MPM‐2 epitope sequence is not sufficient for recognition and phosphorylation by ME kinase‐H

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Abstract
Monoclonal antibody MPM‐2 recognizes a large family of mitotic phosphoproteins in a phosphorylation‐dependent manner. The antigenic phosphoepitope, designated the MPM‐2 epitope, putatively consists of hydrophobic residue‐Thr/Ser‐Pro‐hydrophobic residue‐uncharged/basic residue. In this study, we addressed whether this sequence motif contains all the information necessary for recognition and phosphorylation by the kinase that phosphorylates most MPM‐2 antigens. A fusion protein between glutathione S‐transferase and a 19‐residue peptide that contained two representative MPM‐2 epitope sequences overlapping with two potential MAP kinase phosphorylation sites was constructed. Both the MPM‐2 epitope sequences in the fusion protein (GST‐MPM2) were phosphorylated by Xenopus egg extract, making the fusion protein MPM‐2 reactive. However, while MAP kinase phosphorylated both the MPM‐2 epitope sequences, neither ME kinase‐H, a good candidate for a major MPM‐2 epitope kinase, nor mitotic cdc2 kinase, which is known to phosphorylate certain MPM‐2 antigens in vitro, phosphorylated GST‐MPM2 to any significant extent. Furthermore, depletion of MAP kinase activity removed most, if not all, of the GST‐MPM2 phosphorylating activity from crude Xenopus egg extracts. These results suggest that additional or different structural information than that provided by the deduced MPM‐2 epitope sequence is required for recognition and phosphorylation by ME kinase‐H or other major MPM‐2 epitope kinases. They also offer a valid explanation for selective phosphorylation of certain MPM‐2 antigens by MAP kinase as well as selective recognition of certain phosphorylated MAP kinase substrates by MPM‐2.