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Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK.

ABSTRACT: Macroautophagy (herein referred to as autophagy) is an evolutionarily conserved self-digestive process cells adapt to starvation and other stress responses. Upon starvation, autophagy is induced, providing cells with needed nutrient supplies. We report here that Unc-51-like kinase 1 (Ulk1), a key initiator for mammalian autophagy, undergoes dramatic dephosphorylation upon starvation, particularly at serine 638 and serine 758. Phosphorylations of Ulk1 are mediated by mammalian target-of-rapamycin (mTOR) kinase and adenosine monophosphate activated protein kinase (AMPK). AMPK interacts with Ulk1 in a nutrient-dependent manner. Proper phosphorylations on Ulk1 are crucial for Ulk1/AMPK association, as a single serine-to-alanine mutation (S758A) at Ulk1 impairs this interaction. Compared to the wild-type ULK1, this Ulk1-S758A mutant initiates starvation-induced autophagy faster at an early time point, but does not alter the maximum capacity of autophagy when starvation prolongs. This study therefore revealed previously unnoticed acute autophagy response to environmental changes.

SUBMITTER: Shang L

PROVIDER: S-EPMC3064373 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK.

Shang Libin L Chen She S Du Fenghe F Li Shen S Zhao Liping L Wang Xiaodong X

Proceedings of the National Academy of Sciences of the United States of America 20110307 12

Macroautophagy (herein referred to as autophagy) is an evolutionarily conserved self-digestive process cells adapt to starvation and other stress responses. Upon starvation, autophagy is induced, providing cells with needed nutrient supplies. We report here that Unc-51-like kinase 1 (Ulk1), a key initiator for mammalian autophagy, undergoes dramatic dephosphorylation upon starvation, particularly at serine 638 and serine 758. Phosphorylations of Ulk1 are mediated by mammalian target-of-rapamycin ...[more]

PMID: 21383122

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Project description:As a member of the cancer-testis antigen family, sperm-associated antigen 6 (SPAG6) has been reported to be associated with the pathogenesis of myelodysplastic syndromes (MDS). Previous studies have demonstrated that SPAG6 is upregulated in bone marrow from patients with MDS and MDS-transformed acute myeloid leukemia and that knockdown of SPAG6 expression levels suppressed proliferation and promote apoptosis and differentiation in SKM-1 cells. However, the association between SPAG6 and autophagy in SKM-1 cells remains unclear. Hence, the aim of the present study was to investigate this association and its underlying mechanism. The present study used a short hairpin RNA (shRNA) lentivirus to silence SPAG6 expression levels in SKM-1 cells and demonstrated that SPAG6 knockdown increased autophagy and apoptosis. Furthermore, pharmacologically inhibiting autophagy with chloroquine and 3-methyladenine decreased SPAG6 knockdown-mediated apoptosis, indicating that SPAG6 knockdown-mediated autophagy promoted apoptosis in SKM-1 cells. Additionally, compared with the expression levels in negative control-shRNA lentivirus-transfected SKM-1 cells, the protein expression levels of phosphorylated AMP-activated protein kinase (p-AMPK) and phosphorylated unc-51-like autophagy activating kinase 1 (p-ULK1) were upregulated, while phosphorylated mammalian target of rapamycin (p-mTOR) protein expression was downregulated in SPAG6-shRNA lentivirus-transfected cells. Moreover, inhibiting AMPK expression levels with Compound C, a specific inhibitor of AMPK, attenuated SPAG6 knockdown-induced autophagy and apoptosis, suggesting that AMPK-mediated autophagy enhanced the pro-apoptotic effect of SPAG6 knockdown in SKM-1 cells. Taken together, the results of the present study demonstrated that SPAG6 silencing triggered autophagy via regulation of the AMPK/mTOR/ULK1 signaling pathway, which further contributed to the apoptosis of SKM-1 cells induced by SPAG6 knockdown. Thus, the current results indicate that SPAG6 may be a potential therapeutic target against MDS, and that autophagy may represent a potential mechanism for the treatment of MDS.

Project description:Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.AbbreviationsACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1.

Dataset Information

Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK.

Publications

Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK.

Similar Datasets

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