Identification of an 11-residue portion of CTP–phosphocholine cytidylyltransferase that is required for enzyme–membrane interactions

Yang, Jilin; Wang, Jinxia; Tseu, Irene; Kuliszewski, Michael A.; Lee, Wen-Su; Post, Martin

doi:10.1042/bj3250029

Cited by 22 publications

(18 citation statements)

References 33 publications

(50 reference statements)

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“…Adding to this complexity, two more isoforms of CT (CT␤1 and -␤2) without the N-terminal nuclear targeting signal were found in animal tissues, including liver, and were present in the cytoplasm (11). It is not known how the participation of CT␣ in the CDP-choline pathway is regulated, because the amino acid sequences responsible for several previously suspected mechanisms of CT␣ activation can be deleted without affecting its involvement in the CDP-choline pathway (12)(13)(14). In a recent study by Cornell and co-workers (15), a novel mechanism for CT␣ activation was proposed.…”

Section: Ctp:phosphocholine Cytidylyltransferase (Ct)mentioning

confidence: 99%

Nuclear Localization of Enzymatically Active Green Fluorescent Protein-CTP:Phosphocholine Cytidylyltransferase α Fusion Protein Is Independent of Cell Cycle Conditions and Cell Types

DeLong

Qin

Cui

2000

Journal of Biological Chemistry

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To address the recent controversy about the subcellular localization of CTP:phosphocholine cytidylyltransferase ␣ (CT␣), this study was designed to visualize green fluorescent protein (GFP)⅐CT␣ fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP⅐CT␣ fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP⅐CT␣ fusion protein was localized to the nucleus in all cell lines and required the N-terminal nuclear targeting sequence. Serum depletion/replenishment did not cause shuttling of CT␣ between the nucleus and cytoplasm. Moreover, the subcellular localization of CT␣ was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CT␣ between the nucleus and cytoplasm was observed at any stage of the cell cycle. Stimulation of cells with oleate had no effect on the localization of CT␣. The GFP⅐CT␣ lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP⅐CT␣ fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CT␣ is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis. CTP:phosphocholine cytidylyltransferase (CT)1 is the regulatory enzyme of the CDP-choline pathway for de novo synthesis of PC (1). CT catalyzes the second reaction between choline kinase and cholinephosphotransferase. The CDP-choline pathway is present in all mammalian cells and is essential for cell survival (2). Complete inactivation of CT by a temperaturesensitive mutation leads to apoptosis (3). CT␣ was first purified by Weinhold and co-workers in 1986 (4), and its cDNA was first cloned from rat liver by Cornell and co-workers in 1990 (5). PC synthesis is nearly normal even when over 90% of CT is inactivated (2), suggesting that most cellular CT remains inactive for PC synthesis. However, nearly all cellular CT␣ has been found localized in the nucleus (6) although the major site of PC synthesis was found associated with the endoplasmic reticulum (7-9). Because of this physical separation of CT␣ localization and PC synthesis, the precise localization of CT␣ and universality of CT␣ localization in the nucleus have become interesting topics for investigation.Since the nuclear localization of CT␣ was first reported by Kent and co-workers (6), the subcellular localization of CT␣ has become increasingly controversial. A study by Houweling et. al (10) demonstrated that significant staining of CT was detected in both the cytoplasm and nucleus of primary rat hepatocytes and of rat liver thin slices. Adding to this complexity, two more isoforms of CT (CT␤1 and -␤2) without the N-terminal nuclear targeting signal were found in animal tissues, including liver, and were present in the cytoplasm (11). It is not known how the participation of CT␣ in the CDP-choline pat...

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Section: Ctp:phosphocholine Cytidylyltransferase (Ct)mentioning

confidence: 99%

Nuclear Localization of Enzymatically Active Green Fluorescent Protein-CTP:Phosphocholine Cytidylyltransferase α Fusion Protein Is Independent of Cell Cycle Conditions and Cell Types

DeLong

Qin

Cui

2000

Journal of Biological Chemistry

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“…Tissue was prepared for EM by either routine fixation for basic histology or freeze substitution for immunogold examination. A rabbit polyclonal antibody against the NH 2-terminal domain of CCT-␣ was raised as previously described (31,51). Rabbit polyclonal FAS antibody was a generous gift from S. Rooney (Yale University, New Haven, CT).…”

Section: Methodsmentioning

confidence: 99%

Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells

Ridsdale

Post

2004

American Journal of Physiology-Lung Cellular and Molecular Phys

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Pulmonary surfactant is a lipoprotein complex that functions to reduce surface tension at the air liquid interface in the alveolus of the mature lung. In late gestation glycogen-laden type II cells shift their metabolic program toward the synthesis of surfactant, of which phosphatidylcholine (PC) is by far the most abundant lipid. To investigate the cellular site of surfactant PC synthesis in these cells we determined the subcellular localization of two key enzymes for PC biosynthesis, fatty acid synthase (FAS) and CTP:phosphocholine cytidylyltransferase-␣ (CCT-␣), and compared their localization with that of surfactant storage organelles, the lamellar bodies (LBs), and surfactant proteins (SPs) in fetal mouse lung. Ultrastructural analysis showed that immature and mature LBs were present within the glycogen pools of fetal type II cells. Multivesicular bodies were noted only in the cytoplasm. Immunogold electron microscopy (EM) revealed that the glycogen pools were the prominent cellular sites for FAS and CCT-␣. Energy-filtering EM demonstrated that CCT-␣ bound to phosphorus-rich (phospholipid) structures in the glycogen. SP-B and SP-C, but not SP-A, localized predominantly to the glycogen stores. Collectively, these data suggest that the glycogen stores in fetal type II cells are a cellular site for surfactant PC synthesis and LB formation/maturation consistent with the idea that the glycogen is a unique substrate for surfactant lipids.CTP:phosphocholine cytidylyltransferase; pulmonary surfactant; immunogold; fatty acid synthase SHORTLY BEFORE BIRTH, alveolar type II cells must increase their surfactant lipid and protein synthesis to generate sufficient pulmonary surfactant. This material reduces the surface tension at the air/liquid interface and ample amounts are required to prepare the lung for extrauterine life (30). Surfactant production appears to be a carefully timed event because a preterm infant can experience respiratory distress and atelectasis as a direct result of pulmonary surfactant insufficiency.Pulmonary surfactant is composed of a mixture of lipids and proteins. The most abundant lipid species is phosphatidylcholine (PC) with dipalmitoyl-PC being the surface-active reagent (17). Whereas alveolar type II cells in the postpartum lung need to synthesize lipids and proteins for pulmonary surfactant maintenance, the fetal type II cells need to generate enough material to establish a functioning air-liquid barrier immediately at birth (10, 29). It is evident that the surge of production of surfactant at late fetal gestation is a daunting metabolic task for the type II cells. Morphologically, large amounts of glycogen accumulate in the distal undifferentiated epithelium before surfactant synthesis, and these glycogen stores are depleted as surfactant is synthesized (6, 32). Biochemically, temporal relationships between glycogen and phospholipids (5, 7) have suggested that glycogen is used as a carbon source to generate the glycerol backbone and acyl chains of the surfactant lipids, but a direct precu...

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“…The HIF-1a template was used as template to generate the HIF-1aDODD mutant. The deletion mutant (HIF1a D401-603 ) was constructed by overlap extension using PCR as previously described (36). All constructs were verified by sequencing.…”

Section: Expression Vectormentioning

confidence: 99%

Mouse Snail Is a Target Gene for HIF

Luo

Wang

et al. 2011

Molecular Cancer Research

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102

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The transcriptional inhibitor Snail is a critical regulator for epithelial-mesenchymal transition (EMT). Although low oxygen induces Snail transcription, thereby stimulating EMT, a direct role of hypoxia-inducible factor (HIF) in this process remains to be demonstrated. Here we show that hypoxia induces the expression of Snail via HIF. In silico analysis identified a potential hypoxia-response element (HRE) close to the minimal promoter of the human and mouse genome of the snail gene. Gel shift assays demonstrated that a specific hypoxiainducible complex is formed with the putative HRE and that the complex contains HIF proteins. ChIP assays confirmed the interaction of HIF proteins with the putative HRE in vivo. Reporter gene analyses showed that the putative HRE responds to hypoxia in its natural position as well as in front of a heterologous promoter and that the HRE is directly activated by HIF-1a or HIF-2a. HIF knockdown with siRNA at 2% oxygen and overexpression of an oxygen-insensitive HIF (HIF-DODD) mutant at 21% oxygen showed that HIF regulates Snail activation and subsequent cell migration. Our findings identify snail as a HIF target gene and provide novel insights into the regulation of snail and hypoxia-induced EMT. Mol Cancer Res; 9(2); 234-45. Ó2011 AACR.

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Identification of an 11-residue portion of CTP–phosphocholine cytidylyltransferase that is required for enzyme–membrane interactions

Cited by 22 publications

References 33 publications

Nuclear Localization of Enzymatically Active Green Fluorescent Protein-CTP:Phosphocholine Cytidylyltransferase α Fusion Protein Is Independent of Cell Cycle Conditions and Cell Types

Nuclear Localization of Enzymatically Active Green Fluorescent Protein-CTP:Phosphocholine Cytidylyltransferase α Fusion Protein Is Independent of Cell Cycle Conditions and Cell Types

Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells

Mouse Snail Is a Target Gene for HIF

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