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Proteomic analysis of the nuclear phosphorylated proteins in dairy cow mammary epithelial cells treated with estrogen

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Abstract

Estrogen regulates a variety of physiological processes, including mammary gland growth, morphogenesis of the mammary gland, proliferation and differentiation, and elevating the expression of milk proteins. Many nuclear phosphorylated proteins such as pStat5 and mTOR regulate milk protein synthesis. But the detail of milk protein synthesis controlled at the transcript level and posttranslational level is not well-known. To contribute to the understanding of the molecular mechanism underlying estrogen action on the dairy cow mammary epithelial cells (DCMECs), nuclear phosphorylated proteins regulated by estrogen in DCMECs were identified. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization/time of flight mass spectrometry were used to identify the changes of nuclear phosphorylated proteins in DCMECs treated with estrogen. Seven proteins were identified differentially up-expressed in DCMECs after 24-h estrogen exposure: including glycyl-tRNA synthetase, previously reported in milk protein synthesis of DCMECs, belonging to the class-II aminoacyl-tRNA synthetase family; proteins involved in other cellular functions, such as translation initiation factors, GTP-binding nuclear proteins, heat-shock proteins, and proteins belonging to ubiquitin-proteasome system. This screening reveals that estrogen influences the levels of nuclear phosphorylated proteins of DCMECs which opens new avenue for the study of the molecular mechanism linking to milk synthesis.

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References

  • Akakura S.; Yoshida M.; Yoneda Y.; Horinouchi S. A role for Hsc70 in regulating nucleocytoplasmic transport of a temperature-sensitive p53 (p53Val-135). J. Biol. Chem. 276: 14649–14657; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Alessi D. R.; Andjelkovic M.; Caudwell B.; Cron P.; Morrice N.; Cohen P.; Hemmings B. A. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15(23): 6541–6551; 1996.

    PubMed  CAS  Google Scholar 

  • Antonellis A.; Lee-Lin S.-Q.; Wasterlain A.; Leo P.; Quezado M.; Goldfarb L. G.; Myung K.; Burgess S.; Fischbeck K. H.; GreenGreen E. D. Functional analyses of glycyl-tRNA synthetase mutations suggest a key role for tRNA-charging enzymes in peripheral axons. J. Neurosci. 26(41): 10397–10406; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Asakawa H.; Hiraoka Y.; Haraguchi T. Nuclear translocation of RanGAP1 coincides with virtual nuclear envelope breakdown in fission yeast meiosis. Commun. Integr. Biol. 4(3): 312–314; 2011.

    Article  PubMed  CAS  Google Scholar 

  • Azuma K.; Sasada T.; Takedatsu H.; Shomura H.; Koga M.; Maeda Y.; Yao A.; Hirai T.; Takabayashi A.; Shichijo S.; Itoh K. Ran, a small GTPase gene, encodes cytotoxic T lymphocyte (CTL) epitopes capable of inducing HLA-A33-restricted and tumor-reactive CTLs in cancer patients. Clin. Cancer Res. 10: 6695–6702; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Banks G. T.; Bros-Facer V.; Williams H. P.; Chia R.; Achilli F.; Bryson J. B.; Greensmith L.; Fisher E. M. Mutant glycyl-tRNA synthetase (Gars) ameliorates SOD1(G93A) motor neuron degeneration phenotype but has little affect on Loa dynein heavy chain mutant mice. PLoS One 4(7): 6218–6230; 2009.

    Article  Google Scholar 

  • Bansal N.; Kadamb R.; Mittal S.; Vig L.; Sharma R.; Dwarakanath B. S.; Saluja D. Tumor suppressor protein p53 recruits human Sin3B/HDAC1 complex for down-regulation of its target promoters in response to genotoxic stress. PLoS One 6(10): 26156–26162; 2011.

    Article  Google Scholar 

  • Bassuk J. A.; Kaot W. W. Y.; Herzer P.; Kedersha N. L.; Seyer J.; Demartino J. A.; Daugherty B. L.; III Mark G. E.; Berg R. A. Prolyl 4-hydroxylase: molecular cloning and the primary structure of the a subunit from chicken embryo. Proc. Natl. Acad. Sci. U. S. A. 86: 7382–7386; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Bhui-Kaur A.; Therwath A.; Henry L.; Chiesa J.; Kurkure A.; Scherrer K.; Bureau J. P. Increased prosomal proteins in breast cancer cells and in neighboring normal cells in Parsi and non-Parsi populations. J. Cancer Res. Clin. Oncol. 124(2): 117–126; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Bodo J.; Durkin L.; Hsi E. D. Quantitative in situ detection of phosphoproteins in fixed tissues using quantum dot technology. J. Histochem. Cytochem. 57(7): 701–708; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Bodo J.; Hsi E. D. Phosphoproteins and the dawn of functional phenotyping. Pathobiology 78(2): 115–121; 2011.

    Article  PubMed  CAS  Google Scholar 

  • Bompard G.; Rabeharivelo G.; Frank M.; Cau J.; Delsert C.; Morin N. Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis. J. Cell Biol. 190(5): 807–822; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Brunet A.; Bonni A.; Zigmond M. J.; Lin M. Z.; Juo P.; Hu L. S.; Anderson M. J.; Arden K. C.; Blenis J.; Greenberg M. E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96(6): 857–868; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Cantin G. T.; Venable J. D.; Cociorva D.; Yates III J. R. Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway. J. Proteome Res. 5(1): 127–134; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Cimica V.; Chen H.-C.; Iyer J. K.; Reich N. C. Dynamics of the STAT3 transcription factor: nuclear import dependent on ran and importin-b1. PLoS One 6(5): 20188–20199; 2011.

    Article  Google Scholar 

  • Clarke P. R.; Zhang C. Spatial and temporal coordination of mitosis by Ran GTPase. Nat. Rev. Mol. Cell Biol. 9: 464–477; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Connor E. E.; Meyer M. J.; Li R. W.; Van Amburgh M. E.; Boisclair Y. R.; Capuco A. V. Regulation of gene expression in the bovine mammary gland by ovarian steroids. Dairy Sci. 90: E55–E65; 2007.

    Article  Google Scholar 

  • Cross D. A.; Alessi D. R.; Cohen P.; Andjelkovich M.; Hemmings B. A. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378(6559): 785–789; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Dasso M. Running on ran: nuclear transport and the mitotic spindle. Cell 104: 321–324; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Demasi M. A.; Montor W. R.; Ferreira G. B. Differential proteomic analysis of the anti-proliferative effect of glucocorticoid hormones in ST1 rat glioma cells. Steroid Biochem. Mol. Biol. 103: 137–148; 2007.

    Article  CAS  Google Scholar 

  • Desrivières S.; Kuhn K.; Müller J.; Gläser M.; Laria N. C.; Korder J.; Sonnentag M.; Neumann T.; Schwarz J.; Schäfer J.; Hamon C.; Groner B.; Prinz T. Comparison of the nuclear proteomes of mammary epithelial cells at different stages of functional differentiation. Proteomics 7(12): 2019–2037; 2007.

    Article  PubMed  Google Scholar 

  • Elenich L. A.; Nandi D.; Kent A. E.; McCluskey T. S.; Cruz M.; Iyer M. N. The complete primary structure of mouse 20S proteasomes. Immunogenetics 49: 835–842; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Fan S.; Whiteman E. L.; Hurd T. W.; McIntyre J. C.; Dishinger J. F.; Liu C. J.; Martens J. R.; Verhey K. J.; Sajjan U.; Margolis B. Induction of ran GTP drives ciliogenesis. Mol. Biol. Cell 22(23): 4539–4548; 2011.

    Article  PubMed  CAS  Google Scholar 

  • Feistritzer C.; Wiedermann C. Effects of anticoagulant strategies on activation of inflammation and coagulation. Expert. Opin. Biol. Ther. 7: 855–870; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Feng J.; Park J.; Cron P.; Hess D.; Hemmings B. A. Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J. Biol. Chem. 279: 41189–41196; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Feuermann Y.; Mabjeesh S. J.; Shamay A. Mammary fat can adjust prolactin effect on mammary epithelial cells via leptin and estrogen. Int. J. Endocrinol. 2009: 427260; 2009.

    PubMed  Google Scholar 

  • Guo R.-T.; Chong Y. E.; Guo M.; Yang X.-L. Crystal structures and biochemical analyses suggest a unique mechanism and role for human glycyl-tRNA synthetase in Ap4A homeostasis. J. Biol. Chem. 284(42): 28968–28976; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Hannan R. D.; Jenkins A.; Jenkins A. K.; Brandenburger Y. Cardiac hypertrophy: a matter of translation. Clin. Exp. Pharmacol. Physiol. 30(8): 517–527; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Helguero L. A.; Lindberg K.; Gardmo C.; Schwend T. Different roles of estrogen receptors A and B in the regulation of E-cadherin protein levels in a mouse mammary epithelial cell line. Cancer Res. 68(21): 8695–8704; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Hochstrasser M. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30: 405–439; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Hutchins J. R. A.; Moore W. J.; Clarke P. R. Dynamic localisation of Ran GTPase during the cell cycle. BMC Cell Biol. 10: 66–75; 2009.

    Article  PubMed  Google Scholar 

  • Jastrzebski K.; Hannan K. M.; House C. M.; Hung S. S.-C.; Pearson R. B.; Hannan R. D. A phospho-proteomic screen identifies novel S6K1 and mTORC1 substrates revealing additional complexity in the signaling network regulating cell growth. Cell. Signal. 23: 1338–1347; 2011.

    Article  PubMed  CAS  Google Scholar 

  • Koren 3rd J.; Jinwal U. K.; Jin Y.; O'Leary J.; Jones J. R. et al. Facilitating Akt clearance via manipulation of Hsp70 activity and levels. J. Biol. Chem. 285: 2498–2505; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Kuroda K.; Tajima S. Proliferation of HSP47-positive skin fibroblasts in dermatofibroma. J. Cutan. Pathol. 35(1): 21–27; 2008.

    PubMed  Google Scholar 

  • Kushner P. J.; Agard D. A.; Greene G. L.; Scanlan T. S.; Shiau A. K.; Uht R. M.; Webb P. Estrogen receptor pathways to AP-1. J. Steroid Biochem. Mol. Biol. 74: 311–317; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Laplante A. F.; Moulin V.; Auger F. A.; Landry J.; Li H.; Morrow G.; Tanguay R. M.; Germain L. Expression of heat shock proteins in mouse skin during wound healing. J. Histochem. Cytochem. 46(11): 1291–1301; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Laserna E. J.; Valero M. L.; Sanz L. Proteomic analysis of phosphorylated nuclear proteins underscores novel roles for rapid actions of retinoic acid in the regulation of mRNA splicing and translation. Mol. Endocrinol. 23: 1799–1814; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Lee J. S.; Park S. G.; Park H.; Seol W.; Lee S.; Kim S. Interaction network of human aminoacyl-tRNA synthetases and subunits of elongation factor 1 complex. J. Biochem. Biophys. Res. Commun. 291: 158–164; 2002.

    Article  CAS  Google Scholar 

  • Levin E. R. Cellular functions of plasma membrane estrogen receptors. Steroids 67(33): 471–475; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Li L.; Zhang K.; Cai X. J.; Feng M.; Zhang Y.; Zhang M. Adiponectin upregulates prolyl-4-hydroxylase a1 expression in interleukin 6-stimulated human aortic smooth muscle cells by regulating ERK 1/2 and Sp1. PLoS One 6(7): 22819–22826; 2011.

    Article  Google Scholar 

  • Li C.; Ying H.; Shen; Xinwen W.; Jing W.; Yehua G.; Nanyue C.; Jian W.; Scott A.; LeMaire; Joseph S. C.; Xing Li W. Human prolyl-4-hydroxylase α(I) transcription is mediated by upstream stimulatory factors. Biol. Chem. 281(16): 10849–10855; 2006.

  • Li L. R. W.; Meyer M. J.; Van Tassell C. P.; Sonstegard T. S.; Connor E. E. Identification of estrogen-responsive genes in the parenchyma and fat pad of the bovine mammary gland by microarray analysis. Physiol. Genomics 27: 42–53; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Ma W. J.; Guo X.; Liu J. T.; Liu R. Y.; Hu J. W.; Sun A. G.; Yu Y. X.; Lammi M. J. Proteomic changes in articular cartilage of human endemic osteoarthritis in China. Proteomics 11: 2881–2890; 2011.

    Article  PubMed  CAS  Google Scholar 

  • MacMorris M.; Brocker C.; Blumenthal T. UAP56 levels affect viability and mRNA export in Caenorhabditis elegans. RNA 9(7): 847–857; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Maniatis T.; Reed R. An extensive network of coupling among gene expression machines. Nature 416: 499–506; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Mishin A. S.; Subach F. V.; Yampolsky I. V.; King W.; Lukyanov K. A.; Verkhusha V. V. The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore. Biochemistry 47: 4666–4673; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Proud C. G. eIF2 and the control of cell physiology. Semin. Cell Dev. Biol. 16: 3–12; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Radisavljevic Z. M.; Gonzalez-Flecha B. TOR kinase and ran are downstream from PI3K/Akt in H2O2-induced mitosis. J. Cell. Biochem. 91: 1293–1300; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Raught B.; Gingras A.-C.; Sonenberg N. The target of rapamycin (TOR) proteins. Proc. Natl. Acad. Sci. U. S. A. 98: 7037–7044; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Rawlings N. D.; Tolle D. P.; Barrett A. Evolutionary families of peptidase inhibitors. Biochemistry 378: 705–716; 2004.

    Article  CAS  Google Scholar 

  • Rizzo M. A.; Springer G. H.; Granada B.; Piston D. W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22: 445–449; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Sahni A.; Wang N.; Alexis J. D. UAP56 is an important regulator of protein synthesis and growth in cardiomyocytes. Biochem. Biophys. Res. Commun. 393: 106–110; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Sarbassov D. D.; Guertin D. A.; Ali S. M.; Sabatini D. M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098–1101; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Saville B.; Wormke M.; Wang F.; Nguyen T.; Enmark E.; Kuiper G.; Gustafsson J. A.; Safe S. Ligand, cell, and estrogen receptor subtype (a/h)-dependent activation at GC-rich (Sp1) promoter elements. J. Biol. Chem. 275: 5379–5387; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Schams D.; Kohlenberg S.; Amselgruber W.; Berisha B.; Pfaff M. W. Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution. Endocrinology 177: 305–317; 2003.

    Article  CAS  Google Scholar 

  • Sekimoto T.; Nakajima K.; Tachibana T.; Hirano T.; Yoneda Y. Interferon-gamma-dependent nuclear import of Stat1 is mediated by the GTPase activity of Ran/TC4. J. Biol. Chem. 271(49): 31017–31020; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Sobolewska A.; Gajewska M.; Zarzyńska J. IGF-I, EGF, and sex steroids regulate autophagy in bovine mammary epithelial cells via the mTOR pathway. Eur. J. Cell Biol. 88: 117–130; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Sorokin A. V.; Kim E. R.; Ovchinnikov L. P. Nucleocytoplasmic transport of proteins. Biochemistry (Mosc) 72: 1439–1457; 2007.

    Article  CAS  Google Scholar 

  • Stasyk T.; Morandell S.; Bakry R.; Feuerstein I.; Huck C. W.; Stecher G.; Bonn G. K.; Huber L. A. Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining. Electrophoresis 26(14): 2850–2854; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Stewart M. Molecular mechanism of the nuclear protein import cycle. Nat. Rev. Mol. Cell Biol. 8: 195–208; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Takahashi S.; Dohi N.; Takahashi Y.; Miura T. Cloning and characterization of the 5′-flanking region of the rat P4Halpha gene encoding the prolyl 4-hydroxylase alpha (I) subunit. Biochim. Biophys. Acta 1574(3): 354–358; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Takashima N.; Shioji K.; Kokubo Y. Validation of the association between the gene encoding proteasome subunit αType 6 and myocardial infarction in a Japanese population. Circ. J. 71: 495–498; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Totan S.; Echo A.; Yuksel E. Heat shock proteins modulate keloid formation. Eplasty. 11: e21; 2011.

    PubMed  Google Scholar 

  • van Dijk F. S.; Byers P. H.; Dalgleish R.; Malfait F.; Maugeri A.; Rohrbach M.; Symoens S.; Sistermans E. A.; Pals G. EMQN best practice guidelines for the laboratory diagnosis of osteogenesis imperfecta. Eur. J. Hum. Genet. 20: 11–19; 2012.

    Article  PubMed  Google Scholar 

  • Wan Z.-Y.; Tong H.-L.; Li Q.-Z.; Gao X.-J. Influence on cellular signal transduction pathway in dairy cow mammary gland epithelial cells by galactopoietic compound isolated from Vaccariae segetalis. Agric. Sci. China 10(4): 619–630; 2011.

    Article  CAS  Google Scholar 

  • Whitney J. B.; Asmal M.; Geiben-Lynn R. Serpin induced antiviral activity of prostaglandin synthetase-2 against HIV-1 replication. PLoS One 6(4): e18589; 2011.

    Article  PubMed  CAS  Google Scholar 

  • Yan S. P.; Tang Z. C.; Su W.; Sun W. N. Proteornic analysis of salt stress-responsive proteins in rice root. Proteomics 5: 235–244; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Zhang C.; Zhang M.-X.; Shen Y. H.; Burks J. K.; Zhang Y.; Jian. Nono pathway-JNK-1 expression via the ASK1α suppresses Prolyl-4-hydroxylase αT. Arterioscler. Thromb. Vasc. Biol. 27: 1760–1767; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Zhang L.; Liu X.; Zhang J.; Cao R.; Lin Y.; Xie J.; Chen P.; Sun Y.; Li D.; Liang S. Proteome analysis of combined effects of androgen and estrogen on the mouse mammary gland. Proteomics 6(2): 487–497; 2006.

    Article  PubMed  Google Scholar 

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Acknowledgment

This study was financially supported by Major State Basic Research Development Program of China (973 Program, No. 2011CB100804).

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Correspondence to Xue-jun Gao.

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Huang, Jg., Gao, Xj., Li, Qz. et al. Proteomic analysis of the nuclear phosphorylated proteins in dairy cow mammary epithelial cells treated with estrogen. In Vitro Cell.Dev.Biol.-Animal 48, 449–457 (2012). https://doi.org/10.1007/s11626-012-9531-y

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