The transcription factor Eyes absent is a protein tyrosine phosphatase.

Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.

Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton.

The mammalian TOR (mTOR) pathway integrates nutrient- and growth factor-derived signals to regulate growth, the process whereby cells accumulate mass and increase in size. mTOR is a large protein kinase and the target of rapamycin, an immunosuppressant that also blocks vessel restenosis and has potential anticancer applications. mTOR interacts with the raptor and GbetaL proteins to form a complex that is the target of rapamycin. Here, we demonstrate that mTOR is also part of a distinct complex defined by the novel protein rictor (rapamycin-insensitive companion of mTOR). Rictor shares homology with the previously described pianissimo from D. discoidieum, STE20p from S. pombe, and AVO3p from S. cerevisiae. Interestingly, AVO3p is part of a rapamycin-insensitive TOR complex that does not contain the yeast homolog of raptor and signals to the actin cytoskeleton through PKC1. Consistent with this finding, the rictor-containing mTOR complex contains GbetaL but not raptor and it neither regulates the mTOR effector S6K1 nor is it bound by FKBP12-rapamycin. We find that the rictor-mTOR complex modulates the phosphorylation of Protein Kinase C alpha (PKCalpha) and the actin cytoskeleton, suggesting that this aspect of TOR signaling is conserved between yeast and mammals.

Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL.

The Bcr-Abl fusion protein kinase causes chronic myeloid leukemia and is targeted by the signal transduction inhibitor STI-571/Gleevec/imatinib (STI-571). Sequencing of the BCR-ABL gene in patients who have relapsed after STI-571 chemotherapy has revealed a limited set of kinase domain mutations that mediate drug resistance. To obtain a more comprehensive survey of the amino acid substitutions that confer STI-571 resistance, we performed an in vitro screen of randomly mutagenized BCR-ABL and recovered all of the major mutations previously identified in patients and numerous others that illuminate novel mechanisms of acquired drug resistance. Structural modeling implies that a novel class of variants acts allosterically to destabilize the autoinhibited conformation of the ABL kinase to which STI-571 preferentially binds. This screening strategy is a paradigm applicable to a growing list of target-directed anti-cancer agents and provides a means of anticipating the drug-resistant amino acid substitutions that are likely to be clinically problematic.

GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR.

mTOR and raptor are components of a signaling pathway that regulates mammalian cell growth in response to nutrients and growth factors. Here, we identify a member of this pathway, a protein named GbetaL that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. Like mTOR and raptor, GbetaL participates in nutrient- and growth factor-mediated signaling to S6K1, a downstream effector of mTOR, and in the control of cell size. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Interestingly, nutrients and rapamycin regulate the association between mTOR and raptor only in complexes that also contain GbetaL. Thus, we propose that the opposing effects on mTOR activity of the GbetaL- and raptor-mediated interactions regulate the mTOR pathway.

mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.

mTOR/RAFT1/FRAP is the target of the immunosuppressive drug rapamycin and the central component of a nutrient- and hormone-sensitive signaling pathway that regulates cell growth. We report that mTOR forms a stoichiometric complex with raptor, an evolutionarily conserved protein with at least two roles in the mTOR pathway. Raptor has a positive role in nutrient-stimulated signaling to the downstream effector S6K1, maintenance of cell size, and mTOR protein expression. The association of raptor with mTOR also negatively regulates the mTOR kinase activity. Conditions that repress the pathway, such as nutrient deprivation and mitochondrial uncoupling, stabilize the mTOR-raptor association and inhibit mTOR kinase activity. We propose that raptor is a missing component of the mTOR pathway that through its association with mTOR regulates cell size in response to nutrient levels.