A MAP Kinase Dependent Feedback Mechanism Controls Rho1 GTPase and Actin Distribution in Yeast

In the yeast Saccharomyces cerevisiae the guanosine triphosphatase (GTPase) Rho1 controls actin polarization and cell wall expansion. When cells are exposed to various environmental stresses that perturb the cell wall, Rho1 activates Pkc1, a mammalian Protein Kinase C homologue, and Mpk1, a mitogen activated protein kinase (MAPK), resulting in actin depolarization and cell wall remodeling. In this study, we demonstrate a novel feedback loop in this Rho1-mediated Pkc1-MAPK pathway that involves regulation of Rom2, the guanine nucleotide exchange factor of Rho1, by Mpk1, the end kinase of the pathway. This previously unrecognized Mpk1-depedent feedback is a critical step in regulating Rho1 function. Activation of this feedback mechanism is responsible for redistribution of Rom2 and cell wall synthesis activity from the bud to cell periphery under stress conditions. It is also required for terminating Rho1 activity toward the Pkc1-MAPK pathway and for repolarizing actin cytoskeleton and restoring growth after the stressed cells become adapted

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1α), and other RDM1 isoforms. We show that RDM1α undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response

Rapamycin activates Tap42-associated phosphatases by abrogating their association with Tor complex 1

In Saccharomyces cerevisiae, the Tap42–phosphatase complexes are major targets of the Tor kinases in the rapamycin-sensitive signaling pathway. The immunosuppressive agent, rapamycin, induces a prompt activation of the Tap42-associated phosphatases, which is vitally important in Tor-mediated transcriptional regulation. However, the mechanism for the rapid phosphatase activation is poorly understood. In this study, we show that the Tap42–phosphatase complexes exist mainly on membrane structures through their association with Tor complex 1 (TORC1). Rapamycin abrogates this association and releases the Tap42–phosphatase complexes into the cytosol. Disassembly of the Tap42–phosphatase complexes occurs subsequently, following the release but at a much slower rate, presumably caused by Tap42 dephosphorylation. Release of the Tap42–phosphatase complexes from membrane structures also occurs when cells are deprived of nutrient. These findings suggest that the association of the Tap42–phosphatase complexes with TORC1 represents an important mechanism by which nutrient controls Tor signaling activity. In addition, our data support a model in which rapamycin acts not by inhibiting the kinase activity of Tor but by disrupting its interaction with downstream targets

Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities

The genetic code—the binding specificity of all transfer-RNAs—defines how protein primary structure is determined by DNA sequence. DNA also dictates when and where proteins are expressed, and this information is encoded in a pattern of specific sequence motifs that are recognized by transcription factors. However, the DNA-binding specificity is only known for a small fraction of the ∼1400 human transcription factors (TFs). We describe here a high-throughput method for analyzing transcription factor binding specificity that is based on systematic evolution of ligands by exponential enrichment (SELEX) and massively parallel sequencing. The method is optimized for analysis of large numbers of TFs in parallel through the use of affinity-tagged proteins, barcoded selection oligonucleotides, and multiplexed sequencing. Data are analyzed by a new bioinformatic platform that uses the hundreds of thousands of sequencing reads obtained to control the quality of the experiments and to generate binding motifs for the TFs. The described technology allows higher throughput and identification of much longer binding profiles than current microarray-based methods. In addition, as our method is based on proteins expressed in mammalian cells, it can also be used to characterize DNA-binding preferences of full-length proteins or proteins requiring post-translational modifications. We validate the method by determining binding specificities of 14 different classes of TFs and by confirming the specificities for NFATC1 and RFX3 using ChIP-seq. Our results reveal unexpected dimeric modes of binding for several factors that were thought to preferentially bind DNA as monomers