title: MPM-2 epitope sequence is not sufficient for recognition and phosphorylation by ME kinase-H.
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.
authors.affiliation: Department of Clinical Investigation, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
authors.forename: S
authors.initials: S
authors.lastname: Che
authors.forename: M M
authors.initials: MM
authors.lastname: Weil
authors.forename: M
authors.initials: M
authors.lastname: Nelman-Gonzalez
authors.forename: C L
authors.initials: CL
authors.lastname: Ashorn
authors.forename: J
authors.initials: J
authors.lastname: Kuang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D000939:Epitopes / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D005982:Glutathione Transferase / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016324:Sequence Tagged Sites; D013379:Substrate Specificity; D014982:Xenopus laevis
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase