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High-mobility group (HMG) A1a and A1b proteins are among a family of HMGA proteins that bind to the minor groove of AT-rich regions of DNA. Here we employed tandem mass spectrometry and determined without ambiguity the sites of phosphorylation and the nature of methylation of HMGA1 proteins that were isolated from the PC-3 human prostate cancer cells. We showed by LC-MS/MS that Ser101 and Ser102 were completely phosphorylated in HMGA1a protein, whereas only a portion of the protein was phosphorylated at Ser98. We also found that the HMGA1b protein was phosphorylated at the corresponding sites, that is, Ser90, Ser91 and Ser87. In addition, Arg25, which is within the first DNA-binding AT-hook domain of HMGA1a, was both mono- and dimethylated. Moreover, both symmetric and asymmetric dimethylations were observed. The closely related HMGA1b protein, however, was not methylated. The unambiguous identification of the sites of phosphorylation and the nature of methylation facilitates the future examination of the biological implications of the HMGA1 proteins. 相似文献
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Edberg DD Adkins JN Springer DL Reeves R 《The Journal of biological chemistry》2005,280(10):8961-8973
Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types. 相似文献
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Zeeshan Iqbal Daniel C. Hoessli Afshan Kaleem Jawaria Munir Muhammad Saleem Imran Afzal Abdul Rauf Shakoori Nasir‐ud‐Din 《Journal of cellular biochemistry》2013,114(4):874-887
Proteins function is regulated by co‐translational modifications and post‐translational modifications (PTMs) such as phosphorylation, glycosylation, and acetylation, which induce proteins to perform multiple tasks in a specified environment. Acetylation takes place post‐translationally on the ε‐amino group of Lys in histone proteins, allowing regulation of gene expression. Furthermore, amino group acetylation also occurs co‐translationally on Ser, Thr, Gly, Met, and Ala, possibly contributing to the stability of proteins. In this work, the influence of amino acids next to acetylated sites has been investigated by using MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for PTMs). MAPRes was utilized to examine the sequence patterns vicinal to modified and non‐modified residues, taking into account their charge and polarity. The PTMs data were further sub‐divided according to their sub‐cellular location (nuclear, mitochondrial, and cytoplasmic), and their association patterns were mined. The association patterns mined by MAPRes for acetylated and non‐acetylated residues are consistent with the existing literature but also revealed novel patterns. These rules have been utilized to describe the acetylation and its effects on the protein structure‐function relationship. J. Cell. Biochem. 114: 874–887, 2013. © 2012 Wiley Periodicals, Inc. 相似文献
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Sahara N Lewis J DeTure M McGowan E Dickson DW Hutton M Yen SH 《Journal of neurochemistry》2002,83(6):1498-1508
Transgenic mice (JNPL3), which develop neurofibrillary degeneration and express four-repeat human tau with P301L missense mutation, were characterized biochemically to determine whether the development of aggregated tau from soluble tau involves an intermediate stage. Homogenates from mice of different ages were separated into buffer-soluble (S1), sarkosyl- and salt-extractable (S2) and sarkosyl-insoluble pellet (P3) fractions, and analyzed for human tau distribution, phosphorylation and filament formation. S1 and S2 fractions contained 50-60-kDa tau whereas the S2 fraction also had 64-kDa tau. The level of tau in the P3 fraction increased in an age-dependent manner and correlated positively with the soluble tau concentration. The P3 fraction from 2.5-6.5-month-old mice contained 64- and 50-60-kDa tau, whereas that from 8.5-month and older transgenic animals contained mostly 64-kDa and higher molecular weight tau. The S2 and P3 fractions contained comparable amounts of 64-kDa tau. The 64-kDa tau was predominantly human, and phosphorylated at multiple sites: Thr181, Ser202/Thr205, Thr212, Thr231, Ser262, Ser396/Ser404, Ser409 and Ser422. Most of these sites were phosphorylated to a lesser extent in S2 than in P3 fractions. Tau polymers were detected in P3 fractions from 3-month and older female JNPL3 mice, but not in non-transgenic controls. The results suggest that tau in S2 represents an intermediate from which insoluble tau is derived, and that phosphorylation may play a role in filament formation and/or stabilization. 相似文献
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Annamaria Sandomenico Annalia Focà Luca Sanguigno Andrea Caporale Giuseppina Focà Angelica Pignalosa 《MABS-AUSTIN》2016,8(8):1575-1589
Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24–39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs. 相似文献
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Subcellular distribution of aminoacyl-transfer RNA synthetases in Chinese hamster ovary cell culture 总被引:9,自引:0,他引:9
Chinese hamster ovary cells grown in cell culture were broken and fractionated by differential centrifugation. Four principal fractions: nuclear and membrane, microsomal, postribosomal, and supernatant were obtained. The distribution of aminoacyl-tRNA synthetases in these four fractions was determined for all twenty amino acids.It was shown that there is a differential distribution of synthetases. Activities specific for eight amino acids: Ala, Ser, Gly, Cys, His, Arg, Thr and Pro were found mainly in the supernatant fraction. Activities specific for eleven amino acids: Asp, Asn, Glu, Gln, Ile, Leu, Lys, Met, Phe, Tyr and Val were found mainly in the postribosomal fraction. Four activities were found at significant levels in the microsomal fraction: Asp, Phe, Lys and Pro. The nuclear and membrane fraction contained activity for Lys, His, Asp and Thr.Changes in aminoacyl-tRNA synthetase activities in various fractions from preparations made by breaking cells with a membrane-dissociating detergent showed that some of the aminoacyl-tRNA synthetase activities may be membrane bound. 相似文献
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Geraghty KM Chen S Harthill JE Ibrahim AF Toth R Morrice NA Vandermoere F Moorhead GB Hardie DG MacKintosh C 《The Biochemical journal》2007,407(2):231-241
AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases. 相似文献
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Tokumitsu H Hatano N Inuzuka H Sueyoshi Y Yokokura S Ichimura T Nozaki N Kobayashi R 《The Journal of biological chemistry》2005,280(42):35108-35118
To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts, and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein. 相似文献
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Chen S Murphy J Toth R Campbell DG Morrice NA Mackintosh C 《The Biochemical journal》2008,409(2):449-459
AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking of GLUT4 (glucose transporter 4) storage vesicles to the cell surface. All animal species examined contain TBC1D1, whereas AS160 evolved with the vertebrates. TBC1D1 has two clusters of phosphorylated residues, either side of the second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated in HEK (human embryonic kidney)-293 cells, 14-3-3s bind primarily to pSer237 (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr596 (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer237 and pThr596 contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr596 of TBC1D1, and promotes phosphorylation of AMPK and Ser237 of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser235 prevents subsequent phosphorylation of Ser237. In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser237 and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr596 but not 14-3-3 binding. In contrast, AS160 is phosphorylated on its 14-3-3-binding sites (Ser341 and Thr642) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle. 相似文献
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Shechter D Nicklay JJ Chitta RK Shabanowitz J Hunt DF Allis CD 《The Journal of biological chemistry》2009,284(2):1064-1074
Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status. 相似文献
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N-terminal acetylation in the yeast Saccharomyces cerevisiae is catalysed by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain the catalytic subunits Ard1p, Nat3p and Mak3p, respectively. Yeast 6-phosphofructo-2-kinase (PFK2) was found to be acetylated at the amino acid lysine 3. The Lys3-Arg mutant was not acetylated and the mutation causes a slight decrease in enzyme activity. PFK2 from yeast cells exposed to hypo-osmotic stress is known to be phosphorylated at Ser8 and Ser652 (Dihazi et al., 2001a). We have taken a mass spectrometric approach to investigate the influence of PFK2 acetylation on its phosphorylation. Wild-type PFK2 and the Lys3-Arg mutant were purified from hypo-osmotically stressed cells and analysed with MALDI-TOF MS for phosphorylation. Wild-type PFK2 without any tag sequence was found to be acetylated and two times phosphorylated at the N-terminal peptide T(1-40) carrying the acetylation. The same results were observed with C-terminally His-tagged PFK2. When the His-tag was added to the N-terminus of the protein PFK2, acetylation was found to be incomplete and only one phosphate was incorporated in the peptide T(1-41). The Lys3-Arg mutant of PFK2 was not at all post-translationally modified at the N-terminal peptide. Our data indicate that Lys3 acetylation affects the N-terminal phosphorylation of PFK2 under hypo-osmotic stress. 相似文献
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Udo H Lam CK Mori S Inouye M Inouye S 《Journal of molecular microbiology and biotechnology》2000,2(4):557-563
Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases. 相似文献
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General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a "dead" kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIalpha subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338. 相似文献
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Mueller C Edmiston KH Carpenter C Gaffney E Ryan C Ward R White S Memeo L Colarossi C Petricoin EF Liotta LA Espina V 《PloS one》2011,6(8):e23780
Background
There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.Results
Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.Conclusion
In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research. 相似文献18.
Ching-Yu Lin Chieh Huo Li-Kuo Kuo Richard A. Hiipakka Richard Baker Jones Hui-Ping Lin Yuwen Hung Liang-Cheng Su Jen-Chih Tseng Ying-Yu Kuo Yu-Ling Wang Yasuhisa Fukui Yung-Hsi Kao John M. Kokontis Chien-Chih Yeh Linyi Chen Shiaw-Der Yang Hsiao-Hui Fu Ya-Wen Chen Kelvin K. C. Tsai Jang-Yang Chang Chih-Pin Chuu 《PloS one》2013,8(6)
Oxysterols are oxidation products of cholesterol. Cholestane-3β, 5α, 6β-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10–40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20–40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27Kip. Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of β-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer. 相似文献
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Kaleem A Ahmad I Hoessli DC Walker-Nasir E Saleem M Shakoori AR Nasir-Ud-Din 《Molecular biology reports》2009,36(4):631-639
Post-translational modifications (PTMs) of proteins induce structural and functional changes that are most often transitory and difficult to follow and investigate in vivo. In silico prediction procedures for PTMs are very valuable to foresee and define such transitory changes responsible for the multifunctionality of proteins. Epidermal growth factor receptor (EGFR) is such a multifunctional transmembrane protein with intrinsic tyrosine kinase activity that is regulated primarily by ligand-stimulated transphosphorylation of dimerized receptors. In human EGFR, potential phosphorylation sites on Ser, Thr and Tyr residues including five autophosphorylation sites on Tyr were investigated using in silico procedures. In addition to phosphorylation, O-GlcNAc modifications and interplay between these two modifications was also predicted. The interplay of phosphorylation and O-GlcNAc modification on same or neighboring Ser/Thr residues is termed as Yin Yang hypothesis and the interplay sites are named as Yin Yang sites. Amongst these modification sites, one residue is localized in the juxtamembrane (Thr 654) and two are found in the catalytic domain (Ser 1046/1047) of the EGFR. We propose that, when EGFR is O-GlcNAc modified on Thr 654, EGFR may be transferred from early to late endosomes, whereas when EGFR is O-GlcNAc modified on Ser 1046/1047 desensitization of the receptor may be prevented. These findings suggest a complex interplay between phosphorylation and O-GlcNAc modification resulting in modulation of EGFR's functionality. 相似文献