title: Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn.
abstract: Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204, 1159-1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation.
authors.affiliation: CNR, Department of Organic Chemistry, University of Padua, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Ruzza
authors.forename: A
authors.initials: A
authors.lastname: Calderan
authors.forename: B
authors.initials: B
authors.lastname: Filippi
authors.forename: B
authors.initials: B
authors.lastname: Biondi
authors.forename: A D
authors.initials: AD
authors.lastname: Deana
authors.forename: L
authors.initials: L
authors.lastname: Cesaro
authors.forename: L A
authors.initials: LA
authors.lastname: Pinna
authors.forename: G
authors.initials: G
authors.lastname: Borin
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010456:Peptides, Cyclic / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014270:Trifluoroethanol; D014867:Water; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D010455:Peptides; D010456:Peptides, Cyclic; D014867:Water; D014270:Trifluoroethanol; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D016392:Proto-Oncogene Proteins pp60(c-src); C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases