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1.
J Proteome Res ; 22(3): 768-789, 2023 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-36763541

RESUMO

Phosphorylation-dependent signal transduction plays an important role in regulating the functions and fate of skeletal muscle cells. Central players in the phospho-signaling network are the protein kinases AKT, S6K, and RSK as part of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathways. However, despite their functional importance, knowledge about their specific targets is incomplete because these kinases share the same basophilic substrate motif RxRxxp[ST]. To address this, we performed a multifaceted quantitative phosphoproteomics study of skeletal myotubes following kinase inhibition. Our data corroborate a cross talk between AKT and RAF, a negative feedback loop of RSK on ERK, and a putative connection between RSK and PI3K signaling. Altogether, we report a kinase target landscape containing 49 so far unknown target sites. AKT, S6K, and RSK phosphorylate numerous proteins involved in muscle development, integrity, and functions, and signaling converges on factors that are central for the skeletal muscle cytoskeleton. Whereas AKT controls insulin signaling and impinges on GTPase signaling, nuclear signaling is characteristic for RSK. Our data further support a role of RSK in glucose metabolism. Shared targets have functions in RNA maturation, stability, and translation, which suggests that these basophilic kinases establish an intricate signaling network to orchestrate and regulate processes involved in translation.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Fibras Musculares Esqueléticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa , Proteínas Quinases S6 Ribossômicas 70-kDa
2.
Anal Chem ; 94(31): 10893-10906, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35880733

RESUMO

With increasing sensitivity and accuracy in mass spectrometry, the tumor phosphoproteome is getting into reach. However, the selection of quantitation techniques best-suited to the biomedical question and diagnostic requirements remains a trial and error decision as no study has directly compared their performance for tumor tissue phosphoproteomics. We compared label-free quantification (LFQ), spike-in-SILAC (stable isotope labeling by amino acids in cell culture), and tandem mass tag (TMT) isobaric tandem mass tags technology for quantitative phosphosite profiling in tumor tissue. Compared to the classic SILAC method, spike-in-SILAC is not limited to cell culture analysis, making it suitable for quantitative analysis of tumor tissue samples. TMT offered the lowest accuracy and the highest precision and robustness toward different phosphosite abundances and matrices. Spike-in-SILAC offered the best compromise between these features but suffered from a low phosphosite coverage. LFQ offered the lowest precision but the highest number of identifications. Both spike-in-SILAC and LFQ presented susceptibility to matrix effects. Match between run (MBR)-based analysis enhanced the phosphosite coverage across technical replicates in LFQ and spike-in-SILAC but further reduced the precision and robustness of quantification. The choice of quantitative methodology is critical for both study design such as sample size in sample groups and quantified phosphosites and comparison of published cancer phosphoproteomes. Using ovarian cancer tissue as an example, our study builds a resource for the design and analysis of quantitative phosphoproteomic studies in cancer research and diagnostics.


Assuntos
Neoplasias Ovarianas , Proteômica , Feminino , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Neoplasias Ovarianas/diagnóstico , Proteoma/química , Proteômica/métodos
3.
Cells ; 10(10)2021 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-34685691

RESUMO

Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.


Assuntos
Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mapas de Interação de Proteínas , Aminoácidos/deficiência , Animais , Proteínas Sanguíneas/farmacologia , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Organogênese/efeitos dos fármacos , Análise de Componente Principal , Mapas de Interação de Proteínas/efeitos dos fármacos , Sirolimo/farmacologia
4.
Cell ; 184(3): 655-674.e27, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33497611

RESUMO

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Helicases/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Esclerose Tuberosa/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/química , Evolução Molecular , Feminino , Humanos , Insulina/farmacologia , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismo
5.
Nat Commun ; 7: 13254, 2016 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-27869123

RESUMO

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoácidos/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Modelos Biológicos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética
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