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Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase. [ABSTRACT FROM AUTHOR]

Circadian locomotor output cycles kaput (CLOCK) is a transcription factor that activates transcription of clock-controlled genes by heterodimerizing with BMAL1 and binding to E-box elements on DNA. Although several phosphorylation sites on CLOCK have already been identified, this study characterizes a novel phosphorylation site at serine 845 (Ser-836 in humans). Here, we show that CLOCK is a novel AKT substrate in vitro and in cells, and this phosphorylation site is a negative regulator of CLOCK nuclear localization by acting as a binding site for 14-3-3 proteins. To examine the role of CLOCK phosphorylation in vivo, ClockS845A knockin mice were generated using CRISPR/Cas9 technology. ClockS845A mice are essentially normal with normal central circadian rhythms and hemodynamics. However, examination of core circadian gene expression from peripheral tissues demonstrated that ClockS845A mice have diminished expression of Per2, Reverba, Dbp, and Npas2 in skeletal muscle and Per2, Reverba, Dbp, Per1, Rora, and Npas2 in the liver during the circadian cycle. The reduction in Dbp levels is associated with reduced H3K9ac at E-boxes where CLOCK binds despite no change in total CLOCK levels. Thus, CLOCK phosphorylation by AKT on Ser-845 regulates its nuclear translocation and the expression levels of certain core circadian genes in insulin-sensitive tissues. [ABSTRACT FROM AUTHOR]

The AKT signaling pathway is important for circadian rhythms in mammals and flies (Drosophila). However, AKT signaling in mammals is more complicated since there are 3 isoforms of AKT, each performing slightly different functions. Here we study the most ubiquitous AKT isoform, Akt1, and its role at the organismal level in the central and vascular peripheral clocks. Akt1–/– mice exhibit relatively normal behavioral rhythms with only minor differences in circadian gene expression in the liver and heart. However, circadian gene expression in the Akt1–/– aorta, compared with control aorta, follows a distinct pattern. In the Akt1–/– aorta, positive regulators of circadian transcription have lower amplitude rhythms and peak earlier in the day, and negative circadian regulators are expressed at higher amplitudes and peak later in the day. In endothelial cells, negative circadian regulators exhibit an increased amplitude of expression, while the positive circadian regulators are arrhythmic with a decreased amplitude of expression. This indicates that Akt1 conditions the normal circadian rhythm in the vasculature more so than in other peripheral tissues where other AKT isoforms or kinases might be important for daily rhythms. [ABSTRACT FROM AUTHOR]

The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas non-physiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akfl-deficient, but not Akt2-deficient, ECs, including eNOS and Fork-head box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling. [ABSTRACT FROM AUTHOR]

Premise of the study: Previous studies have shown that polyploidy has pronounced effects on photosynthesis. Most of these studies have focused on synthetic or recently formed autopolyploids, and comparatively little is known about the integrated effects of natural allopolyploidy, which involves hybridity and genome doubling and often incorporates multiple genotypes through recurrent origins and lineage recombination. Methods: Glycine dolichocarpa (designated T2) is a natural allotetraploid with multiple origins. We quantified 21 anatomical, biochemical, and physiological phenotypes relating to photosynthesis in T2 and its diploid progenitors, G. tomentella (D3) and G. syndetika (D4). To assess how direction of cross affects these phenotypes, we included three T2 accessions having D3-1ikc plastids (T2D3) and two accessions having D4-1ike plastids (T2m). Key results: T2 accessions were transgressive (more extreme than any diploid accession) for 17 of 21 phenotypes, and species means differed significantly in T2 vs. both progenitors for four of 21 phenotypes (higher for guard cell length, electron transport capacity [Jmax] per palisade cell, and Jmax. per mesophyll cell; lower for palisade cells per unit leaf area). Within T2, four of 21 parameters differed significantly between T2D3 and T2D3 (palisade cell volume; chloroplast number and volume per unit leaf area; and Jmax per unit leaf area). Conclusions: T2 is characterized by transgressive photosynthesis-related phenotypes (including an ca. 2-fold increase in Jmax per cell), as well as by significant intraspecies variation correlating with plastid type. These data indicate prominent roles for both nucleotypic effects and cytoplasmic factors in photosynthetic responses to allopolyploidy. [ABSTRACT FROM AUTHOR]

* Premise of the study: RNA-seq analysis of plant transcriptomes poses unique challenges due to the highly duplicated nature of plant genomes. We address these challenges in the context of recently formed polyploid species and detail an RNA-seq experiment comparing the leaf transcription profile of an allopolyploids relative of soybean with the diploid species that contributed its homoeologous genomes. * Methods: RNA-seq reads were obtained from the three species and were aligned against the genome sequence of Glycine max. Transcript levels were estimated for each gene, relative contributions of polyploidy-duplicated loci (homoeologues) in the tetraploid were identified, and comparisons of transcript profiles and individual genes were used to analyze the regulation of transcript levels. * Key results: We present a novel metric developed to address issues arising from high degrees of gene space duplication and a method for dissecting a gene's measured transcript level in a polyploid species into the relative contribution of its homocologues. We identify the gene family likely contributing to differences in photosynthetic rate between the allotetraploid and its progenitors and show that the tetraploid appears to be using the "redundant" gene copies in novel ways. * Conclusions: Given the prevalence of polyploidy events in plants, we believe many of the approaches developed here to be applicable, and often necessary, in most plant RNA-seq experiments. The deep sampling provided by RNA-seq allows us to dissect the genetic underpinnings of specific phenotypes as well as examine complex interactions within polyploid genomes. [ABSTRACT FROM AUTHOR]

Impaired wound healing is a major complication of diabetes, and despite the associated risks, treatment strategies for diabetic wounds remain limited. This is due, in part, to an incomplete understanding of the underlying pathological mechanisms, including the effects of hyperglycemia on components of the extracellular matrix (ECM). In the current study, we explored whether the expression of thrombospondin 2 (TSP2), a matricellular protein with a demonstrated role in response to injury, was associated with delayed healing in diabetes. First, we found that TSP2 expression was elevated in diabetic mice and skin from patients with diabetes. Then, to determine the contribution of TSP2 to impaired healing in diabetes, we developed a novel diabetic TSP2-deficient model. Though the TSP2-deficient mice developed obesity and hyperglycemia comparable with diabetic control mice, they exhibited significantly improved healing, characterized by accelerated reepithelialization and increased granulation tissue formation, fibroblast migration, and blood vessel maturation. We further found that hyperglycemia increased TSP2 expression in fibroblasts, the major cellular source of TSP2 in wounds. Mechanistically, high glucose increased activation of the hexosamine pathway and nuclear factor-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes. [ABSTRACT FROM AUTHOR]

The contribution of endothelial-derived miR-17~92 to ischemia-induced arteriogenesis has not been investigated in an in vivomodel. In the present study, we demonstrate a critical role for the endothelial-derived miR-17~92 cluster in shaping physiological and ischemia-triggered arteriogenesis. Endothelial-specific deletion of miR-17~92 results in an increase in collateral density limbs and hearts and in ischemic limbs compared with control mice, and consequently improves blood flow recovery. Individual cluster components positively or negatively regulate endothelial cell (EC) functions in vitro, and, remarkably, ECs lacking the cluster spontaneously form cords in a manner rescued by miR-17a, -18a, and -19a. Using both in vitro and in vivo analyses, we identified FZD4 and LRP6 as targets of miR-19a/b. Both of these targets were up-regulated in 17~92 KO ECs compared with control ECs, and both were shown to be targeted by miR- 19 using luciferase assays. We demonstrate that miR-19a negatively regulates FZD4, its coreceptor LRP6, and WNT signaling, and that antagonism of miR-19a/b in aged mice improves blood flow recovery after ischemia and reduces repression of these targets. Collectively, these data provide insights into miRNA regulation of arterialization and highlight the importance of vascular WNT signaling in maintaining arterial blood flow. [ABSTRACT FROM AUTHOR]

Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of β1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of β1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo. [ABSTRACT FROM AUTHOR]