c-Src tyrosine kinase activity is elevated in several types of human cancer, and this has been attributed to elevated c-Src expression levels, increased c-Src specific activity, and activating mutations in c-Src. We have found a number of human breast cancer cell lines with elevated c-Src specific activity that also possess elevated phosphatase activity directed against the carboxyl-terminal negative regulatory domain of Src family kinases. To identify this phosphatase, cell extracts from MDA-MB-435S cells were chromatographed and the fractions were assayed for phosphatase activity. Four peaks of phosphatase activity directed against the nonspecific substrate poly(Glu/Tyr) were detected. c-Src is a membrane-associated non-receptor tyrosine kinase that plays an important role in relaying signals received by receptors on the cell surface to the cell interior (for a recent review, see Refs.

Elevated levels of Src kinase activity have been reported in a number of human cancers, including colon and breast cancer. We have analysed four human breast tumor cell lines that exhibit high levels of Src kinase activity, and have determined that these cell lines also exhibit a high level of a phosphotyrosine phosphatase activity that recognizes the Src carboxy-terminal PTyr530 negative regulatory site. Total Src kinase activity in these cell lines is elevated as much as 30-fold over activity in normal control cells and speci®c activity is elevated as much as 5.6-fold. When the breast tumor cells were grown in the presence of the tyrosine phosphatase inhibitor vanadate, Src kinase activity was reduced in all four breast tumor cell lines, suggesting that Src was being activated by a phosphatase which could recognize the Tyr530 negative regulatory site. In fractionated cell extracts from the breast tumor cells, we found elevated levels of a membrane associated tyrosine phosphatase activity that preferentially dephosphorylated a Src family carboxy-terminal phosphopeptide containing the regulatory tyrosine 530 site. Src was hypophosphorylated in vivo at tyrosine 530 in at least two of the tumor cell lines, further suggesting that Src was being activated by a phosphatase in these cells. In preliminary immunoprecipitation and antibody depletion experiments, we were unable to correlate the major portion of this phosphatase activity with several known phosphatases.

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to signi¢cantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of interdomain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Signi¢cantly, the in£uence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs. ß

Proteins possessing the same fold may undergo similar motions, particularly if these motions involve large conformational transitions. The increasing amounts of structural data provide a useful starting point with which to test this hypothesis. We have performed a total of 0.29 micros of molecular dynamics across a series of proteins within the same fold family (periplasmic binding proteinlike) in order to address to what extent similarity of motion exists. Analysis of the local conformational space on these timescales (10-20 ns) revealed that the behavior of the proteins could be readily distinguished between an apo-state and a ligand-bound state. Moreover, analysis of the root-mean-square fluctuations reveals that the presence of the ligand exerts a stabilizing effect on the protein, with similar motions occurring, but with reduced magnitude. Furthermore, the conformational space in the presence of the ligand appears to be dictated by sequence but not by the type of ligand present. In contrast, apo-simulations showed considerable overlap of conformational space across the fold as a result of their ability to undergo larger fluctuations. Indeed, we observed several transitions from different simulations between states corresponding to the closed-cleft and open-cleft forms of the fold, with the predominant motions being conserved across the different proteins. Thus, large-scale conformational changes do indeed appear to be conserved across this fold architecture, but smaller conformational motions appear to reflect the differences in sequence and local fold.