A quantitative literature-curated gold standard for kinase-substrate pairs

We describe the Yeast Kinase Interaction Database (KID, http://www.moseslab.csb.utoronto.ca/KID/), which contains high- and low-throughput data relevant to phosphorylation events. KID includes 6,225 low-throughput and 21,990 high-throughput interactions, from greater than 35,000 experiments. By quantitatively integrating these data, we identified 517 high-confidence kinase-substrate pairs that we consider a gold standard. We show that this gold standard can be used to assess published high-throughput datasets, suggesting that it will enable similar rigorous assessments in the future

Bck2 acts through the MADS box protein Mcm1 to activate cell-cycle-regulated genes in budding yeast.

The Bck2 protein is a potent genetic regulator of cell-cycle-dependent gene expression in budding yeast. To date, most experiments have focused on assessing a potential role for Bck2 in activation of the G1/S-specific transcription factors SBF (Swi4, Swi6) and MBF (Mbp1, Swi6), yet the mechanism of gene activation by Bck2 has remained obscure. We performed a yeast two-hybrid screen using a truncated version of Bck2 and discovered six novel Bck2-binding partners including Mcm1, an essential protein that binds to and activates M/G1 promoters through Early Cell cycle Box (ECB) elements as well as to G2/M promoters. At M/G1 promoters Mcm1 is inhibited by association with two repressors, Yox1 or Yhp1, and gene activation ensues once repression is relieved by an unknown activating signal. Here, we show that Bck2 interacts physically with Mcm1 to activate genes during G1 phase. We used chromatin immunoprecipitation (ChIP) experiments to show that Bck2 localizes to the promoters of M/G1-specific genes, in a manner dependent on functional ECB elements, as well as to the promoters of G1/S and G2/M genes. The Bck2-Mcm1 interaction requires valine 69 on Mcm1, a residue known to be required for interaction with Yox1. Overexpression of BCK2 decreases Yox1 localization to the early G1-specific CLN3 promoter and rescues the lethality caused by overexpression of YOX1. Our data suggest that Yox1 and Bck2 may compete for access to the Mcm1-ECB scaffold to ensure appropriate activation of the initial suite of genes required for cell cycle commitment

Regulation of the Yeast Amphiphysin Homologue Rvs167p by Phosphorylation

The yeast amphiphysin homologue Rvs167p plays a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. Rvs167p is a phosphoprotein in vegetatively growing cells and shows increased phosphorylation upon treatment with mating pheromone. Previous work has shown that Rvs167p can be phosphorylated in vitro by the cyclin-dependent kinase Pho85p complexed with its cyclin Pcl2p. Using chymotryptic phosphopeptide mapping, we have identified the sites on which Rvs167p is phosphorylated in vitro by Pcl2p-Pho85p. We have shown that these same sites are phosphorylated in vivo during vegetative growth and that phosphorylation at two of these sites is Pcl-Pho85p dependent. In cells treated with mating pheromone, the MAP kinase Fus3p is needed for full phosphorylation of Rvs167p. Functional genomics and genetics experiments revealed that mutation of other actin cytoskeleton genes compromises growth of a strain in which phosphorylation of Rvs167p is blocked by mutation. Phosphorylation of Rvs167p inhibits its interaction in vitro with Las17p, an activator of the Arp2/3 complex, as well as with a novel protein, Ymr192p. Our results suggest that phosphorylation of Rvs167p by a cyclin-dependent kinase and by a MAP kinase is an important mechanism for regulating protein complexes involved in actin cytoskeleton function

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

Truncation analysis of the <i>BCK2</i> gene.

<p>Fragments of the <i>BCK2</i> gene (black bar) were amplified from genomic DNA using primer positions shown and designated as “amino acid+F (Forward), or R (reverse)”. PCR products were cloned into a yeast two-hybrid vector to create <i>BCK2</i> fragments fused to the N-terminal <i>GAL4</i> DBD (DNA Binding Domain). High density growth spots (in either the <i>ADE2</i> transcription activation assay or the complementation assay) were called “+”, “++”, or “+++” depending on extent of growth. A complete absence of growth was called “−”. Numbers in the β-Gal column represent averaged quantities (per fusion protein) in Miller Units (U).</p

Bck2-interacting proteins identified in a genome-wide yeast two-hybrid screen.

<p>Yeast transformants carrying <i>ADH1</i>-<i>GAL4</i> DBD (vector; <i>LEU2</i>) or <i>ADH1</i>-<i>GAL4</i> DBD-<i>BCK2</i> Fragment 11 (Bck2) in a two-hybrid bait strain (Y8930) were mated to yeast transformants of a two-hybrid prey strain (Y8800) bearing specific gene ORFs fused to the N-terminal <i>GAL4</i> AD (activation domain; <i>TRP1</i>, i.e. <i>ADH1</i>-<i>GAL4</i> AD-ORF plasmid). Diploids were selected by streaking on double plasmid selection medium (SD – Leu – Trp). Strains were grown to equivalent optical density, and spotted in serial 10-fold dilutions on double plasmid selection medium (SD – Leu – Trp) or medium where growth is proportional to transcription of the <i>ADE2</i> gene (SD – Leu – Trp - Ade). Plates were incubated for 48 h at 30°C.</p

Summary of Bck2-dependent regulation of cell cycle gene expression.

<p>In pre-START cells Bck2 binds to Mcm1 and promotes expression of M/G₁ genes such as <i>SWI4</i> and <i>CLN3</i>, which encode important constituents of the transcriptional ‘switch’ for START. Cln3 then contributes to activation of Swi4, a component of SBF, which induces expression of G₁/S genes such as <i>CLN2</i>. Bck2 also induces <i>CLN2</i> expression directly through physical association with Swi4 and with Mcm1. In G₂/M Bck2 promotes expression of <i>CLB2</i>, possibly through interaction with Mcm1. We speculate that Bck2 may be responding to environmental signals such as nutrients in its role of activating cell cycle gene expression (see text for details). Bold lines indicate interactions, both protein-protein (in blue) and protein-DNA (in green) described in this work.</p