The movement of many transcription factors, kinases and replication factors between the nucleus and cytoplasm is important in regulating their activity. In some cases, phosphorylation of a protein regulates its entry into the nucleus; in others, it causes the protein to be exported to the cytoplasm. The mechanism by which phosphorylation promotes protein export from the nucleus is poorly understood. Here we investigate how the export of the yeast transcription factor Pho4 is regulated in response to changes in phosphate availability. We show that phosphorylation of Pho4 by a nuclear complex of a cyclin with a cyclin-dependent kinase, Pho80-Pho85, triggers its export from the nucleus. We also find that the shuttling receptor used by Pho4 for nuclear export is the importin-beta-family member Msn5, which is required for nuclear export of Pho4 in vivo and binds only to phosphorylated Pho4 in the presence of the GTP-bound form of yeast Ran in vitro. Our results reveal a simple mechanism by which phosphorylation can control the nuclear export of a protein.

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) determine tissue and cell polarity in a variety of organisms. In yeast, cells orient polarized growth toward the mating partner along a pheromone gradient by a mechanism that requires Far1p and Cdc24p. Far1p bound Gbetagamma and interacted with polarity establishment proteins, which organize the actin cytoskeleton. Cells containing mutated Far1p unable to bind Gbetagamma or polarity establishment proteins were defective for orienting growth toward their mating partner. In response to pheromones, Far1p moves from the nucleus to the cytoplasm. Thus, Far1p functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling marked by Gbetagamma to polarize assembly of the cytoskeleton in a morphogenetic gradient.

During Caenorhabditis elegans vulval development, an inductive signal from the anchor cell stimulates three of the six vulval precursor cells (VPCs) to adopt vulval rather than nonvulval epidermal fates. Genes necessary for this induction include the lin-3 growth factor, the let-23 receptor tyrosine kinase, and let-60 ras. lin-15 is a negative regulator of this inductive pathway. In lin-15 mutant animals, all six VPCs adopt vulval fates, even in the absence of inductive signal. Previous genetic studies suggested that lin-15 is a complex locus with two independently mutable activities, A and B. We have cloned the lin-15 locus by germline transformation and find that it encodes two nonoverlapping transcripts that are transcribed in the same direction. The downstream transcript encodes the lin-15A function; the upstream transcript encodes the lin-15B function. The predicted lin-15A and lin-15B proteins are novel and hydrophilic. We have identified a molecular null allele of lin-15 and have used it to analyze the role of lin-15 in the signaling pathway. We find that lin-15 acts upstream of let-23 and in parallel to the inductive signal.

Far1p is a bifunctional protein that is required to arrest the cell cycle and to establish cell polarity during yeast mating. Far1p is localized predominantly in the nucleus but accumulates in the cytoplasm in cells exposed to pheromones. Here we show that Far1p functions in both subcellular compartments: nuclear Far1p is required to arrest the cell cycle, whereas cytoplasmic Far1p is involved in the establishment of cell polarity. The subcellular localization of Far1p is regulated by two mechanisms: (1) Far1p contains a functional bipartite nuclear localization signal (NLS), and (2) Far1p is exported from the nucleus by Msn5p/Ste21p, a member of the exportin family. Cells deleted for Msn5p/Ste21p failed to export Far1p in response to pheromones, whereas overexpression of Msn5p/Ste21p was sufficient to accumulate Far1p in the cytoplasm in the absence of pheromones. Msn5p/Ste21p was localized in the nucleus and interacted with Far1p in a manner dependent on GTP-bound Gsp1p. Two-hybrid analysis identified a small fragment within Far1p that is necessary and sufficient for binding to Msn5p/Ste21p, and is also required to export Far1p in vivo. Finally, similar to ⌬msn5/ste21 strains, cells expressing a mutant Far1p, which can no longer be exported, exhibit a mating defect, but are able to arrest their cell cycle in response to pheromones. Taken together, our results suggest that nuclear export of Far1p by Msn5p/Ste21p coordinates the two separable functions of Far1p during mating.

Drug target identification is a crucial step in development, yet is also among the most complex. To address this, we develop BANDIT, a Bayesian machine-learning approach that integrates multiple data types to predict drug binding targets. Integrating public data, BANDIT benchmarked a ~90% accuracy on 2000+ small molecules. Applied to 14,000+ compounds without known targets, BANDIT generated ~4,000 previously unknown molecule-target predictions. From this set we validate 14 novel microtubule inhibitors, including 3 with activity on resistant cancer cells. We applied BANDIT to ONC201—an anti-cancer compound in clinical development whose target had remained elusive. We identified and validated DRD2 as ONC201’s target, and this information is now being used for precise clinical trial design. Finally, BANDIT identifies connections between different drug classes, elucidating previously unexplained clinical observations and suggesting new drug repositioning opportunities. Overall, BANDIT represents an efficient and accurate platform to accelerate drug discovery and direct clinical application.

The late phase of long-term potentiation (LTP) requires activation of the mammalian target of rapamycin (mTOR) pathway and synthesis of new proteins. mTOR regulates protein synthesis via phosphorylation of 4E-binding proteins (4E-BPs) and S6K, and via selective up-regulation of 5′ terminal oligopyrimidine (5′ TOP) mRNAs that encode components of the translational machinery. In this study, we explored the regulation of 5′ TOP mRNAs during late-LTP (L-LTP). Synaptic plasticity was studied at Schaffer collateral -CA1 pyramidal cell synapses in rat organotypic hippocampal slices. Forskolin, an adenylate cyclase activator, induced L-LTP in organotypic slices that was mTOR-dependent. To determine if 5′ TOP mRNAs are specifically up-regulated during L-LTP, we generated a 5′ TOP-myr-dYFP reporter to selectively monitor 5′ TOP translation. Confocal imaging experiments in cultured slices revealed an increase in somatic and dendritic fluorescence after forskolin treatment. This up-regulation was dependent on an intact TOP sequence and was mTOR, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-dependent. Our findings indicate that forskolin induces L-LTP in hippocampal neurons and up-regulates 5′ TOP mRNAs translation via mTOR, suggesting that up-regulation of the translational machinery is a candidate mechanism for the stabilization of LTP.

Green Fluorescent Protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP, and GFPγ, and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of utmost importance. The GenBank accession number for Envy is KM891731, Ivy is KM891732, and the yeast optimized GFPγ described in this paper is KM891733.

Objective: This paper describes the Precision Medicine Knowledge Base (PMKB; https://pmkb.weill.cornell.edu), an interactive online application for collaborative editing, maintenance, and sharing of structured clinical-grade cancer mutation interpretations. Materials and Methods: PMKB was built using the Ruby on Rails Web application framework. Leveraging existing standards such as the Human Genome Variation Society variant description format, we implemented a data model that links variants to tumor-specific and tissue-specific interpretations. Key features of PMKB include support for all major variant types, standardized authentication, distinct user roles including high-level approvers, and detailed activity history. A REpresentational State Transfer (REST) application-programming interface (API) was implemented to query the PMKB programmatically. Results: At the time of writing, PMKB contains 457 variant descriptions with 281 clinical-grade interpretations. The EGFR, BRAF, KRAS, and KIT genes are associated with the largest numbers of interpretable variants. PMKB’s interpretations have been used in over 1500 AmpliSeq tests and 750 whole-exome sequencing tests. The interpretations are accessed either directly via the Web interface or programmatically via the existing API. Discussion: An accurate and up-to-date knowledge base of genomic alterations of clinical significance is critical to the success of precision medicine programs. The open-access, programmatically accessible PMKB represents an important attempt at creating such a resource in the field of oncology. Conclusion: The PMKB was designed to help collect and maintain clinical-grade mutation interpretations and facilitate reporting for clinical cancer genomic testing. The PMKB was also designed to enable the creation of clinical cancer genomics automated reporting pipelines via an API.