title: Solid phase synthesis of pp60src-related phosphopeptides via 'global' phosphorylation and their use as substrates for enzymatic phosphorylation by casein kinase-2.
abstract: The seven phosphopeptide derivatives based on the native -NEYTA- sequence of the pp60src protein kinase family, Asn-Glu-Tyr(P)-Ser-Ala, Ala-Glu-Tyr(P)-Ser-Ala, Ala-Ser-Tyr(P)-Ser-Ala, Ala-Ser(P)-Tyr-Ser-Ala, Ala-Thr-Tyr(P)-Ser-Ala, Ala-Thr(P)-Tyr-Ser-Ala and Ala-Ser(P)-Tyr(P)-Ser-Ala, were prepared in good yield using the "global' "phosphite-triester' phosphorylation method. The peptide resins were assembled using the Fmoc mode of solid phase peptide synthesis (PyBOP coupling method) with specific Ser-, Thr-, or Tyr-residues incorporated as their side chain free Fmoc-derivatives. The final "global' phosphorylation of the peptide resins was accomplished using di-tert-butyl N, N-diethylphosphoramidite followed by m-chloroperoxybenzoic acid oxidation of the resultant di-t-butyl phosphite triester intermediate. Subsequent resin cleavage and deprotection of the phosphorylated peptide resins was effected by treatment with 5% anisole: TFA and gave the seven phosphopeptides in high yield and purity. The use of the seven synthetic phosphopeptides in enzymatic (casein kinase-2) phosphorylation studies showed that, (A) the change of the target Thr site to Ser resulted in markedly improved phosphorylation of the peptide substrates, (B) that the Tyr(P) residue in the - 1 position was significantly more important than the Ser(P)/Thr(P) residue in the - 2 position for efficient seryl phosphorylation, and (C) that an acidic residue in the - 2 position relative to the target site facilitated phosphorylation of the downstream seryl residue irrespective of the nature of the acidic residue in the -Xxx-Tyr(P)-Ser- and -Xxx-Tyr-Ser- sequences {Xxx = Ser(P), Thr(P), Glu}. In addition to the Tyr(P) residue directing phosphorylation to the +1 position, the good phosphorylation of both ASY(P)SA and ATY(P)SA by casein kinase-2 indicated that the Tyr(P) residue was also able to direct phosphorylation to a Ser/Thr in the - 1 position.
authors.affiliation: Centre of Pharmacology-Endocrinology, CNRS-INSERM, Montpellier, France.
authors.forename: J W
authors.initials: JW
authors.lastname: Perich
authors.forename: F
authors.initials: F
authors.lastname: Meggio
authors.forename: L A
authors.initials: LA
authors.lastname: Pinna
mesh_terms: D000595:Amino Acid Sequence; D047390:Casein Kinase II; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000138:chemical synthesis*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D010748:Phosphopeptides; D016392:Proto-Oncogene Proteins pp60(c-src); D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases