RESUMEN
AMP-activated protein kinase (AMPK) is a master sensor and regulator of cellular energy status. Upon metabolic stress, AMPK suppresses anabolic and promotes catabolic processes to regain energy homeostasis. Cancer cells can occasionally suppress the growth-restrictive AMPK pathway by mutation of an upstream regulatory kinase. Here, we describe a widespread mechanism to suppress AMPK through its ubiquitination and degradation by the cancer-specific MAGE-A3/6-TRIM28 ubiquitin ligase. MAGE-A3 and MAGE-A6 are highly similar proteins normally expressed only in the male germline but frequently re-activated in human cancers. MAGE-A3/6 are necessary for cancer cell viability and are sufficient to drive tumorigenic properties of non-cancerous cells. Screening for targets of MAGE-A3/6-TRIM28 revealed that it ubiquitinates and degrades AMPKα1. This leads to inhibition of autophagy, activation of mTOR signaling, and hypersensitization to AMPK agonists, such as metformin. These findings elucidate a germline mechanism commonly hijacked in cancer to suppress AMPK.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Antígenos de Neoplasias/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Metabolismo Energético , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Alineación de Secuencia , Transducción de Señal , Testículo/metabolismoRESUMEN
Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Transporte de Proteínas , Proteínas/metabolismo , Actinas/metabolismo , Proteínas de Unión al ADN/genética , Endosomas/metabolismo , Técnicas de Silenciamiento del Gen , Aparato de Golgi/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/genética , Proteínas/genética , Interferencia de ARN , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
Nitric oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca(2+)](i)), and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells and that inhibition requires an increase in [Ca(2+)](i). Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca(2+)](i), up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca(2+)](i), also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca(2+) remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in K(m) for GTP, which rises to 834 µM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca(2+)](i) in response to NO, inducing vasodilation, but also is inhibited by high [Ca(2+)](i), providing a fine balance between signals for vasodilation and vasoconstriction.
Asunto(s)
Angiotensina II/farmacología , Calcio/metabolismo , Guanilato Ciclasa/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Trombospondina 1/farmacología , Vasoconstrictores/farmacología , Antígeno CD47 , Células Cultivadas , Citometría de Flujo , Guanilato Ciclasa/metabolismo , Humanos , Células Jurkat , Cinética , Óxido Nítrico/metabolismo , Fosforilación , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa Soluble , Vasoconstricción/efectos de los fármacosRESUMEN
Melanoma Antigen Genes (MAGEs) are a family of genes that have piqued the interest of scientists for their unique expression pattern. A subset of MAGEs (Type I) are expressed in spermatogonial cells and in no other somatic tissue, and then re-expressed in many cancers. Type I MAGEs are often referred to as cancer-testis antigens due to this expression pattern, while Type II MAGEs are more ubiquitous in expression. This study determines the cause and consequence of the aberrant expression of the MAGE-A subfamily of cancer-testis antigens. We have discovered that MAGE-A genes are regulated by DNA methylation, as revealed by treatment with 5-azacytidine, an inhibitor of DNA methyltransferases. Furthermore, bioinformatics analysis of existing methylome sequencing data also corroborates our findings. The consequence of expressing certain MAGE-A genes is an increase in cell proliferation and colony formation and resistance to chemo-therapeutic agent 5-fluorouracil and DNA damaging agent sodium arsenite. Taken together, these data indicate that DNA methylation plays a crucial role in regulating the expression of MAGE-A genes which then act as drivers of cell proliferation, anchorage-independent growth and chemo-resistance that is critical for cancer-cell survival.