Activation of Src family tyrosine kinases by ferric ions.

The Src-family tyrosine kinases (SFKs) are oncogenic enzymes that contribute to the initiation and progression of many types of cancer. In normal cells, SFKs are kept in an inactive state mainly by phosphorylation of a consensus regulatory tyrosine near the C-terminus (Tyr(530) in the SFK c-Src). As recent data indicate that tyrosine modification enhances binding of metal ions, the hypothesis that SFKs might be regulated by metal ions was investigated. The c-Src C-terminal peptide bound two Fe(3+) ions with affinities at pH4.0 of 33 and 252µM, and phosphorylation increased the affinities at least 10-fold to 1.4 and 23µM, as measured by absorbance spectroscopy. The corresponding phosphorylated peptide from the SFK Lyn bound two Fe(3+) ions with much higher affinities (1.2pM and 160nM) than the Src C-terminal peptide. Furthermore, when Lyn or Hck kinases, which had been stabilised in the inactive state by phosphorylation of the C-terminal regulatory tyrosine, were incubated with Fe(3+) ions, a significant enhancement of kinase activity was observed. In contrast Lyn or Hck kinases in the unphosphorylated active state were significantly inhibited by Fe(3+) ions. These results suggest that Fe(3+) ions can regulate SFK activity by binding to the phosphorylated C-terminal regulatory tyrosine.

Tyrosine modification enhances metal-ion binding.

Tyrosine sulfation is a common modification of many proteins, and the ability to phosphorylate tyrosine residues is an intrinsic property of many growth-factor receptors. In the present study, we have utilized the peptide hormone CCK(8) (cholecystokinin), which occurs naturally in both sulfated and unsulfated forms, as a model to investigate the effect of tyrosine modification on metal-ion binding. The changes in absorbance and fluorescence emission on Fe(3+) binding indicated that tyrosine sulfation or phosphorylation increased the stoichiometry from 1 to 2, without greatly affecting the affinity (0.6-2.8 microM at pH 6.5). Measurement of Ca(2+) binding with a Ca(2+)-selective electrode revealed that phosphorylated CCK(8) bound two Ca(2+) ions. CCK(8) and sulfated CCK(8) each bound only one Ca(2+) ion with lower affinity. Binding of Ca(2+), Zn(2+) or Bi(3+) to phosphorylated CCK(8) did not cause any change in absorbance, but substantially increased the change in absorbance on subsequent addition of Fe(3+). The results of the present study demonstrate that tyrosine modification may increase the affinity of metal-ion binding to peptides, and imply that metal ions may directly regulate many signalling pathways.

Identification of metabolites of importance in the pathogenesis of pulmonary cryptococcoma using nuclear magnetic resonance spectroscopy.

Primary lung infection with Cryptococcus neoformans is characterised by circumscribed lesions (cryptococcomas). To identify cryptococcal and/or host products of importance in pathogenesis, we applied proton nuclear magnetic resonance (NMR) spectroscopy, which identifies mobile compounds present in complex mixtures, to experimental pulmonary cryptococcomas from rats. Magnetic resonance experiments were performed on cryptococcomas (n = 10) and healthy lungs (n = 8). Signal assignment to key metabolites was confirmed by homo-nuclear and hetero-nuclear NMR correlation spectroscopy. Cryptococcal metabolites, dominating spectra from cryptococcomas included the stress protectants, trehalose and mannitol, acetate, and in some animals, ethanol. Glycerophosphorylcholine was also abundant in cryptococcomas, consistent with hydrolysis of phospholipids in vivo by the cryptococcal enzyme, phospholipase B (PLB). PLB has been identified by molecular studies as a cryptococcal virulence determinant. We propose that PLB secreted by cryptococci promotes tissue invasion by hydrolysing host phospholipids, such as dipalmitoyl phosphatidyl choline, which is abundant in pulmonary surfactant, and lung cell membrane phospholipids. Our results confirm the utility of NMR spectroscopy in studies of microbial pathogenesis.

Biological activity and ferric ion binding of fragments of glycine-extended gastrin.

Nonamidated gastrins such as progastrin and glycine-extended gastrin17 (Ggly) induce cell proliferation and migration in vitro and colonic mucosal proliferation in vivo. Our earlier NMR study defined the structure of Ggly and showed that ferric ions are essential to its biological activity, with the first binding to Glu7 and the second to Glu8 and Glu9 (Pannequin, J. et al. (2002) J. Biol. Chem. 277, 48602-48609). The aims of this study were to define the minimum biologically active fragment of Ggly and to determine whether ferric ions were also required for its activity. Cell-proliferation studies with Ggly fragments containing the five glutamate residues showed that the nonapeptide LE(5)AYG, the octapeptide LE(5)AY, and the heptapeptides E(5)AY and LE(5)A were fully active and that their activity was dependent on the presence of ferric ions. The activity of the hexapeptides LE(5) and E(5)A and the pentapeptide E(5) was reduced and independent of the presence of iron. The stoichiometry of ferric ion binding to LE(5)AYG, LE(5)AY, and E(5)AY, determined by absorption spectroscopy, was 2 mol/mol. NMR spectroscopy showed that the nonapeptide LE(5)AYG and shorter fragments had no defined structure and that the iron-binding sites differed from those in Ggly. We conclude that, in contrast to amidated gastrins where the C-terminal tetrapeptide is the minimum bioactive fragment, the shortest fully active fragments of Ggly are the heptapeptides LE(5)A and E(5)AY. These observations indicate that extensive proteolytic processing may not completely inactivate Ggly and that bioactive forms that are not detected by current radioimmunoassays may be present in tissues and/or plasma.