Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localise to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequiste for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knockin of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, CDK7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knockin mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine resistant breast cancer mediated by ER mutations.

A Calvin cycle multiprotein complex including phosphoribulokinase (PRK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a small protein, CP12, has previously been identified. In this article, we have studied this complex in leaves and have shown that dissociation and reassociation of the PRK/GAPDH/CP12 complex occurs in a time frame of minutes, allowing for rapid regulation of enzyme activity. Furthermore, we have shown that the extent of formation and dissociation of the PRK/GAPDH/CP12 complex correlates with the quantity of light. These data provide evidence linking the status of this complex with the rapid and subtle regulation of GAPDH and PRK activities in response to fluctuations in light availability. We have also demonstrated that dissociation of this complex depends on electron transport chain activity and that the major factor involved in the dissociation of the pea complex was thioredoxin f. We show here that both PRK and GAPDH are present in the reduced form in leaves in the dark, but are inactive, demonstrating the role of the PRK/GAPDH/CP12 complex in deactivating these enzymes in response to reductions in light intensity. Based on our data, we propose a model for thioredoxin f-mediated activation of PRK and GAPDH by two mechanisms: directly through reduction of disulfide bonds within these enzymes and indirectly by mediating the breakdown of the complex in response to changes in light intensity.Calvin cycle ͉ photosynthesis ͉ protein-protein interactions ͉ redox

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Gα subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.

The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical heterotrimeric GTP-binding protein (G protein) and mitogen-activated protein kinase (MAPK) cascade. Although signal transmission by such pathways has been modeled in detail, postreceptor down-regulation is less well understood. The pheromone-responsive G protein alpha subunit (Galpha) of yeast down-regulates the mating signal, but its targets are unknown. We have found that Galpha binds directly to the mating-specific MAPK in yeast cells responding to pheromone. This interaction contributes both to modulation of the mating signal and to the chemotropic response, and it demonstrates direct communication between the top and bottom of a Galpha-MAPK pathway.