Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

BACKGROUND: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. RESULTS: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. CONCLUSION: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics

A novel role for NUAK1 in promoting ovarian cancer metastasis through regulation of fibronectin production in Spheroids

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production

Large-scale mapping of human protein–protein interactions by mass spectrometry

Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein–protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations

Identification of Clk/Sty as a serine/arginine-rich protein kinase

grantor: University of TorontoI have investigated the function of the protein kinase Clk/Sty. To discover proteins that may interact with Clk/Sty, I performed a two-hybrid screen in yeast using Clk/Sty as bait. Twenty-six mouse cDNA clones were identified in the screen. Four of these clones encode known transcription factors c-Myc, ZFP38, N-CoR, as well as a novel zinc finger protein. Other clones identified in the screen suggest a role for Clk/Sty in RNA processing. For instance, one of the clones, CARS-Cyp, encodes a novel arginine/serine-rich (RS) cyclophilin which co-localizes with splicing factors to nuclear speckles. Furthermore, six of the two-hybrid clones encode RNA-binding proteins. One of these clones is a mammalian homologue of the Drosophila splicing regulator tra-2. Three of the remaining five RNA-binding proteins are members of the SR family of essential splicing factors. I focused my studies on one member of the SR family, ASF/SF2, and found that Clk/Sty phosphorylated ASF/SF2 in vitro on sites that are also phosphorylated in vivo. As well, a catalytically inactive Clk/Sty co-localized to nuclear speckles with SR family members. Interestingly, overexpression of wild-type Clk/Sty led to disassembly of nuclear speckles resulting in redistribution of SR proteins. Therefore, Clk/Sty regulates the intranuclear distribution of SR proteins, presumably by phosphorylation. SRPK1 was the first kinase to be cloned and identified as an SR protein kinase. I found that SRPK1 phosphorylated RSRS repeats in ASF/SF2. Clk/Sty, on the other hand, phosphorylated SPR and KSKS repeats as well as RSRS repeats. Therefore, Clk/Sty displays a broader substrate specificity than SRPK1 in vitro, suggesting that Clk/Sty may phosphorylate more substrates than SRPK1 does in vivo. Pre-mRNA splicing is a complex process that is executed with high fidelity. In order to choose splice sites appropriately, the splicing machinery must be responsive to its external environment. I propose that Clk/Sty functions to transduce signals to this machinery enabling proper splice site selection.Ph.D

Interaction of the Saccharomyces cerevisiae Cortical Actin Patch Protein Rvs167p With Proteins Involved in ER to Golgi Vesicle Trafficking

We have used affinity chromatography to identify two proteins that bind to the SH3 domain of the actin cytoskeleton protein Rvs167p: Gyp5p and Gyl1p. Gyp5p has been shown to be a GTPase activating protein (GAP) for Ypt1p, a Rab GTPase involved in ER to Golgi trafficking; Gyl1p is a protein that resembles Gyp5p and has recently been shown to colocalize with and belong to the same protein complex as Gyp5p. We show that Gyl1p and Gyp5p interact directly with each other, likely through their carboxy-terminal coiled-coil regions. In assays of GAP activity, Gyp5p had GAP activity toward Ypt1p and we found that this activity was stimulated by the addition of Gyl1p. Gyl1p had no GAP activity toward Ypt1p. Genetic experiments suggest a role for Gyp5p and Gyl1p in ER to Golgi trafficking, consistent with their biochemical role. Since Rvs167p has a previously characterized role in endocytosis and we have shown here that it interacts with proteins involved in Golgi vesicle trafficking, we suggest that Rvs167p may have a general role in vesicle trafficking