Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process.     

Phosphorylation by Aurora-B Negatively Regulates Survivin Function During Mitosis

Survivin operates in a complex with aurora B kinase and is phosphorylated by it on threonine 117 in vitro. Here we ask whether phosphorylation of survivin by aurora B kinase regulates its function during mitosis in vivo. Using a phospho-specific antibody we first establish that survivin is phosphorylated at T117 during mitosis and is present at the midbody during cytokinesis. Next we use two independent RNAi complementation approaches to investigate threonine 117 mutants in survivin depleted cells. Our data suggest that while non-phosphorylatable survivin, survivinT117A, can substitute for the wild type protein, a phosphomimic, survivinT117E cannot restore viability, nor can it complement chromosome congression and spindle checkpoint defects that arise due to depletion of endogenous survivin. Fluorescence imaging and fluorescence recovery after photobleaching analysis suggest that the phosphomimic has reduced affinity for centromeres compared with the non-phosphorylatable form. We conclude that survivin is phosphorylated at T117 during mitosis, and once phosphorylated, dephosphorylation is crucial for chromosome congression and progression into anaphase.     

SIRT1 Regulates Dendritic Development in Hippocampal Neurons

Dendritic arborization is required for proper neuronal connectivity. SIRT1, a NAD+ dependent histone deacetylase, has been associated to ageing and longevity, which in neurons is linked to neuronal differentiation and neuroprotection. In the present study, the role of SIRT1 in dendritic development was evaluated in cultured hippocampal neurons which were transfected at 3 days in vitro with a construct coding for SIRT1 or for the dominant negative SIRT1H363Y, which lacks the catalytic activity. Neurons overexpressing SIRT1 showed an increased dendritic arborization, while neurons overexpressing SIRT1H363Y showed a reduction in dendritic arbor complexity. The effect of SIRT1 was mimicked by treatment with resveratrol, a well known activator of SIRT1, which has no effect in neurons overexpressing SIRT1H363Y indicating that the effect of resveratrol was specifically mediated by SIRT1. Moreover, hippocampal neurons overexpressing SIRT1 were resistant to dendritic dystrophy induced by Aβ aggregates, an effect that was dependent on the deacetylase activity of SIRT1. Our findings indicate that SIRT1 plays a role in the development and maintenance of dendritic branching in hippocampal neurons, and suggest that these effects are mediated by the ROCK signaling pathway.     

SIRT1 Negatively Regulates the Mammalian Target of Rapamycin

The IGF/mTOR pathway, which is modulated by nutrients, growth factors, energy status and cellular stress regulates aging in various organisms. SIRT1 is a NAD+ dependent deacetylase that is known to regulate caloric restriction mediated longevity in model organisms, and has also been linked to the insulin/IGF signaling pathway. Here we investigated the potential regulation of mTOR signaling by SIRT1 in response to nutrients and cellular stress. We demonstrate that SIRT1 deficiency results in elevated mTOR signaling, which is not abolished by stress conditions. The SIRT1 activator resveratrol reduces, whereas SIRT1 inhibitor nicotinamide enhances mTOR activity in a SIRT1 dependent manner. Furthermore, we demonstrate that SIRT1 interacts with TSC2, a component of the mTOR inhibitory-complex upstream to mTORC1, and regulates mTOR signaling in a TSC2 dependent manner. These results demonstrate that SIRT1 negatively regulates mTOR signaling potentially through the TSC1/2 complex.     

Function of SIRT1 in physiology

Sirtuins were originally defined as a family of oxidized nicotinamide adenine nucleotide (NAD⁺)-dependent enzymes that deacetylate lysine residues on various proteins. The sirtuins are remarkably conserved throughout evolution from archae to eukaryotes. They were named after their homology to the Saccharomyces cerevisiae gene silent information regulator 2 (Sir2). The mammalian sirtuins, SIRT1-7, are implicated in a variety of cellular functions ranging from gene silencing, control of the cell cycle and apoptosis, and energy homeostasis. As SIRT1 is a nuclear protein and is the mammalian homolog most highly related to Sir2, it has been the focus of a large number of recent studies. Here we review some of the current data related to SIRT1 and discuss its mode of action and biological role in cellular and organismal models. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 869–876.     

Multisite Phosphorylation Regulates GpsB Function in Cephalosporin Resistance of Enterococcus faecalis

Enterococci are normal human commensals and major causes of hospital-acquired infections. Enterococcal infections can be difficult to treat because enterococci harbor intrinsic and acquired antibiotic resistance, such as resistance to cephalosporins. In Enterococcus faecalis, the transmembrane kinase IreK, a member of the bacterial PASTA kinase family, is essential for cephalosporin resistance. The activity of IreK is boosted by the cytoplasmic protein GpsB, which promotes IreK autophosphorylation and signaling to drive cephalosporin resistance. A previous phosphoproteomics study identified eight putative IreK-dependent phosphorylation sites on GpsB, but the functional importance of GpsB phosphorylation was unknown. Here we used genetic and biochemical approaches to define three sites of phosphorylation on GpsB that functionally impact IreK activity and cephalosporin resistance. Phosphorylation at two sites (S80 and T84) serves to impair the ability of GpsB to activate IreK in vivo, suggesting phosphorylation of these sites acts as a means of negative feedback for IreK. The third site of phosphorylation (T133) occurs in a segment of GpsB termed the C-terminal extension that is unique to enterococcal GpsB homologs. The C-terminal extension is highly mobile in solution, suggesting it is largely unstructured, and phosphorylation of T133 appears to enable efficient phosphorylation at S80 / T84. Overall our results are consistent with a model in which multisite phosphorylation of GpsB impairs its ability to activate IreK, thereby diminishing signal transduction through the IreK-dependent pathway and modulating phenotypic cephalosporin resistance.     

Phosphorylation of HuR by Chk2 regulates SIRT1 expression

The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.     

SIRT1 Regulates the Chemoresistance and Invasiveness of Ovarian Carcinoma Cells

BACKGROUND: SIRT1 is a longevity gene that forestalls aging and age-related diseases including cancer, and has recently attracted widespread attention due to its overexpression in some cancers. We previously identified the overexpression of SIRT1 in ovarian carcinoma (OvCa) as a poor prognostic factor. However, mechanistic insights into the function of SIRT1 in OvCa have yet to be elucidated. METHODS: Quantitative real-time reverse PCR (qRT-PCR) and Western blotting were employed to examine the expression of SIRT1 in a panel of human OvCa cell lines. si-RNA or sh-RNA and cDNA technologies were utilized to knockdown or overexpress SIRT1, respectively. The effects of SIRT1 on proliferation and chemoresistance were examined using a WST-1 assay, and the underlying mechanisms were confirmed using an apoptotic assay, and the quantification of glutathione (GSH), and reactive oxygen species (ROS). The aggressiveness of SIRT1 was analyzed using in vitro invasion and migration assays. RESULTS: SIRT1 was more strongly expressed in OvCa cell lines than in the immortalized ovarian epithelium at the gene and protein levels. Stress up-regulated the expression of SIRT1 in dose- and time-dependent manners. SIRT1 significantly enhanced the proliferation (P < .05), chemoresistance (P < .05), and aggressiveness of OvCa cells by up-regulating multiple antioxidant pathways to inhibit oxidative stress. Further study into the overexpression of SIRT1 demonstrated the up-regulation of several stemness-associated genes and enrichment of CD44v9 via an as-yet-unidentified pathway. CONCLUSIONS: Our results suggest that SIRT1 plays a role in the acquisition of aggressiveness and chemoresistance by OvCa, and has potential as a therapeutic target for OvCa.     

Plk1-mediated Phosphorylation of Topors Regulates p53 Stability

Polo-like kinase 1 (Plk1) overexpression is associated with tumorigenesis by an unknown mechanism. Likewise, Plk1 was suggested to act as a negative regulator of tumor suppressor p53, but the mechanism remains to be determined. Herein, we have identified topoisomerase I-binding protein (Topors), a p53-binding protein, as a Plk1 target. We show that Plk1 phosphorylates Topors on Ser⁷¹⁸ in vivo. Significantly, expression of a Plk1-unphosphorylatable Topors mutant (S718A) leads to a dramatic accumulation of p53 through inhibition of p53 degradation. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase. Plk1-mediated phosphorylation of Topors inhibits Topors-mediated sumoylation of p53, whereas p53 ubiquitination is enhanced, leading to p53 degradation. These results demonstrate that Plk1 modulates Topors activity in suppressing p53 function and identify a likely mechanism for the tumorigenic potential of Plk1.Polo-like kinase-1 (Plk1)³ has multiple functions required for cell cycle progression, and overexpression of Plk1 is observed in various types of human tumors (1, 2). Thus, Plk1 has been proposed as a novel diagnostic marker for cancers. Accumulating evidence suggests that Plk1 negatively regulates the function of the tumor suppressor p53, whose loss-of-function mutations have been observed in nearly 50% of human tumors (1). In our earlier studies, we were the first to demonstrate that Plk1 depletion results in increased p53 level in HeLa cells (3) and that human cells with different levels of p53 respond to Plk1 depletion differently (4). Subsequently, it was shown that Plk1 directly binds to the DNA-binding domain of p53 through its N-terminal kinase domain and inhibits the transactivation as well as the proapoptotic function of p53 (5). Although it has been suggested that Plk1 might regulate p53 through direct phosphorylation (5), our repeated efforts to prove p53 as a direct target of Plk1 have been unsuccessful.Topors was discovered in a screen searching for proteins that bind to DNA topoisomerase I (6) and was also identified as a p53-binding protein (7). Although Topors is widely expressed in normal human tissues, its expression is decreased or undetectable in colon, lung, and brain adenocarcinomas, indicating that it might function as a tumor suppressor (8). Topors contains an N-terminal C3HC4-type RING domain that is closely related in sequence to the RING domains of known E3 ligases (see Fig. 1A) and is the first example of a protein that has both ubiquitin and SUMO-1 E3 ligase activity. Topors functions as an E3 ubiquitin ligase for p53 and NKX3.1, and Topors-mediated ubiquitination leads to the degradation of these proteins (9, 10). Substrates of the SUMO-1 E3 ligase activity of Topors include DNA topoisomerase I and p53 (11, 12). In contrast to ubiquitination-induced protein degradation, Topors-induced p53 sumoylation is accompanied by an increase in the level of p53 protein (11). Taken together, these studies indicate that Topors functions both as an ubiquitin and as a SUMO-1 E3 ligase for p53. Therefore, it is likely that the effects of Topors on p53 depend on cellular context (10).Open in a separate windowFIGURE 1.Plk1 phosphorylates Topors at Ser⁷¹⁸in vitro and in vivo. A, schematic representation of the domain structure of Topors. Two separate regions encoding putative p53-binding domains are aa 456–731 and 854–916. Amino acid residues in the putative Ring finger motif are shown in a black box. PEST, sequences rich in Pro, Glu, Ser, and Thr; RS domain, Arg- and Ser-rich domain; NLS, nuclear localization sequence; NB, nuclear bodies. B, purified Plk1 was incubated with purified GST-Topors (aa 1–510) or GST-Topors (aa 511–1045) for 30 min at 30 °C in the presence of [γ-³²P]ATP (³²P). Reaction mixtures were resolved by SDS-PAGE followed by autoradiography. Coom., Coomassie Blue. C and D, Plk1 phosphorylates Topors (aa 679–760). Purified Plk1 was incubated with purified GST-Topors fragments (aa 1–250, 251–510, 511–760, 756–1045, 511–596, 597–678, and 679–760). Kinase assays were performed as described in B. E, Ser⁷¹⁸ of Topors is a Plk1 phosphorylation site in vitro. Purified Plk1 was incubated with the indicated serine to alanine Topors (aa 679–760) mutants and analyzed as in B. F, Topors is phosphorylated in vivo at Ser⁷¹⁸ by Plk1. HEK293T cells were transfected with WT-Topors-Myc (lanes 1 and 3) or S718A-Topors-Myc (lane 2) and depleted of Plk1 by using double-stranded RNA targeting Plk1 (lane 3). After overnight incubation, cells were treated with nocodazole for 10 h and metabolically labeled with [³²P]orthophosphate. Phosphoproteins were immunoprecipitated with anti-Myc antibodies, resolved by SDS-PAGE, and subjected to autoradiography. Relative ³²P (Rel. ³²P) incorporations of Topors are indicated on the bottom.In this study, we provide evidence that Plk1 phosphorylates Topors on Ser⁷¹⁸. Significantly, we demonstrate that the Plk1-mediated phosphorylation of Topors results in reduced sumoylation of p53, whereas the ubiquitination activity toward p53 is increased, thereby facilitating p53 degradation.     

Resveratrol Boosts Cognitive Function by Targeting SIRT1

Cognitive decline is among the most devastating age-related conditions and is rapidly becoming an important cause of disease burdens worldwide. New strategies for the prevention and management of cognitive decline are needed. Resveratrol, a polyphenolic compound, has been found to enhance brain health through multiple signaling pathways. Optimal SIRT1 activation is the most crucial step in the neuroprotection provided by resveratrol against cognitive impairment. This review discusses several recent developments in our understanding of the mechanisms by which resveratrol delay age-related cognitive decline through SIRT1. The regulatory mechanisms include anti-oxidative, anti-inflammatory, anti-apoptotic processes and autophagy regulation, as well as increases in cerebral blood flow and improvements in the plasticity of synaptic pathways. Resveratrol, as well as novel SIRT1 activators, is likely to provide promising therapeutic strategies for impeding cognitive decline, repairing brain functions, and supporting healthy aging.     

PICK1蛋白C端酸性区域对其功能的调节

蛋白激酶C相互作用蛋白1(protein interacting with Ckinase1,PICK1)是调节AMPA(alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)受体在细胞膜上的数量与分布,引起LTP与LTD现象的重要蛋白.本文利用基因克隆、荧光光谱以及免疫分析等方法,分析了PICK1蛋白C末端酸性区对BAR结构域与膜脂结合能力以及PICK1分子内BAR(Bin/amphiphysin/RVS)结构域与PDZ结构域相互作用的影响,研究了钙离子结合C末端酸性区后对上述相互作用的调节.结果显示,C末端酸性区的存在使BAR结构域与膜脂的结合能力减弱大约10倍,但PICK1分子内的BAR与PDZ结构域的相互作用与不含C末端的酸性区相比增强了大约4倍.另一方面,C末端酸性区的存在,伴随钙离子浓度的提高,有助于增强BAR与膜脂的结合,却削弱了PDZ和BAR结构域的作用.当钙离子浓度增加到500μmol/L时,BARC的脂质结合能力以及和PDZ的亲和力与不含酸性区相当.