Hydroxylation of aspartic acid to erythro-beta-aspartic acid (Hya) occurs in epidermal growth factor (EGF)-like modules in numerous extracellular proteins with diverse functions. Several EGF-like modules with the consensus sequence for hydroxylation bind Ca2+, and it has therefore been suggested that the hydroxyl group is essential for Ca2+ binding. To determine directly the influence of beta-hydroxylation on calcium binding in the EGF-like modules from coagulation factors IX and X, we have now measured calcium binding to both the fully beta-hydroxylated and the non-beta-hydroxylated modules of the two proteins. At low ionic strength, the Hya-containing module of factor X has a slightly higher Ca2+ affinity, but at physiological salt concentrations this difference is no longer significant for either factor IX or X. Analysis of the 1H NMR chemical shift differences between the hydroxylated and nonhydroxylated factor X modules show that hydroxylation has no effect on the domain fold. Furthermore, measurements on factor IX show that hydroxylation has no effect on the Ca2+/Mg2+ specificity of the ion binding site. We conclude that the hydroxyl group is not a direct ligand for the calcium ion in these EGF-like modules, nor is it essential for high-affinity Ca2+ binding.
Abstract Introduction Akt1, Akt2 and Akt3 kinases are downstream components of phosphoinositol 3-kinase derived signals from receptor tyrosine kinases, which influence cell growth, proliferation and survival. Akt2 overexpression and amplification have been described in breast, ovarian and pancreatic cancers. The present study was designed to investigate the prognostic significance of activated Akt in primary breast cancer and its association with other tumour biomarkers. Methods Using a two-site chemiluminescence-linked immunosorbent assay, we measured the quantitative expression levels of total phosphorylated (P-S473) Akt (Akt1/Akt2/Akt3) on cytosol fractions obtained from fresh frozen tissue samples of 156 primary breast cancer patients. Results Akt phosphorylation was not associated with nodal status or ErbB-2 protein expression levels. High levels of phosphorylated Akt correlated ( P < 0.01) with poor prognosis, and the significance of this correlation increased ( P < 0.001) in the subset of patients with ErbB-2 overexpressing tumours. In addition, phosphorylated Akt was found to be associated with mRNA expression levels of several proliferation markers (e.g. thymidylate synthase), measured using quantitative real-time RT-PCR. Conclusion Our findings demonstrate that, in breast cancer patients, Akt activation is associated with tumour proliferation and poor prognosis, particularly in the subset of patients with ErbB2-overexpressing tumours.
Crystals of a calcium-binding epidermal growth factor (EGF)-like domain of human clotting factor IX suitable for X-ray diffraction analysis have been obtained by vapour diffusion (sitting drop) against 48% PEG 400. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit-cell dimensions a = b = 40.3, c = 98.2 A. The crystals diffract beyond 1.5 A resolution and are relatively stable in the X-ray beam. This is the first reported crystallization of a calcium-binding EGF-like domain.
The process of cell motility involves coordinate signaling events among proteins associated in interactive integrin-linked networks. Mass spectrometric analysis of immunoprecipitation-derived protein mixtures have provided efficient means of identifying proteomes. In this study, we investigate strategies to enhance the detection of interactome proteins for the known signaling module: PAK1, betaPIX, GIT1, and paxillin. Our results indicate that near-endogenous expression levels of bait protein enhances the identification of associated proteins, and that phosphatase inhibition augments the detection of specific protein interactions. Following the analysis of a large pool of spectral data, we have identified and mapped clusters of proteins that either share common interactions among the four bait proteins of interest or are exclusive to single bait proteins. Taken together, these data indicate that biochemical manipulations can enhance the ability for LC-MS/MS to identify interactome proteins, and that qualitative screening of multiple samples leads to the compilation of proteins associated with a known plexus.
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