Limited exposure of aminophospholipids on the outer leaflet of the plasma membrane is a fundamental feature of eukaryotic cells and is maintained by the action of inward-directed P-type ATPases ("flippases"). Yeast S. cerevisiae has five flippases (Dnf1, Dnf2, Dnf3, Drs2, and Neo1), but their regulation is poorly understood. Two paralogous plasma membrane-associated protein kinases, Pkh1 and Pkh2 (orthologs of mammalian PDK1), are required for viability of S. cerevisiae cells because they activate several essential downstream protein kinases by phosphorylating a critical Thr in their activation loops. Two such targets are related protein kinases Ypk1 and Ypk2 (orthologs of mammalian SGK1), which have been implicated in multiple processes, including endocytosis and coupling of membrane expansion to cell wall remodeling, but the downstream effector(s) of these kinases have been elusive. Here we show that a physiologically relevant substrate of Ypk1 is another protein kinase, Fpk1, a known flippase activator. We show that Ypk1 phosphorylates and thereby down-regulates Fpk1, and further that a complex sphingolipid counteracts the down-regulation of Fpk1 by Ypk1. Our findings delineate a unique regulatory mechanism for imposing a balance between sphingolipid content and aminophospholipid asymmetry in eukaryotic plasma membranes.Fpk1 | plasma membrane | signal transduction | sphingolipids | Ypk1

Communication between neoplastic cells and cells of their microenvironment is critical to cancer progression. To investigate the role of cytoneme-mediated signaling as a mechanism for distributing growth factor signaling proteins between tumor and tumor-associated cells, we analyzed EGFR and RET Drosophila tumor models and tested several genetic loss-of-function conditions that impair cytoneme-mediated signaling. Neuroglian, capricious, Irk2, SCAR, and diaphanous are genes that cytonemes require during normal development. Neuroglian and Capricious are cell adhesion proteins, Irk2 is a potassium channel, and SCAR and Diaphanous are actin-binding proteins, and the only process to which they are known to contribute jointly is cytoneme-mediated signaling. We observed that diminished function of any one of these genes suppressed tumor growth and increased organism survival. We also noted that EGFR-expressing tumor discs have abnormally extensive tracheation (respiratory tubes) and ectopically express Branchless (Bnl, a FGF) and FGFR. Bnl is a known inducer of tracheation that signals by a cytoneme-mediated process in other contexts, and we determined that exogenous over-expression of dominant negative FGFR suppressed tumor growth. Our results are consistent with the idea that cytonemes move signaling proteins between tumor and stromal cells and that cytoneme-mediated signaling is required for tumor growth and malignancy.

Communication between neoplastic cells and cells of their microenvironment is critical to cancer progression. To investigate the role of cytoneme-mediated signaling as a mechanism for distributing growth factor signaling proteins between tumor and tumor-associated cells, we analyzed EGFR and RET Drosophila tumor models. We tested several genetic loss-of-function conditions that impair cytoneme-mediated signaling. diaphanous, Neuroglian, SCAR, capricious are genes that cytonemes require during normal development. Genetic inhibition of cytonemes restored apical basal polarity to tumor cells, reduced tumor growth, and increased organism survival. These findings suggest that cytonemes traffic the signaling proteins that move between tumor and stromal cells, and that cytoneme-mediated signaling is required for tumor growth and malignancy.

Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities we analyzed the function of Drosophila dRYBP, a conserved PcG- and trxG-associated protein. We show that dRYBP is itself ubiquitylated and binds ubiquitylated proteins. Additionally we show that dRYBP maintains H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further we show that dRYBP interacts with the repressive SCE and dKDM2 proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, our results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. Our findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development.