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Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody

Authors
 Mi Young Koo  ;  Jiyoung Park  ;  Jung Mi Lim  ;  Sei Yoon Joo  ;  Seung-Pil Shin  ;  Hyun Bo Shim  ;  Junho Chung  ;  Dongmin Kang  ;  Hyun Ae Woo  ;  Sue Goo Rhee 
Citation
 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol.111(17) : 6269-6274, 2014 
Journal Title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN
 0027-8424 
Issue Date
2014
MeSH
Animals ; Antibodies, Phospho-Specific/pharmacology* ; Antibody Specificity/drug effects ; Antibody Specificity/immunology ; Cell Nucleus/drug effects ; Cell Nucleus/metabolism ; Green Fluorescent Proteins/metabolism ; Humans ; Interleukin-6/pharmacology ; Lipopolysaccharides/pharmacology ; Liver/drug effects ; Liver/metabolism ; Mice ; Mitochondria/drug effects ; Mitochondria/metabolism ; Phosphorylation/drug effects ; Phosphoserine/metabolism ; Phosphotyrosine/metabolism* ; Protein Transport/drug effects ; Proto-Oncogene Proteins c-fos/genetics ; Proto-Oncogene Proteins c-fos/metabolism ; STAT3 Transcription Factor/antagonists & inhibitors* ; STAT3 Transcription Factor/metabolism ; Single-Chain Antibodies/metabolism ; Tetradecanoylphorbol Acetate/pharmacology
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.
Files in This Item:
T201403304.pdf Download
DOI
10.1073/pnas.1316815111
Appears in Collections:
1. College of Medicine (의과대학) > BioMedical Science Institute (의생명과학부) > 1. Journal Papers
Yonsei Authors
Rhee, Sue Goo(이서구)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/99846
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