 phosphorylation sites. Human MARK3 amino acid sequence was aligned using Clustal W, with the indicated MARK homologues (human: hMARK1, hMARK2, hMARK4; Xenopus: xPAR-1; C. elegans: cePAR-1; Drosophila: dPAR-1; S. cerevisiae KIN1: yPAR-1. Conserved residues are indicated by a tick, no conservation by a cross. E, phosphorylation site is Gln; S, phosphorylation site is Ser rather than Thr; T, phosphorylation site is Thr rather than Ser.