LINEAR AND CYCLIC SYNTHETIC PEPTIDES RELATED TO THE MAIN AUTOPHOSPHORYLATION SITE OF THE SRC TYROSINE KINASES AS SUBSTRATES AND INHIBITORS OF LYN

Tyrosine protein kinases (TPKs) of the src family contain two major ph osphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60(c-src). The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased k inase 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 repro duces the main autophosphorylation site of the Src family enzymes [Don ella-Deana, A., Marin, O., Brunati, A.M. and Pinna, L.A. (1992) fur. J . Biochem. 204, 1159-1163]. Our study was addressed to the synthesis o f some derivatives of this sequence in order to obtain both peptide su bstrates suitable for the detection of the Src-like tyrosine kinase ac tivity 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 ami dated analogs. The correlation between the different structural proper ties induced by the modifications of the native sequence and the prope nsity of the peptides to act as Lyn substrates was examined. The kinet ic data obtained indicate that the extent of the peptide phosphorylati on varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the hep tapeptide are detrimental for the Lyn activity, dimeric derivatives di splay very favourable kinetic constants. In particular the cyclic dime r is an especially suitable substrate for the tyrosine kinase and a po werful inhibitor of both the phosphorylation activity of Lyn and the e nzyme autophosphorylation. (C) Munksgaard 1995.