Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Protection of Pyruvate against Glutamate Excitotoxicity Is Mediated by Regulating DAPK1 Protein Complex

The neuroprotective activity of pyruvate has been confirmed in previous in vivo and in vitro studies. Here, we report a novel mechanism that pyruvate prevents SH-SY5Y cells from glutamate excitotoxicity by regulating death-associated protein kinase 1 (DAPK1) protein complex. Our results showed pyruvate regulated DAPK1 protein complex to protect cells by two ways. First, pyruvate induced the dissociation of DAPK1 with NMDA receptors. The disruption of the DAPK1-NMDA receptors complex resulted in a decrease in NMDA receptors phosphorylation. Then the glutamate-stimulated Ca²⁺ influx was inhibited and intracellular Ca²⁺ overload was alleviated, which blocked the release of cytochrome c and cell death. In addition, increased Bcl-xL induced by pyruvate regulated Bax/Bak dependent death by inhibiting the release of cytochrome c from the mitochondrial inter-membrane space into the cytosol. As a result, the cytochrome c-initiated caspase cascade, including caspase-3 and caspase-9, was inhibited. Second, pyruvate promoted the association between DAPK1 and Beclin-1, which resulted in autophagy activation. The autophagy inhibitor 3-methyladenine reversed the protection afforded by pyruvate. Furthermore, the attenuation of mitochondrial damage induced by pyruvate was partly reduced by 3-methyladenine. This suggested autophagy mediated pyruvate protection by preventing mitochondrial damage. Taken together, pyruvate protects cells from glutamate excitotoxicity by regulating DAPK1 complexes, both through dissociation of DAPK1 from NMDA receptors and association of DAPK1 with Beclin-1. They go forward to protect cells by attenuating Ca²⁺ overload and activating autophagy. Finally, a convergence of the two ways protects mitochondria from glutamate excitotoxicity, which leads to cell survival.     

DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn’t rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.     

DAPK3 inhibits gastric cancer progression via activation of ULK1-dependent autophagy

Dysregulation of the balance between cell proliferation and cell death is a central feature of malignances. Death-associated protein kinase 3 (DAPK3) regulates programmed cell death including apoptosis and autophagy. Our previous study showed that DAPK3 downregulation was detected in more than half of gastric cancers (GCs), which was related to tumor invasion, metastasis, and poor prognosis. However, the precise molecular mechanism underlying DAPK3-mediated tumor suppression remains unclear. Here, we showed that the tumor suppressive function of DAPK3 was dependent on autophagy process. Mass spectrometry, in vitro kinase assay, and immunoprecipitation revealed that DAPK3 increased ULK1 activity by direct ULK1 phosphorylation at Ser556. ULK1 phosphorylation by DAPK3 facilitates the ULK1 complex formation, the VPS34 complex activation, and autophagy induction upon starvation. The kinase activity of DAPK3 and ULK1 Ser556 phosphorylation were required for DAPK3-modulated tumor suppression. The coordinate expression of DAPK3 with ULK1 Ser556 phosphorylation was confirmed in clinical GC samples, and this co-expression was correlated with favorable survival outcomes in patients. Collectively, these findings indicate that the tumor-suppressor roles of DAPK3 in GC are associated with autophagy and that DAPK3 is a novel autophagy regulator, which can directly phosphorylate ULK1 and activate ULK1. Thus, DAPK3 might be a promising prognostic autophagy-associated marker.Subject terms: Tumour-suppressor proteins, Macroautophagy     

Melatonin attenuates MPTP-induced neurotoxicity via preventing CDK5-mediated autophagy and SNCA/α-synuclein aggregation

Autophagy is involved in the pathogenesis of neurodegenerative diseases including Parkinson disease (PD). However, little is known about the regulation of autophagy in neurodegenerative process. In this study, we characterized aberrant activation of autophagy induced by neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and demonstrated that melatonin has a protective effect on neurotoxicity. We found an excessive activation of autophagy in monkey brain tissues and C6 cells, induced by MPTP, which is mediated by CDK5 (cyclin-dependent kinase 5). MPTP treatment significantly reduced total dendritic length and dendritic complexity of cultured primary cortical neurons and melatonin could reverse this effect. Decreased TH (tyrosine hydroxylase)-positive cells and dendrites of dopaminergic neurons in the substantia nigra pars compacta (SNc) were observed in MPTP-treated monkeys and mice. Along with decreased TH protein level, we observed an upregulation of CDK5 and enhanced autophagic activity in the striatum of mice with MPTP injection. These changes could be salvaged by melatonin treatment or knockdown of CDK5. Importantly, melatonin or knockdown of CDK5 reduced MPTP-induced SNCA/α-synuclein aggregation in mice, which is widely thought to trigger the pathogenesis of PD. Finally, melatonin or knockdown of CDK5 counteracted the PD phenotype in mice induced by MPTP. Our findings uncover a potent role of CDK5-mediated autophagy in the pathogenesis of PD, and suggest that control of autophagic pathways may provide an important clue for exploring potential target for novel therapeutics of PD.     

Peptide combinatorial libraries identify TSC2 as a death-associated protein kinase (DAPK) death domain-binding protein and reveal a stimulatory role for DAPK in mTORC1 signaling

Death-associated protein kinase (DAPK) is a multidomain enzyme that plays a central role in autophagic and apoptotic signaling, although the protein-protein interactions regulating DAPK functions are not well defined. Peptide aptamer libraries were used to identify the tumor suppressor protein tuberin (TSC2) as a novel DAPK death domain-binding protein, and we evaluated whether DAPK is a positive or negative effector of the TSC2-regulated mammalian target of rapamycin (mTORC1) signaling pathway. Binding studies using death domain miniproteins in vitro and deletion analysis in vivo determined that the death domain of DAPK is the major site for the interaction with TSC2. Recombinant DAPK phosphorylates TSC2 in vitro, and DAPK kinase activity is stimulated by growth factor signaling. Transfection of DAPK promotes phosphorylation of TSC2 in vivo, whereas short interfering RNA-mediated attenuation of DAPK reduces growth factor-stimulated phosphorylation of TSC2. DAPK-dependent phosphorylation leads to TSC1-TSC2 complex dissociation, and consequently manipulation of DAPK by transfection or short interfering RNA demonstrated that DAPK is a positive regulator of mTORC1 in response to growth factor activation. Epistatic studies suggest that DAPK functions downstream from the RAS-MEK-ERK and phosphatidylinositol 3-kinase-AKT growth factor signaling pathways. DAPK(+/-) mouse embryo fibroblasts have attenuated mTORC1 signaling compared with DAPK+/+ counterparts, and overexpression of DAPK in DAPK(+/-) MEFs stimulates mTORC1 activity. These data uncover a novel interaction between DAPK and TSC2 proteins that has revealed a positive link between growth factor stimulation of DAPK and mTORC1 signaling that may ultimately affect autophagy, cell survival, or apoptosis.     

ITPRIP promotes glioma progression by linking MYL9 to DAPK1 inhibition

Epigenetic gene silencing of the tumor suppressor death-associated protein kinase 1 (DAPK1) is implicated in the progression of malignant gliomas. However, the mechanism underlying the repression of DAPK1 in gliomas remains elusive. In this study, we identified the existence of DAPK1-inositol 1,4,5-trisphosphate receptor (IP₃R)-interacting protein (ITPRIP) -myosin regulatory light polypeptide 9 (MYL9) complex in malignant glioma cells. Lentivirus co-infection and coimmunoprecipitation showed that ITPRIP bound with the death domain (DD) of DAPK1 in vitro. Further, dissociating ITPRIP-DAPK1 interaction inhibited glioma tumor growth in vitro but not in vivo. Moreover, knockdown of ITPRIP or DAPK1 impaired the ternary complex formation, whereas MYL9 knockdown did not affect ITPRIP-DAPK1 association. We further found that ITPRIP recruited MYL9 to the kinase domain (KD) of DAPK1, and in turn impeded the phosphorylation of MYL9. Accordingly, interference of ITPRIP enhanced the suppressive effects of DAPK1-KD on glioma progression both in vitro and in vivo. Our results demonstrate that ITPRIP plays a crucial role in the inhibition of DAPK1 and enhancement of tumorigenic properties of malignant glioma cells.     

死亡相关蛋白激酶（DAPK）是负调控还是正调控自噬?

自噬(autophagy)是1个严谨调控的代谢途径. 哺乳动物细胞通过自噬能够降解和循环利用大分子和某些细胞器.自噬作为一种适应的机制,保护有机体对抗各种病理病变,包括感染、癌症、退行性病变、衰老和心脏疾病等.在移除蛋白聚集体、以及受损或过剩细胞器时,自噬发挥着很关键的作用,从而能够维持细胞能量平衡并适应环境压力.当自噬不足或过剩时,自噬也可作为一种促死亡的机制.在不同的情况下,自噬活化的程度通过自噬信号通路网络而精确地调节.死亡相关蛋白激酶(DAPK)是1个刺激调节蛋白激酶,它由几个功能结构域组成.这些结构域能让它参与广泛的信号通路中,包括凋亡、自噬和膜泡.DAPK在调节自噬中发挥着关键的作用.本文综述了在不同的情况下DAPK负调控或是正调控自噬的路径.     

Sestrin2 Protects Dopaminergic Cells against Rotenone Toxicity through AMPK-Dependent Autophagy Activation

Dysfunction of the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) was thought to be an important pathogenic mechanism in synuclein pathology and Parkinson''s disease (PD). In the present study, we investigated the role of sestrin2 in autophagic degradation of α-synuclein and preservation of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were increased in a TP53-dependent manner in Mes 23.5 cells after treatment with rotenone. Genetic knockdown of sestrin2 compromised the autophagy induction in response to rotenone, while overexpression of sestrin2 increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent fashion, as sestrin2 overexpression activated AMPK, and genetic knockdown of AMPK abrogated autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown recovered the autophagy activity. Sestrin2 overexpression ameliorated α-synuclein accumulation, inhibited caspase 3 activation, and reduced the cytotoxicity of rotenone. These results suggest that sestrin2 upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation of AMPK, and that sestrin2 exerts a protective effect on dopaminergic cells.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

The emerging interrelation between ROCO and related kinases,intracellular Ca2+ signaling,and autophagy

ROCO kinases form a family of proteins characterized by kinase activity in addition to the presence of the so-called ROC (Ras of complex proteins)/COR (C-terminal of ROC) domains having a role in their GTPase activity. These are the death-associated protein kinase (DAPK) 1 and the leucine-rich repeat kinases (LRRK) 1 and 2. These kinases all play roles in cellular life and death decisions and in autophagy in particular. Related to the ROCO kinases is DAPK 2 that however cannot be classified as a ROCO protein due to the absence of the ROC/COR domains. This review aims to bring together what is known about the relation between these proteins and intracellular Ca²⁺ signals in the induction and regulation of autophagy. Interestingly, DAPK 1 and 2 and LRRK2 are all linked to Ca²⁺ signaling in their effects on autophagy, though in various ways. Present evidence supports an upstream role for LRRK2 that via lysosomal and endoplasmic reticulum Ca²⁺ release can trigger autophagy induction. In contrast herewith, DAPK1 and 2 react on existing Ca²⁺ signals to stimulate the autophagic pathway. Further research will be needed for obtaining a full understanding of the role of these various kinases in autophagy and to assess their exact relation with intracellular Ca²⁺ signaling as this would be helpful in the development of novel therapeutic strategies against neurodegenerative disorders, cancer and auto-immune diseases.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

DAPK2 is a novel regulator of mTORC1 activity and autophagy

Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca²⁺-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction. DAPK2 knockdown reduced autophagy triggered either by amino acid deprivation or by increases in intracellular Ca²⁺ levels. At the molecular level, DAPK2 depletion interfered with mTORC1 inhibition caused by these two stresses, as reflected by the phosphorylation status of mTORC1 substrates, ULK1 (unc-51-like kinase 1), p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1. An increase in mTORC1 kinase activity was also apparent in unstressed cells that were depleted of DAPK2. Immunoprecipitated mTORC1 from DAPK2-depleted cells showed increased kinase activity in vitro, an indication that DAPK2 regulation of mTORC1 is inherent to the complex itself. Indeed, we found that DAPK2 associates with components of mTORC1, as demonstrated by co-immunoprecipitation with mTOR and its complex partners, raptor (regulatory-associated protein of mTOR) and ULK1. DAPK2 was also able to interact directly with raptor, as shown by recombinant protein-binding assay. Finally, DAPK2 was shown to phosphorylate raptor in vitro. This phosphorylation was mapped to Ser721, a site located within a highly phosphorylated region of raptor that has previously been shown to regulate mTORC1 activity. Thus, DAPK2 is a novel kinase of mTORC1 and is a potential new member of this multiprotein complex, modulating mTORC1 activity and autophagy levels under stress and steady-state conditions.Macroautophagy (hereafter referred to as autophagy) is a highly regulated intracellular bulk degradation process found ubiquitously in eukaryotes. During autophagy a double-membrane vesicle, termed an autophagosome, engulfs cytoplasmic materials, including whole organelles. The autophagosome is later fused with the lysosome and its content degraded by hydrolases.¹ Basal levels of autophagy are maintained within the cell during steady state, and are involved in cell homeostasis activities such as turnover of long-lived proteins, preventing accumulation of protein aggregates, and removal of damaged cellular structures.² Beyond this homeostatic function, autophagy is stimulated during various stress conditions, such as nutrient deprivation, intracellular Ca²⁺ increase, hypoxia, ER stress and oxidative stress, to ensure continuous cell survival under stress.³A critical step in the induction of autophagy comprises the inactivation of a key negative regulator of the process, the mammalian target of rapamycin (mTOR).⁴ mTOR is a conserved serine/threonine protein kinase that acts as a master regulator in the cell. mTOR forms a rapamycin-sensitive complex named mTORC1 with its binding partner raptor (regulatory-associated protein of mTOR), which mediates mTOR''s substrate presentation.⁵ mTORC1 senses nutrient availability, growth factors and energy levels, and, in response, regulates cell growth, metabolism and protein synthesis, mainly by phosphorylation of substrates involved in protein translation: the p70 ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Under nutrient-rich conditions, mTORC1 suppresses autophagy to basal levels by phosphorylating and inhibiting the autophagy proteins ULK1 (unc-51-like kinase 1) and Atg13. Upon autophagic stimulus, mTORC1 activity is inhibited and the ULK1 complex is activated, leading to autophagy induction.⁶ The activity levels of mTORC1 are regulated by several mechanisms, such as interacting proteins, cellular localization and phosphorylation events. Raptor phosphorylation has been suggested as a mechanism by which upstream kinases such as AMPK,⁷ RSK⁸ and ULK1⁹ can regulate mTORC1 activity.Death-associated protein kinase 2 (DAPK2; also named DRP-1) is a 42-kDa Ca²⁺/calmodulin (CaM)-regulated serine/threonine kinase,¹⁰ and a closely related homolog of DAPK, a gene originally discovered in an attempt to find positive regulators of cell death.¹¹ DAPK2 was identified based on homology to the catalytic domain of DAPK. DAPK2 is a soluble cytoplasmatic protein, which triggers massive membrane blebbing and appearance of double-membrane autophagic vesicles upon its overexpression (for a review see Shiloh et al.¹²). DAPK2''s substrates and interacting proteins are mostly unknown, with the exception of the myosin II regulatory light chain, which has been shown to be an in vitro and in vivo substrate.¹³ Although many publications have studied DAPK, its substrates and its role in cell death and autophagy,^{14, 15} very little is known about DAPK2 substrates, cellular functions or the molecular pathways that it regulates.In this work, we studied the involvement of DAPK2 in the autophagic module. We identified DAPK2 as a novel interacting protein of mTORC1, and as a negative regulator of the complex both during steady-state growth conditions and in response to different stress autophagic signals. We identified mTOR''s binding partner, raptor, as a substrate of DAPK2, and found Ser721 as its phosphorylation site.     

Death-associated protein kinase 2: Regulator of apoptosis,autophagy and inflammation

Death-associated protein kinase 2 (DAPK2/DRP-1) belongs to a family of five related serine/threonine kinases that mediate a range of cellular processes, including membrane blebbing, apoptosis, and autophagy, and possess tumour suppressive functions. The three most conserved family members DAPK1/DAPK, DAPK2 and DAPK3/ZIPK share a high degree of homology in their catalytic domain, but differ significantly in their extra-catalytic structures and tissue-expression profiles. Hence, each orthologue binds to various unique interaction partners, localizes to different subcellular regions and controls some dissimilar cellular functions. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms that activate DAPK2 and that execute DAPK2-mediated apoptosis, autophagy and inflammation. In this “molecules in focus” review on DAPK2, the structure, modes of regulation and various cellular functions of DAPK2 will be summarized and discussed.     

The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.     

PSPH induces cell autophagy and promotes cell proliferation and invasion in the hepatocellular carcinoma cell line Huh7 via the AMPK/mTOR/ULK1 signaling pathway

Phosphoserine phosphatase (PSPH), a key enzyme of the l -serine synthesis pathway, has been involved in cancer progression and survival. However, limited evidence revealed the PSPH influence on hepatocellular carcinoma (HCC). Herein, we observed that PSPH expression was upregulated in both HCC tissues and cell lines, which was determined by western blotting. TCGA database showed that the PSPH protein levels were significantly upregulated and affected patient survival rates in HCC. Then gain- and loss-of-function manipulations were performed by transfection with a pcDNA-PSPH expression vector or a specific short interfering RNA against PSPH in Huh7 cells. Huh7 cell proliferation, stemness, invasion, and apoptosis were assessed by using CCK-8 test, colony formation assay, Transwell assay, and Flow cytometry analysis, respectively, and levels of autophagy-related proteins were detected by using western blotting. The results showed that PSPH could induce Huh7 cell autophagy, promote cell proliferation and invasion, and inhibit apoptosis. The knockdown of PSPH could inhibit Huh7 cell proliferation, invasion, and autophagy. Furthermore, PSPH activated Liver kinase B1 (LKB1) and TGF beta-activated kinase 1 (TAK1), affected the adenosine 5′-monophosphate-activated protein kinase (AMPK)/mTOR/ULK1 signaling pathway, but could not activate calcium/calmodulin-dependent protein kinase kinase (CaMKK) in Huh7 cells. Inhibition of either LKB1, TAK1, or AMPK could eliminate the effect of PSPH overexpression on Huh7 cell behaviors. However, inhibition of CaMKK could not influence the effect of PSPH overexpression on Huh7 cell behaviors. In conclusion, PSPH could induce autophagy, promote proliferation and invasion, and inhibit apoptosis in HCC cells via the AMPK/mTOR/ULK1 signaling pathway.     

Astrocyte elevated gene 1 (AEG-1) promotes anoikis resistance and metastasis by inducing autophagy in hepatocellular carcinoma

Astrocyte elevated gene 1 (AEG-1) is overexpressed in hepatocellular carcinoma (HCC) and is strongly associated with tumor metastasis. Anoikis resistance and autophagy may play an important role in the survival of circulating tumor cells. However, the relationship among AEG-1, anoikis resistance, autophagy, and metastasis in HCC is still not clear. The results of this study indicate that AEG-1 expression is increased in HCC cell lines grown in suspension culture. AEG-1 could enhance anoikis resistance to promote the survival of detached HCC cells. Moreover, the anoikis resistance appears to be partly dependent on autophagy. Regulating AEG-1 expression changed the autophagy levels to modulate anoikis resistance, likely acting via the protein kinase RNA-like ER kinase (PERK)-eIF2α-ATF4-CHOP signaling axis. Finally, inhibiting autophagy by RNA interference prevented the AEG-1-promoted metastasis of HCC xenografts to the liver and lungs of nude mice. Taken together, AEG-1 is a key contributor to anoikis resistance and metastasis by inducing autophagy in vitro and in vivo, and it may be a potential target for therapeutic intervention in HCC.     

Novel insights into DAPK autophagic signalling using peptide aptamer combinatorial protein-interaction screens

DAPK represents a relatively unique enzyme in the protein kinase superfamily whose major biological functions are linked to both autophagy and signal-mediated apoptosis. However, genetic studies have not yet uncovered how DAPK integrates into the core autophagy-related (Atg) machinery since DAPK is not present in a genetically tractable eukaryotic cell such as yeast. Furthermore, there have been no definitive DAPK binding proteins identified in metazoan systems that play a direct role in cooperating with DAPK in autophagy. We have utilized a growing concept in systems biology that invokes linear peptide-motifs as a fundamental mechanism driving protein-protein interactions and as a key switch underlying the dynamics of a signal transduction pathway. By using peptide combinatorial libraries as an assay that reflects the diversity of the linear peptide motif repertoire in the mammalian proteome, we identified microtubule-associated protein 1B (MAP1B) as a novel DAPK interacting protein that stimulates DAPK-dependent membrane blebbing and autophagy. MAP1B has previously been shown to form a functional interaction with the autophagosomal protein Atg8 (LC3). Together these studies define a genetic interaction between DAPK-MAP1B in the regulation of autophagy that may have particular relevance to cellular signalling pathways that regulate cell survival or cell death under distinct environmental stresses.