Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.
Among the interactions that stabilize the native state of proteins, the role of electrostatic interactions has been difficult to quantify precisely. Surface salt bridges or ion pairs between acidic and basic side chains have only a modest stabilizing effect on the stability of helical peptides or proteins: estimates are roughly 0.5 kcal/mol or less. On the other hand, theoretical arguments and the occurrence of salt bridge networks in thermophilic proteins suggest that multiple salt bridges may exert a stronger stabilizing effect. We show here that triads of charged side chains, Arg(+)-Glu(-)-Arg(+) spaced at i,i+4 or i,i+3 intervals in a helical peptide stabilize alpha helix by more than the additive contribution of two single salt bridges. The free energy of the triad is more than 1 kcal/mol in excess of the sum of the individual pairs, measured in low salt concentration (10 mM). The effect of spacing the three groups is severe; placing the charges at i,i+4 or i,i+3 sites has a strong effect on stability relative to single bridges; other combinations are weaker. A conservative calculation suggests that interactions of this kind between salt bridges can account for much of the stabilization of certain thermophilic proteins.
We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcall mol, an apparent elevation of the melting temperature relative to wild type of about 22 "C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences. Keywords: GCN4; leucine zipper; salt bridge; thermal stabilityInteractions that stabilize the native state of proteins include the hydrophobic effect, van der Waals interactions, hydrogen bonds and ionic effects, including dipole interactions and salt bridges (Creighton, 1993). The question of which of these are most important in protein stabilization has been debated since the review by Kauzmann (1959). One aspect of the problem concerns how to account for the additional stabilization of proteins from thermophiles, which can have very high thermal stabilities (see Hiller et al., 1997). Since the pioneering work of Matthews et al. (1974) on thermolysin, structures of thermophilic and mesophilic proteins have been compared in a search for clues to what accounts for the higher stability of the former (Korndorfer et al., 1995;Yip et al., 1995;Hatanaka et al., 1997;Robb & Maeder, 1998). In 1978, Perutz (1978 observed that the main discernible difference between a thermophilic and mesophilic version of ferrodoxin lay in the greater number of salt bridges on the surface of the thermophile. As more crystal structures of thermophilic proteins have become available, other mechanisms have been proposed to explain their stability (Vogt & Argos, 1997): improved internal packing, burial of a greater hydrophobic area (Chan et al., 1995; Delboni et al., 1995), and networks of complex salt bridges (Yip et al., 1995;Pappenberger et al., 1997 Yip et al., 1995;Robb & Maeder, 1998).Here, we consider the role of complex salt bridges in stabilizing a simplified model protein structure. The strength of a salt bridge can be estimated by different experimental methodologies: changes in the helicity of model peptides (Merutka & Stellwagen, 1990;Lyu et al., 1992;Scholtz et al., 1993), shifts in pK, of interacting side chains (Anderson et al., 1990;, or T, differences in model proteins Dao-Pin et al., 1991). Using the first method, Smi...
The Bcr-Abl fusion kinase drives oncogenesis in chronic myeloid leukemia (CML). CML patients are currently treated with the Abl tyrosine kinase inhibitor imatinib, which is effective in early stages of the disease. However, resistance to imatinib arises in later disease stages primarily because of a Bcr-Abl mutation. To gain deeper insight into Bcr-Abl signaling pathways, we generated phosphotyrosine profiles for 6 cell lines that represent 3 BcrAbl fusion types by using immunoaffinity purification of tyrosine phosphopeptides followed by tandem mass spectrometry. We identified 188 nonredundant tyrosinephosphorylated sites, 77 of which are novel. By comparing the profiles, we found a number of phosphotyrosine sites common to
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