RESUMO
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.
Assuntos
Fator 1 de Ribosilação do ADP , Mapas de Interação de Proteínas , Proteína Companheira de mTOR Insensível à Rapamicina , Serina-Treonina Quinases TOR , Humanos , Fator 1 de Ribosilação do ADP/metabolismo , Insulina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Mapeamento de Interação de Proteínas/métodosRESUMO
A "tug-of-war" between kinases and phosphatases establishes the phosphorylation states of proteins. While serine and threonine phosphorylation can be catalyzed by more than 400 protein kinases, the majority of serine and threonine dephosphorylation is carried out by seven phosphoprotein phosphatases (PPPs). The PPP family consists of protein phosphatases 1 (PP1), 2A (PP2A), 2B (PP2B), 4 (PP4), 5 (PP5), 6 (PP6), and 7 (PP7). The imbalance in numbers between serine- and threonine-directed kinases and phosphatases led to the early belief that PPPs are unspecific and that kinases are the primary determinants of protein phosphorylation. However, it is now clear that PPPs achieve specificity through association with noncatalytic subunits to form multimeric holoenzymes, which expands the number of functionally distinct signaling entities to several hundred. Although there has been great progress in deciphering signaling by kinases, much less is known about phosphatases.We have developed a chemical proteomic strategy for the systematic interrogation of endogenous PPP catalytic subunits and their interacting proteins, including regulatory and scaffolding subunits (the "PPPome"). PP1, PP2A, PP4, PP5, and PP6 were captured using an immobilized, specific but nonselective PPP inhibitor microcystin-LR (MCLR), followed by protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a single analysis. Here, we combine this approach of phosphatase inhibitor bead profiling and mass spectrometry (PIB-MS) with label-free and tandem mass tag (TMT) quantification to map the PPPome in human cancer cell lines, mouse tissues, and yeast species, through which we identify cell- and tissue-type-specific PPP expression patterns and discover new PPP interacting proteins.
Assuntos
Domínio Catalítico , Microcistinas/farmacologia , Neoplasias/enzimologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Proteômica/métodos , Saccharomyces cerevisiae/enzimologia , Animais , Cromatografia Líquida , Células HeLa , Humanos , Células MCF-7 , Toxinas Marinhas , Camundongos , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
It is unknown how cues from the tumor microenvironment can regulate post-transcriptional mechanisms, such as alternative splicing, that control genes that drive malignant growth. The induction of cyclooxygenase 2 (Cox-2) by integrin α3ß1 in breast cancer cells can promote tumor progression. We have used RNAi to suppress α3ß1 in human MDA-MB-231 breast cancer cells and then investigated changes in global gene expression. Numerous mRNAs, including Cox-2, show altered expression and/or alternative exon usage (AEU) in α3ß1-deficient cells. AEU included patterns predicted to render an mRNA susceptible to degradation, such as 3'-UTR variations or retention of elements that target an mRNA for nonsense-mediated decay (NMD). PCR-based analysis of α3ß1-deficient cells confirmed changes in Cox-2 mRNA that might target it for NMD, including retention of an intron that harbors premature termination codons and changes within the 3'-UTR. Moreover, Cox-2 mRNA has reduced stability in α3ß1-deficient cells, which is partially reversed by knockdown of the essential NMD factor UPF1. Our study identifies α3ß1-mediated AEU as a novel paradigm of integrin-dependent gene regulation that has potential for exploitation as a therapeutic target.
Assuntos
Ciclo-Oxigenase 2/genética , Integrina alfa3beta1/fisiologia , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Neoplasias da Mama , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Íntrons , Laminina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , RNA Helicases , RNA Mensageiro/metabolismo , Transativadores/genética , Transativadores/metabolismo , TranscriptomaRESUMO
Time-restricted feeding (TRF) has gained attention as a dietary regimen that promotes metabolic health. This study questioned if the health benefits of an intermittent TRF (iTRF) schedule require ketone flux specifically in skeletal and cardiac muscles. Notably, we found that the ketolytic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1) is uniquely enriched in isolated mitochondria derived from heart and red/oxidative skeletal muscles, which also have high capacity for fatty acid oxidation (FAO). Using mice with BDH1 deficiency in striated muscles, we discover that this enzyme optimizes FAO efficiency and exercise tolerance during acute fasting. Additionally, iTRF leads to robust molecular remodeling of muscle tissues, and muscle BDH1 flux does indeed play an essential role in conferring the full adaptive benefits of this regimen, including increased lean mass, mitochondrial hormesis, and metabolic rerouting of pyruvate. In sum, ketone flux enhances mitochondrial bioenergetics and supports iTRF-induced remodeling of skeletal muscle and heart.
Assuntos
Cetonas , Miocárdio , Camundongos , Animais , Cetonas/metabolismo , Miocárdio/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Coração , Músculo Esquelético/metabolismoRESUMO
STIM1 is an ER/SR transmembrane protein that interacts with ORAI1 to activate store operated Ca2+ entry (SOCE) upon ER/SR depletion of calcium. Normally highly expressed in skeletal muscle, STIM1 deficiency causes significant changes to mitochondrial ultrastructure that do not occur with loss of ORAI1 or other components of SOCE. The datasets in this article are from large-scale proteomics and phosphoproteomics experiments in an inducible mouse model of skeletal muscle-specific STIM1 knock out (KO). These data reveal statistically significant changes in the relative abundance of specific proteins and sites of protein phosphorylation in STIM1 KO gastrocnemius. Protein samples from five biological replicates of each condition (+/- STIM1) were enzymatically digested, the resulting peptides labeled with tandem mass tag (TMT) reagents, mixed, and fractionated. Phosphopeptides were enriched and a small amount of each input retained for protein abundance analysis. All phosphopeptide and input fractions were analyzed by nano LC-MS/MS on a Q Exactive Plus Orbitrap mass spectrometer, searched with Proteome Discoverer software, and processed with in-house R-scripts for data normalization and statistical analysis. Article published in Molecular Metabolism [1].
RESUMO
OBJECTIVE: Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane endoplasmic/sarcoplasmic reticulum (E/SR) protein recognized for its role in a store operated Ca2+ entry (SOCE), an ancient and ubiquitous signaling pathway. Whereas STIM1 is known to be indispensable during development, its biological and metabolic functions in mature muscles remain unclear. METHODS: Conditional and tamoxifen inducible muscle STIM1 knock-out mouse models were coupled with multi-omics tools and comprehensive physiology to understand the role of STIM1 in regulating SOCE, mitochondrial quality and bioenergetics, and whole-body energy homeostasis. RESULTS: This study shows that STIM1 is abundant in adult skeletal muscle, upregulated by exercise, and is present at SR-mitochondria interfaces. Inducible tissue-specific deletion of STIM1 (iSTIM1 KO) in adult muscle led to diminished lean mass, reduced exercise capacity, and perturbed fuel selection in the settings of energetic stress, without affecting whole-body glucose tolerance. Proteomics and phospho-proteomics analyses of iSTIM1 KO muscles revealed molecular signatures of low-grade E/SR stress and broad activation of processes and signaling networks involved in proteostasis. CONCLUSION: These results show that STIM1 regulates cellular and mitochondrial Ca2+ dynamics, energy metabolism and proteostasis in adult skeletal muscles. Furthermore, these findings provide insight into the pathophysiology of muscle diseases linked to disturbances in STIM1-dependent Ca2+ handling.
Assuntos
Tolerância ao Exercício , Proteostase , Molécula 1 de Interação Estromal , Animais , Cálcio/metabolismo , Metabolismo Energético , Camundongos , Músculo Esquelético/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as "methylation"). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3ß bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Espectrometria de Massas , Metilação , Camundongos , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Fosforilação , Domínios Proteicos , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteômica/métodosRESUMO
Proteolytic processing of the laminin-γ2 chain is a hallmark of basement membrane maturation in the skin. Integrin α3ß1, a major receptor for epidermal adhesion to laminin-332, is critical for proper basement membrane organization during skin development and wound healing. Previously, we identified a role for α3ß1 in promoting the processing of laminin-γ2 in cultured keratinocytes in vitro and in wound epidermis in vivo. In this study we identify the Bmp1 gene, which encodes variants of the mTLD/BMP-1 metalloproteases, as a critical regulator of α3ß1-dependent laminin-γ2 processing, thereby expanding the role of this integrin in controlling the secretion by the epidermis of factors that modulate the tissue microenvironment. Because our previous studies identified another epidermal integrin, α9ß1, as a suppressive regulator of α3ß1-dependent wound angiogenesis, we investigated whether α9ß1 has a similar cross-suppressive effect on the ability of α3ß1 to promote basement membrane organization. Here, we show that, rather than a cross-suppressive role, α9ß1 has an opposing role in basement membrane assembly/maturation through reduced laminin-γ2 processing via mTLD/BMP-1. Although α3ß1 promotes this process during wound healing, α9ß1 has an inhibitory role, suggesting that regulation of basement membrane assembly requires a complex interplay between these distinct epidermal integrins.
Assuntos
Proteína Morfogenética Óssea 1/metabolismo , Integrina alfa3beta1/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Cicatrização/fisiologia , Ferimentos e Lesões/patologia , Animais , Membrana Basal/metabolismo , Proteína Morfogenética Óssea 1/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Epiderme/lesões , Epiderme/metabolismo , Humanos , Integrina alfa3beta1/genética , Integrinas/genética , Queratinócitos , Camundongos , Camundongos Knockout , Proteólise , RNA Interferente Pequeno/metabolismo , Ferimentos e Lesões/etiologia , CalininaRESUMO
Polo-like kinase 1 (Plk1) is an essential protein kinase that promotes faithful mitotic progression in eukaryotes. The subcellular localization and substrate interactions of Plk1 are tightly controlled and require its binding to phosphorylated residues. To identify phosphorylation-dependent interactions within the Plk1 network in human mitotic cells, we performed quantitative proteomics on HeLa cells cultured with kinase inhibitors or expressing a Plk1 mutant that was deficient in phosphorylation-dependent substrate binding. We found that many interactions were abolished upon kinase inhibition; however, a subset was protected from phosphatase opposition or was unopposed, resulting in persistent interaction of the substrate with Plk1. This subset includes phosphoprotein phosphatase 6 (PP6), whose activity toward Aurora kinase A (Aurora A) was inhibited by Plk1. Our data suggest that this Plk1-PP6 interaction generates a feedback loop that coordinates and reinforces the activities of Plk1 and Aurora A during mitotic entry and is terminated by the degradation of Plk1 during mitotic exit. Thus, we have identified a mechanism for the previously puzzling observation of the Plk1-dependent regulation of Aurora A.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Fosfoproteínas Fosfatases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Quinase 1 Polo-LikeRESUMO
Glycogen is the primary storage form of glucose. Glycogen synthesis and breakdown are tightly controlled by glycogen synthase (GYS) and phosphorylase, respectively. The enzyme responsible for dephosphorylating GYS and phosphorylase, which results in their activation (GYS) or inactivation (phosphorylase) to robustly stimulate glycogen synthesis, is protein phosphatase 1 (PP1). However, our understanding of how PP1 recruits these substrates is limited. Here, we show how PP1, together with its muscle glycogen-targeting (GM) regulatory subunit, recruits and selectively dephosphorylates its substrates. Our molecular data reveal that the GM carbohydrate binding module (GM CBM21), which is amino-terminal to the GM PP1 binding domain, has a dual function in directing PP1 substrate specificity: It either directly recruits substrates (i.e., GYS) or recruits them indirectly by localization (via glycogen for phosphorylase). Our data provide the molecular basis for PP1 regulation by GM and reveal how PP1-mediated dephosphorylation is driven by scaffolding-based substrate recruitment.
Assuntos
Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Músculo Esquelético/enzimologia , Proteína Fosfatase 1/metabolismo , Animais , Glicogênio Sintase/química , Humanos , Fosforilação , Conformação Proteica , Proteína Fosfatase 1/química , Coelhos , Especificidade por SubstratoRESUMO
Development of wound therapies is hindered by poor understanding of combinatorial integrin function in the epidermis. In this study, we generated mice with epidermis-specific deletion of α3ß1, α9ß1, or both integrins as well as keratinocyte lines expressing these integrin combinations. Consistent with proangiogenic roles for α3ß1, α3-null keratinocytes showed reduced paracrine stimulation of endothelial cell migration and survival, and wounds of epidermis-specific α3 knockout mice displayed impaired angiogenesis. Interestingly, α9ß1 in keratinocytes suppressed α3ß1-mediated stimulation of endothelial cells, and wounds of epidermis-specific α9 knockout mice displayed delayed vascular normalization and reduced endothelial apoptosis, indicating that α9ß1 cross-suppresses α3ß1 proangiogenic functions. Moreover, α9ß1 inhibited α3ß1 signaling downstream of focal adhesion kinase (FAK) autoactivation at the point of Src-mediated phosphorylation of FAK Y861/Y925. Finally, α9ß1 cross-suppressed many α3ß1-dependent genes, including the gene that encodes MMP-9, which we implicated as a regulator of integrin-dependent cross talk to endothelial cells. Our findings identify a novel physiological context for combinatorial integrin signaling, laying the foundation for therapeutic strategies that manipulate α9ß1 and/or α3ß1 during wound healing.