title: Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody.
abstract: Signal transducer and activator of transcription 3 (STAT3) is a multifunctional protein that participates in signaling pathways initiated by various growth factors and cytokines. It exists in multiple forms including those phosphorylated on Tyr(705) (pYSTAT3) or Ser(727) (pSSTAT3) as well as the unphosphorylated protein (USTAT3). In addition to the canonical transcriptional regulatory role of pYSTAT3, both USTAT3 and pSSTAT3 function as transcriptional regulators by binding to distinct promoter sites and play signaling roles in the cytosol or mitochondria. The roles of each STAT3 species in different biological processes have not been readily amenable to investigation, however. We have now prepared an intrabody that binds specifically and with high affinity to the tyrosine-phosphorylated site of pYSTAT3. Adenovirus-mediated expression of the intrabody in HepG2 cells as well as mouse liver blocked both the accumulation of pYSTAT3 in the nucleus and the production of acute phase response proteins induced by interleukin-6. Intrabody expression did not affect the overall accumulation of pSSTAT3 induced by interleukin-6 or phorbol 12-myristate 13-acetate (PMA), the PMA-induced expression of the c-Fos gene, or the PMA-induced accumulation of pSSTAT3 specifically in mitochondria. In addition, it had no effect on interleukin-6-induced expression of the gene for IFN regulatory factor 1, a downstream target of STAT1. Our results suggest that the engineered intrabody is able to block specifically the downstream effects of pYSTAT3 without influencing those of pSSTAT3, demonstrating the potential of intrabodies as tools to dissect the cellular functions of specific modified forms of proteins that exist as multiple species.
authors.affiliation: Division of Life and Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, and Global Top 5 Research Program, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea.
authors.forename: Mi Young
authors.initials: MY
authors.lastname: Koo
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Park
authors.forename: Jung Mi
authors.initials: JM
authors.lastname: Lim
authors.forename: Sei Yoon
authors.initials: SY
authors.lastname: Joo
authors.forename: Seung-Pil
authors.initials: SP
authors.lastname: Shin
authors.forename: Hyun Bo
authors.initials: HB
authors.lastname: Shim
authors.forename: Junho
authors.initials: J
authors.lastname: Chung
authors.forename: Dongmin
authors.initials: D
authors.lastname: Kang
authors.forename: Hyun Ae
authors.initials: HA
authors.lastname: Woo
authors.forename: Sue Goo
authors.initials: SG
authors.lastname: Rhee
mesh_terms: D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000494:pharmacology*; D000918:Antibody Specificity / Q000187:drug effects / Q000276:immunology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D045163:Antibodies, Phospho-Specific; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016760:Proto-Oncogene Proteins c-fos; D050796:STAT3 Transcription Factor; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D019000:Phosphotyrosine; D013755:Tetradecanoylphorbol Acetate
references.citation: Cell Res. 2008 Apr;18(4):443-51
references.pmid: 18364677
references.citation: Nat Rev Cancer. 2009 Nov;9(11):798-809
references.pmid: 19851315
references.citation: Cell. 2011 Sep 30;147(1):185-98
references.pmid: 21962515
references.citation: Science. 2009 Feb 6;323(5915):793-7
references.pmid: 19131594
references.citation: Proc Natl Acad Sci U S A. 2005 Jun 7;102(23):8150-5
references.pmid: 15919823
references.citation: Cell Res. 2011 Jan;21(1):159-68
references.pmid: 21187858
references.citation: Chem Biol. 2006 Nov;13(11):1235-42
references.pmid: 17114005
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11107-12
references.pmid: 9736697
references.citation: Immunol Today. 1994 Feb;15(2):74-80
references.pmid: 7512342
references.citation: Science. 2009 Jun 26;324(5935):1713-6
references.pmid: 19556508
references.citation: Science. 1995 Mar 3;267(5202):1349-53
references.pmid: 7871433
references.citation: Ann N Y Acad Sci. 2009 Aug;1171:59-76
references.pmid: 19723038
references.citation: Annu Rev Med. 1987;38:417-32
references.pmid: 3555304
references.citation: Oncogene. 2000 May 15;19(21):2628-37
references.pmid: 10851062
references.citation: Science. 1997 Sep 12;277(5332):1630-5
references.pmid: 9287210
references.citation: J Biol Chem. 2012 Apr 20;287(17):14192-200
references.pmid: 22378781
references.citation: Trends Mol Med. 2003 Sep;9(9):390-6
references.pmid: 13129705
references.citation: J Immunol. 1997 Sep 1;159(5):2212-21
references.pmid: 9278309
references.citation: Cell Signal. 2005 Dec;17(12):1542-50
references.pmid: 15935617
references.citation: Sci Signal. 2009 Sep 29;2(90):pe61
references.pmid: 19797267
references.citation: J Biol Chem. 1998 Nov 20;273(47):31327-36
references.pmid: 9813041
references.citation: J Biol Chem. 2003 May 2;278(18):15794-9
references.pmid: 12600988