1. The extraordinary active site substrate specificity of pp60c-src. A multiple specificity protein kinase
T R Lee, J Niu, D S Lawrence J Biol Chem. 1995 Mar 10;270(10):5375-80. doi: 10.1074/jbc.270.10.5375.
We report the first active site substrate specificity analysis of a tyrosine-specific protein kinase, namely pp60c-src. Like the cAMP-dependent protein kinase and protein kinase C, pp60c-src will phosphorylate an assortment of achiral residues attached to active site-directed peptides. Furthermore, pp60c-src phosphorylates both aromatic and aliphatic alcohols. However, the substrate specificity of pp60c-src is much broader than that of the two previously examined serine/threonine-specific protein kinases. We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine (Kwon, Y.-G., Mendelow, M., and Lawrence, D. S. (1994) J. Biol. Chem. 269, 4839-4844). In marked contrast, pp60c-src does not discriminate against substrates on the basis of chain length, catalyzing the phosphorylation of residues that contain anywhere from 2-12 carbons between the alcohol functional group and the adjacent peptide bond. In addition, pp60c-src phosphorylates L-serine in an active site-directed peptide. The possible structural basis for the multiple specificity of pp60c-src is discussed. Finally, the active site specificity of pp60c-src is not just limited to L-amino acid residues, but also extends into the realm of D-amino acids as well.
2. The design, synthesis and activity of pentapeptide pp60c-src inhibitors containing L-phosphotyrosine mimics
J H Lai, T H Marsilje, S Choi, S A Nair, D G Hangauer J Pept Res. 1998 Apr;51(4):271-81. doi: 10.1111/j.1399-3011.1998.tb00424.x.
Efficient syntheses of 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine and 4-carboxy-L-phenylalanine within the context of the pentapeptide Ac-Ile-X-Gly-Glu-Phe-NH2 (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide-based tyrosine kinase inhibitors. The key intermediate, Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2, was synthesized by a facile palladium-catalyzed carbonylation of Ac-Ile-Phe(4-iodo)-Gly-Glu(O-tBu)-Phe-NH2. Oxidation of Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2 with tetrabutylammonium permanganate or addition of di-t-butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4-carboxy-L-phenylalanine or 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60c-src than the corresponding pentapeptide wherein X = L-phenylalanine, demonstrating that appended functionalities at the 4-position are accepted and can enhance binding through added interactions within the catalytic region of the active site.
3. Cyclic peptide substrates of pp60c-src. Synthesis and evaluation
J S McMurray, R J Budde, D F Dyckes Int J Pept Protein Res. 1993 Sep;42(3):209-15. doi: 10.1111/j.1399-3011.1993.tb00134.x.
To study the effects of constrained conformation and amino acid sequence on their kinetic parameters, a series of cyclic peptides were synthesized and each was tested as both a substrate and an inhibitor of pp60c-src, the product of the src proto-oncogene. The amino acid sequences were derived from Glu-Leu-Pro-Tyr-Ala-Gly and from the autophosphorylation site of pp60c-src (Ile-Glu-Asp-Asn-Glu-Tyr-Ala-Ala-Arg-Gln-Gly). Linear precursor peptides were synthesized by SPPS on aminomethylated polystyrene resin using the Fmoc-tert-butyl protection scheme with 4-hydroxymethyl-3-methoxyphenoxyacetic acid as the linkage agent. The peptides were cleaved from the support with 1% TFA in dichloromethane with the N-terminal Fmoc and the side-chain protecting groups in place. Removal of the Fmoc group with diethylamine and cyclization with BOP afforded cyclic peptides in 55-78% yield. Side-chain deprotection and further purification gave the final products in 25-48% yields based on their linear precursors. Based on the activities of the linear analogues, cyclization had little effect on the binding (Ki and Km) and rate of phosphorylation (Vmax) of cyclo(Glu-Leu-Pro-Tyr-Ala-Gly) and cyclo(Ile-Glu-Asp-Asn-Glu-Tyr-Ala-Ala-Arg-Gln). A series of cyclic decapeptides that contained the dipeptide D-Phe-Pro inserted in various positions in the autophosphorylation sequence showed marked differences in Ki, Km and Vmax. Compared to the well characterized linear substrate Val-5 angiotensin II, the D-Phe-Pro-containing cyclic peptides have higher Vmax values but differ little in Km, with values in the millimolar range.
