bims-pimaco 2022-08-21 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–08–21
nine papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Proximity labeling of endogenous RICTOR identifies mTOR Complex 2 regulation by ADP ribosylation factor ARF1.
AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.
Structural keys unlock RAS-MAPK cellular signalling pathway.
PH domain-mediated autoinhibition and oncogenic activation of Akt.
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy.
Direct control of lysosomal catabolic activity by mTORC1 through regulation of V-ATPase assembly.
Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.
Ordered and deterministic cancer genome evolution after p53 loss.
TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle.

J Biol Chem. 2022 Aug 13. pii: S0021-9258(22)00822-5. [Epub ahead of print] 102379

Proximity labeling of endogenous RICTOR identifies mTOR Complex 2 regulation by ADP ribosylation factor ARF1.

Amelia K Luciano, Ekaterina Korobkina, Scott P Lyons, John A Haley, Shelagh Fluharty, Su Myung Jung, Arminja N Kettenbach, David A Guertin.

  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- 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.

Keywords:  ADP ribosylation factor (ARF); Akt/PKB; BioID2; RICTOR; cell signaling; growth factor; mTORC2; mammalian target of rapamycin (mTOR); proximity labeling

DOI:  https://doi.org/10.1016/j.jbc.2022.102379
Nat Commun. 2022 Aug 17. 13(1): 4836

AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.

George K E Umanah, Leire Abalde-Atristain, Mohammed Repon Khan, Jaba Mitra, Mohamad Aasif Dar, Melissa Chang, Kavya Tangella, Amy McNamara, Samuel Bennett, Rong Chen, Vasudha Aggarwal, Marisol Cortes, Paul F Worley, Taekjip Ha, Ted M Dawson, Valina L Dawson.

The mechanistic target of rapamycin (mTOR) signals through the mTOR complex 1 (mTORC1) and the mTOR complex 2 to maintain cellular and organismal homeostasis. Failure to finely tune mTOR activity results in metabolic dysregulation and disease. While there is substantial understanding of the molecular events leading mTORC1 activation at the lysosome, remarkably little is known about what terminates mTORC1 signaling. Here, we show that the AAA + ATPase Thorase directly binds mTOR, thereby orchestrating the disassembly and inactivation of mTORC1. Thorase disrupts the association of mTOR to Raptor at the mitochondria-lysosome interface and this action is sensitive to amino acids. Lack of Thorase causes accumulation of mTOR-Raptor complexes and altered mTORC1 disassembly/re-assembly dynamics upon changes in amino acid availability. The resulting excessive mTORC1 can be counteracted with rapamycin in vitro and in vivo. Collectively, we reveal Thorase as a key component of the mTOR pathway that disassembles and thus inhibits mTORC1.

DOI: https://doi.org/10.1038/s41467-022-32365-2
Nature. 2022 Aug 15.

Structural keys unlock RAS-MAPK cellular signalling pathway.

Hugo Lavoie, Marc Therrien.



Keywords:  Molecular biology; Structural biology

DOI:  https://doi.org/10.1038/d41586-022-02189-7
Elife. 2022 Aug 15. pii: e80148. [Epub ahead of print]11

PH domain-mediated autoinhibition and oncogenic activation of Akt.

Hwan Bae, Thibault Viennet, Eunyoung Park, Nam Chu, Antonieta Salguero, Michael J Eck, Haribabu Arthanari, Philip A Cole.

  Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.

Keywords:  biochemistry; chemical biology; human; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.80148
EMBO J. 2022 Aug 15. e110398

Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy.

Samuel J Rodgers, Emily I Jones, Senthil Arumugam, Sabryn A Hamila, Jill Danne, Rajendra Gurung, Matthew J Eramo, Randini Nanayakkara, Georg Ramm, Meagan J McGrath, Christina A Mitchell.

  Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells co-ordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient-rich conditions, is unknown. Here, we identify an endosome-dependent phosphoinositide pathway that links PI3Kα signaling to lysosome repopulation during basal autophagy. We show that PI3Kα-derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvation-induced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5-kinase, PIKfyve, to generate PI(3,5)P2 . The SNX-BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyve-dependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient-rich conditions, PI3Kα, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P2 -dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Kα signaling to lysosome reformation during basal autophagy.

Keywords:  INPP4B; PI3Kα; PIKfyve; autophagy; lysosome

DOI:  https://doi.org/10.15252/embj.2021110398
Nat Commun. 2022 Aug 17. 13(1): 4848

Direct control of lysosomal catabolic activity by mTORC1 through regulation of V-ATPase assembly.

Edoardo Ratto, S Roy Chowdhury, Nora S Siefert, Martin Schneider, Marten Wittmann, Dominic Helm, Wilhelm Palm.

Mammalian cells can acquire exogenous amino acids through endocytosis and lysosomal catabolism of extracellular proteins. In amino acid-replete environments, nutritional utilization of extracellular proteins is suppressed by the amino acid sensor mechanistic target of rapamycin complex 1 (mTORC1) through an unknown process. Here, we show that mTORC1 blocks lysosomal degradation of extracellular proteins by suppressing V-ATPase-mediated acidification of lysosomes. When mTORC1 is active, peripheral V-ATPase V1 domains reside in the cytosol where they are stabilized by association with the chaperonin TRiC. Consequently, most lysosomes display low catabolic activity. When mTORC1 activity declines, V-ATPase V1 domains move to membrane-integral V-ATPase Vo domains at lysosomes to assemble active proton pumps. The resulting drop in luminal pH increases protease activity and degradation of protein contents throughout the lysosomal population. These results uncover a principle by which cells rapidly respond to changes in their nutrient environment by mobilizing the latent catabolic capacity of lysosomes.

DOI: https://doi.org/10.1038/s41467-022-32515-6
Elife. 2022 Aug 17. pii: e73747. [Epub ahead of print]11

Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.

Scott D Hansen, Albert A Lee, Benjamin R Duewell, Jay T Groves.

  The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2 containing membranes and dimerize in a protein density dependent manner. Although dispensable for cooperative PI(4,5)P2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane bound kinase.

Keywords:  biochemistry; chemical biology; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.73747
Nature. 2022 Aug 17.

Ordered and deterministic cancer genome evolution after p53 loss.

Timour Baslan, John P Morris, Zhen Zhao, Jose Reyes, Yu-Jui Ho, Kaloyan M Tsanov, Jonathan Bermeo, Sha Tian, Sean Zhang, Gokce Askan, Aslihan Yavas, Nicolas Lecomte, Amanda Erakky, Anna M Varghese, Amy Zhang, Jude Kendall, Elena Ghiban, Lubomir Chorbadjiev, Jie Wu, Nevenka Dimitrova, Kalyani Chadalavada, Gouri J Nanjangud, Chaitanya Bandlamudi, Yixiao Gong, Mark T A Donoghue, Nicholas D Socci, Alex Krasnitz, Faiyaz Notta, Steve D Leach, Christine A Iacobuzio-Donahue, Scott W Lowe.

Although p53 inactivation promotes genomic instability1 and presents a route to malignancy for more than half of all human cancers2,3, the patterns through which heterogenous TP53 (encoding human p53) mutant genomes emerge and influence tumorigenesis remain poorly understood. Here, in a mouse model of pancreatic ductal adenocarcinoma that reports sporadic p53 loss of heterozygosity before cancer onset, we find that malignant properties enabled by p53 inactivation are acquired through a predictable pattern of genome evolution. Single-cell sequencing and in situ genotyping of cells from the point of p53 inactivation through progression to frank cancer reveal that this deterministic behaviour involves four sequential phases-Trp53 (encoding mouse p53) loss of heterozygosity, accumulation of deletions, genome doubling, and the emergence of gains and amplifications-each associated with specific histological stages across the premalignant and malignant spectrum. Despite rampant heterogeneity, the deletion events that follow p53 inactivation target functionally relevant pathways that can shape genomic evolution and remain fixed as homogenous events in diverse malignant populations. Thus, loss of p53-the 'guardian of the genome'-is not merely a gateway to genetic chaos but, rather, can enable deterministic patterns of genome evolution that may point to new strategies for the treatment of TP53-mutant tumours.

DOI: https://doi.org/10.1038/s41586-022-05082-5
J Cell Sci. 2022 Aug 17. pii: jcs.260378. [Epub ahead of print]

TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle.

Paolo Guerra, Luc-Alban P E Vuillemenot, Yulan B van Oppen, Marije Been, Andreas Milias-Argeitis.

  Recent studies have revealed that the growth rate of budding yeast and mammalian cells varies during the cell cycle. By linking a multitude of signals to cell growth, the highly conserved Target of Rapamycin Complex 1 (TORC1) and Protein Kinase A (PKA) pathways are prime candidates for mediating the dynamic coupling between growth and division. However, measurements of TORC1 and PKA activity during the cell cycle are still lacking. Following the localization dynamics of two TORC1 and PKA targets via time-lapse microscopy in hundreds of yeast cells, we found that the activity of these pathways towards ribosome biogenesis fluctuates in synchrony with the cell cycle even under constant external conditions. Mutations of upstream TORC1 and PKA regulators suggested that internal metabolic signals partially mediate these activity changes. Our study reveals a new aspect of TORC1 and PKA signaling, which will be important for understanding growth regulation during the cell cycle.

Keywords:  Cell growth; PKA Signaling; Ribosome biogenesis; Single-cell microscopy; TOR signaling; TORC1

DOI:  https://doi.org/10.1242/jcs.260378