An AMPKa2-specific phospho-switch controls lysosomal targeting for activation

AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are metabolic kinases that co-ordinate nutrient supply with cell growth. AMPK negatively regulates mTORC1, and mTORC1 reciprocally phosphorylates S345/7 in both AMPK α-isoforms. We report that genetic or torin1-induced loss of α2-S345 phosphorylation relieves suppression of AMPK signaling; however, the regulatory effect does not translate to α1-S347 in HEK293T or MEF cells. Dephosphorylation of α2-S345, but not α1-S347, transiently targets AMPK to lysosomes, a cellular site for activation by LKB1. By mass spectrometry, we find that α2-S345 is basally phosphorylated at 2.5-fold higher stoichiometry than α1-S347 in HEK293T cells and, unlike α1, phosphorylation is partially retained after prolonged mTORC1 inhibition. Loss of α2-S345 phosphorylation in endogenous AMPK fails to sustain growth of MEFs under amino acid starvation conditions. These findings uncover an α2-specific mechanism by which AMPK can be activated at lysosomes in the absence of changes in cellular energy

Advances in understanding and treating ADHD

Attention deficit hyperactivity disorder (ADHD) is a neurocognitive behavioral developmental disorder most commonly seen in childhood and adolescence, which often extends to the adult years. Relative to a decade ago, there has been extensive research into understanding the factors underlying ADHD, leading to far more treatment options available for both adolescents and adults with this disorder. Novel stimulant formulations have made it possible to tailor treatment to the duration of efficacy required by patients, and to help mitigate the potential for abuse, misuse and diversion. Several new non-stimulant options have also emerged in the past few years. Among these, cognitive behavioral interventions have proven popular in the treatment of adult ADHD, especially within the adult population who cannot or will not use medications, along with the many medication-treated patients who continue to show residual disability

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The contribution of X-linked coding variation to severe developmental disorders

Abstract: Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders

Elevated basal AMP-activated protein kinase activity sensitizes colorectal cancer cells to growth inhibition by metformin

Expression and activity of the AMP-activated protein kinase (AMPK) α1 catalytic subunit of the heterotrimeric kinase significantly correlates with poor outcome for colorectal cancer patients. Hence there is considerable interest in uncovering signalling vulnerabilities arising from this oncogenic elevation of AMPKα1 signalling. We have therefore attenuated mammalian target of rapamycin (mTOR) control of AMPKα1 to generate a mutant colorectal cancer in which AMPKα1 signalling is elevated because AMPKα1 serine 347 cannot be phosphorylated by mTORC1. The elevated AMPKα1 signalling in this HCT116 α1.S347A cell line confers hypersensitivity to growth inhibition by metformin. Complementary chemical approaches confirmed this relationship in both HCT116 and the genetically distinct HT29 colorectal cells, as AMPK activators imposed vulnerability to growth inhibition by metformin in both lines. Growth inhibition by metformin was abolished when AMPKα1 kinase was deleted. We conclude that elevated AMPKα1 activity modifies the signalling architecture in such a way that metformin treatment compromises cell proliferation. Not only does this mutant HCT116 AMPKα1-S347A line offer an invaluable resource for future studies, but our findings suggest that a robust biomarker for chronic AMPKα1 activation for patient stratification could herald a place for the well-tolerated drug metformin in colorectal cancer therapy

A TOR (target of rapamycin) and nutritional phosphoproteome of fission yeast reveals novel targets in networks conserved in humans

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe. Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPKα (ssp2::ura4+), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated

Supplementary Figure 1; Supplementary Figure 2; Supplementary Figure 3; from Elevated basal AMP-activated protein kinase activity sensitizes colorectal cancer cells to growth inhibition by metformin

Validation of CRISPR-edited ATP1A1 HCT116 cell lines for use as control lines.; Genotypic validation of CRISPR-edited HCT116 cells lines; AMPK activation sensitises HCT116 AMPKα1 wild type but not AMPKα1-KO edited colorectal cancer cells to metformin

Personalized phosphoproteomics identifies functional signaling

Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce ‘personalized phosphoproteomics’, a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure individual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology