Genome-wide CRISPR screens in spheroid culture reveal that the tumor suppressor LKB1 inhibits growth via the PIKFYVE lipid kinase.

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.

mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation.

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

Regulation of GSK3 cellular location by FRAT modulates mTORC1-dependent cell growth and sensitivity to rapamycin.

The mTORC1 pathway regulates cell growth and proliferation by properly coupling critical processes such as gene expression, protein translation, and metabolism to the availability of growth factors and hormones, nutrients, cellular energetics, oxygen status, and cell stress. Although multiple cytoplasmic substrates of mTORC1 have been identified, how mTORC1 signals within the nucleus remains incompletely understood. Here, we report a mechanism by which mTORC1 modulates the phosphorylation of multiple nuclear events. We observed a significant nuclear enrichment of GSK3 when mTORC1 was suppressed, which promotes phosphorylation of several proteins such as GTF2F1 and FOXK1. Importantly, nuclear localization of GSK3 is sufficient to suppress cell proliferation. Additionally, expression of a nuclear exporter of GSK3, FRAT, restricts the nuclear localization of GSK3, represses nuclear protein phosphorylation, and prevents rapamycin-induced cytostasis. Finally, we observe a correlation between rapamycin resistance and FRAT expression in multiple-cancer cell lines. Resistance to Food and Drug Administration (FDA)-approved rapamycin analogs (rapalogs) is observed in many tumor settings, but the underling mechanisms remain incompletely understood. Given that FRAT expression levels are frequently elevated in various cancers, our observations provide a potential biomarker and strategy for overcoming rapamycin resistance.

ERK2 regulates epithelial-to-mesenchymal plasticity through DOCK10-dependent Rac1/FoxO1 activation.

ERK is a key coordinator of the epithelial-to-mesenchymal transition (EMT) in that a variety of EMT-inducing factors activate signaling pathways that converge on ERK to regulate EMT transcription programs. However, the mechanisms by which ERK controls the EMT program are not well understood. Through an analysis of the global changes of gene expression mediated by ERK2, we identified the transcription factor FoxO1 as a potential mediator of ERK2-induced EMT, and thus we investigated the mechanism by which ERK2 regulates FoxO1. Additionally, our analysis revealed that ERK2 induced the expression of Dock10, a Rac1/Cdc42 GEF, during EMT. We demonstrate that the activation of the Rac1/JNK signaling axis downstream of Dock10 leads to an increase in FoxO1 expression and EMT. Taken together, our study uncovers mechanisms by which epithelial cells acquire less proliferative but more migratory mesenchymal properties and reveals potential therapeutic targets for cancers evolving into a metastatic disease state.

Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells.

Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4-mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4-mediated phosphoproteome, including construction of kinase-substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.

Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5.

Cell differentiation requires the ability to detect and respond appropriately to a variety of extracellular signals. Here we investigate a differentiation switch induced by changes in the concentration of a single stimulus. Yeast cells exposed to high doses of mating pheromone undergo cell division arrest. Cells at intermediate doses become elongated and divide in the direction of a pheromone gradient (chemotropic growth). Either of the pheromone-responsive MAP kinases, Fus3 and Kss1, promotes cell elongation, but only Fus3 promotes chemotropic growth. Whereas Kss1 is activated rapidly and with a graded dose-response profile, Fus3 is activated slowly and exhibits a steeper dose-response relationship (ultrasensitivity). Fus3 activity requires the scaffold protein Ste5; when binding to Ste5 is abrogated, Fus3 behaves like Kss1, and the cells no longer respond to a gradient or mate efficiently with distant partners. We propose that scaffold proteins serve to modulate the temporal and dose-response behavior of the MAP kinase.

Checkpoints in a yeast differentiation pathway coordinate signaling during hyperosmotic stress.

All eukaryotes have the ability to detect and respond to environmental and hormonal signals. In many cases these signals evoke cellular changes that are incompatible and must therefore be orchestrated by the responding cell. In the yeast Saccharomyces cerevisiae, hyperosmotic stress and mating pheromones initiate signaling cascades that each terminate with a MAP kinase, Hog1 and Fus3, respectively. Despite sharing components, these pathways are initiated by distinct inputs and produce distinct cellular behaviors. To understand how these responses are coordinated, we monitored the pheromone response during hyperosmotic conditions. We show that hyperosmotic stress limits pheromone signaling in at least three ways. First, stress delays the expression of pheromone-induced genes. Second, stress promotes the phosphorylation of a protein kinase, Rck2, and thereby inhibits pheromone-induced protein translation. Third, stress promotes the phosphorylation of a shared pathway component, Ste50, and thereby dampens pheromone-induced MAPK activation. Whereas all three mechanisms are dependent on an increase in osmolarity, only the phosphorylation events require Hog1. These findings reveal how an environmental stress signal is able to postpone responsiveness to a competing differentiation signal, by acting on multiple pathway components, in a coordinated manner.

Proper protein glycosylation promotes mitogen-activated protein kinase signal fidelity.

The ability of cells to sense and respond appropriately to changing environmental conditions is often mediated by signal transduction pathways that employ mitogen-activated protein kinases (MAPKs). In the yeast Saccharomyces cerevisiae, the high-osmolarity glycerol (HOG) and filamentous growth (FG) pathways are activated following hyperosmotic stress and nutrient deprivation, respectively. Whereas the HOG pathway requires the MAPK Hog1, the FG pathway employs the MAPK Kss1. We conducted a comprehensive screen of nearly 5000 gene deletion strains for mutants that exhibit inappropriate cross-talk between the HOG and FG pathways. We identified two novel mutants, mnn10Δ and mnn11Δ, that allow activation of Kss1 under conditions that normally stimulate Hog1. MNN10 and MNN11 encode mannosyltransferases that are part of the N-glycosylation machinery within the Golgi apparatus; deletion of either gene results in N-glycosylated proteins that have shorter mannan chains. Deletion of the cell surface mucin Msb2 suppressed the mnn11Δ phenotype, while mutation of a single glycosylation site within Msb2 was sufficient to confer inappropriate activation of Kss1 by salt stress. These findings reveal new components of the N-glycosylation machinery needed to ensure MAPK signaling fidelity.

Positive roles for negative regulators in the mating response of yeast.

All cells must detect and respond to changes in their environment, often through changes in gene expression. The yeast pheromone pathway has been extensively characterized, and is an ideal system for studying transcriptional regulation. Here we combine computational and experimental approaches to study transcriptional regulation mediated by Ste12, the key transcription factor in the pheromone response. Our mathematical model is able to explain multiple counterintuitive experimental results and led to several novel findings. First, we found that the transcriptional repressors Dig1 and Dig2 positively affect transcription by stabilizing Ste12. This stabilization through protein-protein interactions creates a large pool of Ste12 that is rapidly activated following pheromone stimulation. Second, we found that protein degradation follows saturating kinetics, explaining the long half-life of Ste12 in mutants expressing elevated amounts of Ste12. Finally, our model reveals a novel mechanism for robust perfect adaptation through protein-protein interactions that enhance complex stability. This mechanism allows the transcriptional response to act on a shorter time scale than upstream pathway activity.

Nutrient signaling: Starvation flips a phosphoinositide switch on lysosomal catabolism.

Lysosomes are highly dynamic organelles that rapidly respond to changes in cellular nutrient status. A new study identifies a phosphoinositide switch that dictates lysosome function during nutrient starvation.

Balancing lysosome abundance in health and disease.

Lysosomes are catabolic organelles that govern numerous cellular processes, including macromolecule degradation, nutrient signalling and ion homeostasis. Aberrant changes in lysosome abundance are implicated in human diseases. Here we outline the mechanisms of lysosome biogenesis and turnover, and discuss how changes in the lysosome pool impact physiological and pathophysiological processes.

LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase.

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1- mediated growth suppression we developed a spheroid-based cell culture assay to study LKB1- dependent growth. Using this assay, along with genome-wide CRISPR screens and validation with orthogonal methods, we discovered that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase, which promotes the internalization of wild-type EGFR. Our findings reveal a new mechanism of regulation of EGFR, which may have implications for the treatment of LKB1 -mutant LUAD.

Altered propionate metabolism contributes to tumour progression and aggressiveness.

The alteration of metabolic pathways is a critical strategy for cancer cells to attain the traits necessary for metastasis in disease progression. Here, we find that dysregulation of propionate metabolism produces a pro-aggressive signature in breast and lung cancer cells, increasing their metastatic potential. This occurs through the downregulation of methylmalonyl coenzyme A epimerase (MCEE), mediated by an extracellular signal-regulated kinase 2-driven transcription factor Sp1/early growth response protein 1 transcriptional switch driven by metastatic signalling at its promoter level. The loss of MCEE results in reduced propionate-driven anaplerotic flux and intracellular and intratumoral accumulation of methylmalonic acid, a by-product of propionate metabolism that promotes cancer cell invasiveness. Altogether, we present a previously uncharacterized dysregulation of propionate metabolism as an important contributor to cancer and a valuable potential target in the therapeutic treatment of metastatic carcinomas.

Rap1-GTPases control mTORC1 activity by coordinating lysosome organization with amino acid availability.

The kinase mTOR complex 1 (mTORC1) promotes cellular growth and is frequently dysregulated in cancers. In response to nutrients, mTORC1 is activated on lysosomes by Rag and Rheb guanosine triphosphatases (GTPases) and drives biosynthetic processes. How limitations in nutrients suppress mTORC1 activity remains poorly understood. We find that when amino acids are limited, the Rap1-GTPases confine lysosomes to the perinuclear region and reduce lysosome abundance, which suppresses mTORC1 signaling. Rap1 activation, which is independent of known amino acid signaling factors, limits the lysosomal surface available for mTORC1 activation. Conversely, Rap1 depletion expands the lysosome population, which markedly increases association between mTORC1 and its lysosome-borne activators, leading to mTORC1 hyperactivity. Taken together, we establish Rap1 as a critical coordinator of the lysosomal system, and propose that aberrant changes in lysosomal surface availability can impact mTORC1 signaling output.

Dynamic Incorporation of Histone H3 Variants into Chromatin Is Essential for Acquisition of Aggressive Traits and Metastatic Colonization.

Metastasis is the leading cause of cancer mortality. Chromatin remodeling provides the foundation for the cellular reprogramming necessary to drive metastasis. However, little is known about the nature of this remodeling and its regulation. Here, we show that metastasis-inducing pathways regulate histone chaperones to reduce canonical histone incorporation into chromatin, triggering deposition of H3.3 variant at the promoters of poor-prognosis genes and metastasis-inducing transcription factors. This specific incorporation of H3.3 into chromatin is both necessary and sufficient for the induction of aggressive traits that allow for metastasis formation. Together, our data clearly show incorporation of histone variant H3.3 into chromatin as a major regulator of cell fate during tumorigenesis, and histone chaperones as valuable therapeutic targets for invasive carcinomas.

Crystal structure of group A streptococcus Mac-1: insight into dimer-mediated specificity for recognition of human IgG.

Group A Streptococcus secretes cysteine proteases named Mac-1 and Mac-2 that mediate host immune evasion by targeting both IgG and Fc receptors. Here, we report the crystal structures of Mac-1 and its catalytically inactive C94A mutant in two different crystal forms. Despite the lack of sequence homology, Mac-1 adopts the canonical papain fold. Alanine mutations at the active site confirmed the critical residues involved in a papain-like catalytic mechanism. Mac-1 forms a symmetric dimer in both crystal forms and displays the unique dimer interface among papain superfamily members. Mutations at the dimer interface resulted in a significant reduction in IgG binding and catalysis, suggesting that the dimer contributes to both IgG specificity and enzyme cooperativity. A tunnel observed at the dimer interface constitutes a target for designing potential Mac-1-specific antimicrobial agents. The structures also offer insight into the functional difference between Mac-1 and Mac-2.

Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK.

Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5(ND)) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or "synthetic," supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling.

Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity.

Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.

Analysis of the aerobactin and ferric hydroxamate uptake systems of Yersinia pestis.

Yersinia pestis genomes contain genes homologous to the aerobactin receptor (iutA) and biosynthetic genes (iucABCD) as well as the ferric hydroxamate uptake system (fhuCDB) of Escherichia coli. However, iucA is disrupted by a frameshift mutation. An E. coli strain carrying the cloned Y. pestis aerobactin region was unable to produce aerobactin, but could use the siderophore as an iron source. Repair of the frameshift mutation in iucA did not allow aerobactin production in E. coli or Y. pestis. In contrast, a Y. pestis strain with a plasmid encoding the iucABCD-iutA genes from Shigella flexneri or pColV-K30 did produce and secrete the siderophore. In addition, Yersinia pseudotuberculosis PB1, which encodes the iucABCD-iutA locus without the Y. pestis-specific frameshift mutation, also failed to produce aerobactin. The Y. pestis fhuCDB operon, encoding an ABC transporter for a range of hydroxamate siderophores, was able to complement a strain of E. coli with a transposon insertion in fhuC, allowing utilization of aerobactin and ferrichrome. Y. pestis KIM6, a strain deficient in the production of the siderophore yersiniabactin, was able to use both the ferrichrome and the aerobactin siderophores as a source of iron. Mutations in iutA or the fhu operon abolished the ability of KIM6 to use aerobactin. Mutations in the fhu operon, but not in iutA, affected the ability of KIM6 to use ferrichrome. This demonstrates that Y. pestis uses both ferrichrome and aerobactin, but has lost the ability to synthesize aerobactin.

Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2.

The molecular basis of pathogen clone emergence is relatively poorly understood. Acquisition of a bacteriophage encoding a previously unknown secreted phospholipase A(2) (designated SlaA) has been implicated in the rapid emergence in the mid-1980s of a new hypervirulent clone of serotype M3 group A Streptococcus. Although several lines of circumstantial evidence suggest that SlaA is a virulence factor, this issue has not been addressed experimentally. We found that an isogenic DeltaslaA mutant strain was significantly impaired in ability to adhere to and kill human epithelial cells compared with the wild-type parental strain. The mutant strain was less virulent for mice than the wild-type strain, and immunization with purified SlaA significantly protected mice from invasive disease. Importantly, the mutant strain was significantly attenuated for colonization in a monkey model of pharyngitis. We conclude that transductional acquisition of the ability of a GAS strain to produce SlaA enhanced the spread and virulence of the serotype M3 precursor strain. Hence, these studies identified a crucial molecular event underlying the evolution, rapid emergence, and widespread dissemination of unusually severe human infections caused by a distinct bacterial clone.