Malonyl-CoA is a conserved endogenous ATP-competitive mTORC1 inhibitor.

Cell growth is regulated by the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here we show that mTORC1 senses the capacity of a cell to synthesise fatty acids by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When fatty acid synthase (FASN) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with acetyl-CoA carboxylase 1 (ACC1) and FASN. Our findings represent a conserved and unique homeostatic mechanism whereby impaired fatty acid biogenesis leads to reduced mTORC1 activity to coordinately link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the existence of a physiological metabolite that directly inhibits the activity of a signalling kinase in mammalian cells by competing with ATP for binding.

Cdk4 and Cdk6 Couple the Cell-Cycle Machinery to Cell Growth via mTORC1.

Cell growth is coupled to cell-cycle progression in mitotically proliferating mammalian cells, but the underlying molecular mechanisms are not well understood. CyclinD-Cdk4/6 is known to phosphorylate RB to promote S-phase entry, but recent work suggests they have additional functions. We show here that CyclinD-Cdk4/6 activates mTORC1 by binding and phosphorylating TSC2 on Ser1217 and Ser1452. Pharmacological inhibition of Cdk4/6 leads to a rapid, TSC2-dependent reduction of mTORC1 activity in multiple human and mouse cell lines, including breast cancer cells. By simultaneously driving mTORC1 and E2F, CyclinD-Cdk4/6 couples cell growth to cell-cycle progression. Consistent with this, we see that mTORC1 activity is cell cycle dependent in proliferating neural stem cells of the adult rodent brain. We find that Cdk4/6 inhibition reduces cell proliferation partly via TSC2 and mTORC1. This is of clinical relevance, because Cdk4/6 inhibitors are used for breast cancer therapy.

Cooperation of axial and sex specific information controls Drosophila female genitalia growth by regulating the Decapentaplegic pathway.

The specification and morphogenesis of an organ requires the coordinate deployment and integration of regulatory information, including sex specific information when the organ is sex specific. Only a few gene networks controlling size and pattern development have been deciphered, which limits the emergence of principles, general or not, underlying the organ-specifying gene networks. Here we elucidate the genetic and molecular network determining the control of size in the Drosophila abdominal A9 primordium, contributing to the female genitalia. This network requires axial regulatory information provided by the Hox protein Abdominal-BR (Abd-BR), the Hox cofactors Extradenticle (Exd) and Homothorax (Hth), and the sex specific transcription factor Doublesex Female (DsxF). These factors synergize to control size in the female A9 by the coordinate regulation of the Decapentaplegic (Dpp) growth pathway. Molecular dissection of the dpp regulatory region and in vivo protein interaction experiments suggest that Abd-BR, Exd, Hth and DsxF coordinately regulate a short dpp enhancer to repress dpp expression and restrict female A9 size. The same regulators can also suppress dpp expression in the A8, but this requires the absence of the Abd-BM isoform, which specifies A8. These results delineate the network controlling female A9 growth in Drosophila.

CycD/Cdk4 and Discontinuities in Dpp Signaling Activate TORC1 in the Drosophila Wing Disc.

The molecular mechanisms regulating animal tissue size during development are unclear. This question has been extensively studied in the Drosophila wing disc. Although cell growth is regulated by the kinase TORC1, no readout exists to visualize TORC1 activity in situ in Drosophila. Both the cell cycle and the morphogen Dpp are linked to tissue growth, but whether they regulate TORC1 activity is not known. We develop here an anti-phospho-dRpS6 antibody that detects TORC1 activity in situ. We find, unexpectedly, that TORC1 activity in the wing disc is patchy. This is caused by elevated TORC1 activity at the cell cycle G1/S transition due to CycD/Cdk4 phosphorylating TSC1/2. We find that TORC1 is also activated independently of CycD/Cdk4 when cells with different levels of Dpp signaling or Brinker protein are juxtaposed. We thereby characterize the spatial distribution of TORC1 activity in a developing organ.

The guanine-exchange factor Ric8a binds to the Ca²âº sensor NCS-1 to regulate synapse number and neurotransmitter release.

The conserved Ca(2+)-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gαs regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2-Ric8a-Gαs, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.

Multiple roles for frequenin/NCS-1 in synaptic function and development.

The calcium-binding protein frequenin (Frq), discovered in the fruit fly Drosophila, and its mammalian homologue neuronal calcium sensor 1 (NCS-1) have been reported to affect several aspects of synaptic transmission, including basal levels of neurotransmission and short- and long-term synaptic plasticities. However, discrepant reports leave doubts about the functional roles of these conserved proteins. In this review, we attempt to resolve some of these seemingly contradictory reports. We discuss how stimulation protocols, sources of calcium (voltage-gated channels versus internal stores), and expression patterns (presynaptic versus postsynaptic) of Frq may result in the activation of various protein targets, leading to different synaptic effects. In addition, the potential interactions of Frq's C-terminal and N-terminal domains with other proteins are discussed. Frq also has a role in regulating neurite outgrowth, axonal regeneration, and synaptic development. We examine whether the effects of Frq on neurotransmitter release and neurite outgrowth are distinct or interrelated through homeostatic mechanisms. Learning and memory are affected by manipulations of Frq probably through changes in synaptic transmission and neurite outgrowth, raising the possibility that Frq may be implicated in human pathological conditions, including schizophrenia, bipolar disorder, and X-linked mental retardation.

Two frequenins in Drosophila: unveiling the evolutionary history of an unusual neuronal calcium sensor (NCS) duplication.

BACKGROUND: Drosophila frequenin (Frq), the homolog of the mammalian neuronal calcium sensor-1 (NCS-1), is a high affinity calcium-binding protein with ubiquitous expression in the nervous system. This protein has an important role in the regulation of neurotransmitter release per synapse, axonal growth and bouton formation. In D. melanogaster, frequenin is encoded by two genes (frq1 and frq2), a very unexpected feature in the Frq/NCS-1 subfamily. These genes are located in tandem in the same genomic region, and their products are 95% identical in their amino acid sequence, clearly indicating their recent origin by gene duplication. Here, we have investigated the factors involved in this unusual feature by examining the molecular evolution of the two frq genes in Drosophila and the evolutionary dynamics of NCS family in a large set of bilaterian species. RESULTS: Surprisingly, we have found no amino acid replacements fixed across the twelve Drosophila species surveyed. In contrast, synonymous substitutions have been prevalent in the evolution of the coding region of frq1 and frq2, indicating the presence of strong functional constraints following gene duplication. Despite that, we have detected that significant evolutionary rate acceleration had occurred in Frq1 in early times from the duplication, in which positive selection (likely promoting functional diversification) had probably an important role. The analysis of sequence conservation and DNA topology at the non-coding regions of both genes has allowed the identification of DNA regions candidates to be cis-regulatory elements. The results reveal a possible mechanism of regulatory diversification between frq1 and frq2. CONCLUSIONS: The presence of two frequenins in Drosophila and the rapid accumulation of amino acid substitutions after gene duplication are very unusual features in the evolution of the Frq/NCS-1 subfamily. Here we show that the action of positive selection in concordance with some extent of regulatory diversification might explain these findings. Selected amino acid substitutions in Frq1 likely contributed to the functional divergence between the two duplicates, which, in turn, should have diverged in their regulation by ecdysone-induced early genes.

Frequenin/NCS-1 and the Ca2+-channel alpha1-subunit co-regulate synaptic transmission and nerve-terminal growth.

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca(2+)-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca(2+) entry and enhanced nerve-terminal growth. The impaired Ca(2+) entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca(2+) channels, or that it regulates the PI4Kbeta pathway as described in other organisms. To determine whether Frq interacts with PI4Kbeta with consequent effects on Ca(2+) channels, we first characterized a PI4Kbeta-null mutant and found that PI4Kbeta was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kbeta-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kbeta. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the alpha(1)-subunit of voltage-gated Ca(2+) channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca(2+) entry through a functional interaction with the alpha(1) voltage-gated Ca(2+)-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.

Chronic and acute alterations in the functional levels of Frequenins 1 and 2 reveal their roles in synaptic transmission and axon terminal morphology.

Frequenin (Frq) and its mammalian homologue, neuronal calcium sensor 1 (NCS-1), are important calcium-binding proteins which enhance neurotransmitter release and facilitation. Here, we report the discovery of a second Frq-encoding gene (frq2) in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Frq1 is more abundant than Frq2, and is most highly expressed in larva. Loss-of-function phenotypes were studied using dominant negative peptides to prevent Frq target binding, RNAi to reduce gene transcription, or both methods. To discriminate chronic from acute loss-of-function effects, we compared the effects of transgenic expression and forward-filling the dominant-negative peptide into presynaptic terminals. In both cases, a 70% reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations of the synapse during development. The chronic treatment also produced more synaptic boutons from MNSNb/d-Is motorneurons, but fewer active zones per bouton. By contrast, excess-of-function conditions yielded a 1.4- to 2-fold increase in quantal content and fewer boutons in the same motorneuron. These synaptic effects resulted in behavioural changes in the Buridan locomotion assay, showing that walking speed is dependent on Frq activity in the nervous system. All the effects were identical for both Frqs, and consistent with excess- and loss-of-function genotypes. We conclude that Frqs have two distinct functions: one in neurotransmission, regulating the probability of release per synapse, and another in axonal growth and bouton formation.