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.

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX.” Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions.

A combinatorial genetic perturbation strategy was applied to interrogate the yeast kinome on a genome-wide scale. We assessed the global effects of gene overexpression or gene deletion to map an integrated genetic interaction network of synthetic dosage lethal (SDL) and loss-of-function genetic interactions (GIs) for 92 kinases, producing a meta-network of 8700 GIs enriched for pathways known to be regulated by cognate kinases. Kinases most sensitive to dosage perturbations had constitutive cell cycle or cell polarity functions under standard growth conditions. Condition-specific screens confirmed that the spectrum of kinase dosage interactions can be expanded substantially in activating conditions. An integrated network composed of systematic SDL, negative and positive loss-of-function GIs, and literature-curated kinasesubstrate interactions revealed kinase-dependent regulatory motifs predictive of novel gene-specific phenotypes. Our study provides a valuable resource to unravel novel functional relationships and pathways regulated by kinases and outlines a general strategy for deciphering mutant phenotypes from large-scale GI networks.

Akt (also known as protein kinase B or PKB) is the major downstream nodal point of the PI3K signaling pathway. This pathway is a promising anticancer therapeutic target, because constitutive activation of the PI3K-Akt pathway is correlated with tumor development, progression, poor prognosis, and resistance to cancer therapies. The Akt serine/threonine kinase regulates diverse cellular functions including cell growth, proliferation, glucose metabolism, and survival. Although all three known Akt isoforms (Akt1-3) are encoded by separate genes, their amino acid sequences show a high degree of similarity. For this and other reasons, it has long been assumed that all three Akt isoforms are activated in the same way, and their functions largely overlap. However, accumulating lines of evidence now suggest that the three Akt isoforms might have unique modes of activation and many distinct functions. In particular, it has recently been found that the Akt isoforms are localized at different subcellular compartments in both adipocytes and cancer cells. In this review, we highlight the unique roles of each Akt isoform by introducing published data obtained from both in vitro and in vivo studies. We also discuss the significant potential of the Akt isoforms as effective anticancer therapeutic targets.

The PI3K-Akt signaling pathway is involved in the regulation of cell growth, proliferation, metabolism, and death by apoptosis and autophagy. There are three isoforms of Akt (1, 2, and 3) that share over 70% sequence identity and two activating phosphorylation sites. The activation of Akt by insulin or insulin-like growth factor (IGF-1) results in rapid phosphorylation of two amino acid residues that are conserved among all three Akt isoforms. Phosphorylation of threonine 308 (Thr308) leads to roughly 100-fold higher activity, while phosphorylation at serine 474 (Ser474) potentiates the activity another 10-fold. The two phosphorylation events are independent of one another and while phosphoinositide-dependent kinase 1 (PDK1) is accepted as the activating kinase for Thr308, the kinase that phosphorylates Ser474 has only recently been confirmed as the target of rapamycin in complex with Rictor and Sin1 (mTORC2). Among the three Akt isoforms, Akt2 is a particularly promising breast cancer therapeutic target, as its ablation most profoundly affects the long-term cell survival rate. Ablation of Akt2 induces autophagy, which increases cancer cell survival and resistance in response to chemotherapy. In this work, we examined the possibility that Akt2 may specifically regulate autophagic destruction of the mitochondria (mitophagy) via the mitophagy activator PINK1. This point of regulation may represent an attractive therapeutic target to decrease autophagy and increase cancer cell death. Insulin signaling promotes the phosphorylation of Akt by mTORC2 via the PTEN-induced putative kinase (PINK1). PINK1 is a serine/threonine mitochondrial kinase that has been linked to familial Parkinson's disease. PINK1 was originally classified in a screen for transcriptional upregulation of genes upon overexpression of PTEN in two endometrial cancer cell lines, but in the same publication high levels of PINK1 mRNA were present in ovarian tumours that were lacking PTEN. No further investigations into this relationship have been described and it remains unclear as to the relationship between PTEN, Akt, and PINK1, and how regulation occurs at the transcriptional or posttranslational level. Normally PINK1 regulates mitochondrial quality control by targeting defective mitochondria for autophagy (referred to as mitophagy) via the ubiquitin E3 ligase Parkin. We have identified an interaction between Akt2 and the major regulator of mitophagy (PINK1). We suggest that Akt2 normally inhibits mitophagy, and that inhibition of Akt leads to selective autophagic destruction of the mitochondria. We next wanted to determine whether Akt2 activation is required for its mitochondrial localization. Mitochondria isolations were performed using magnetic labelling of the mitochondria with Anti-TOM22 microbeads. The results suggest that the key activating residues of Akt2 may not be required for mitochondrial localization. Citation Format: Alison C. Douglas, James Knockleby, Hoyun Lee. Akt2 regulates mitophagy in breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 550. doi:10.1158/1538-7445.AM2013-550