Document Detail


Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha in the living pituitary cell nucleus.
MedLine Citation:
PMID:  12554785     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPalpha) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPalpha was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that coexpressed Pit-1 redistributed C/EBPalpha to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPalpha was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm. A mutation in the Pit-1 homeodomain, or truncation of the C/EBPalpha transactivation domain disrupted the redistribution of C/EBPalpha by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBPalpha, and the truncated C/EBPalpha still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPalpha that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPalpha to DNA is a critical first step in specifying its association with Pit-1. These findings indicated that the protein domains that specify the interaction of Pit-1 and C/EBPalpha are separable from the protein domains that direct the positioning of the associated proteins within the nucleus. The intimate association of Pit-1 and C/EBPalpha at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression.
Authors:
Richard N Day; Ty C Voss; John F Enwright; Cynthia F Booker; Ammasi Periasamy; Fred Schaufele
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.     Date:  2002-12-12
Journal Detail:
Title:  Molecular endocrinology (Baltimore, Md.)     Volume:  17     ISSN:  0888-8809     ISO Abbreviation:  Mol. Endocrinol.     Publication Date:  2003 Mar 
Date Detail:
Created Date:  2003-02-28     Completed Date:  2003-10-16     Revised Date:  2014-09-14    
Medline Journal Info:
Nlm Unique ID:  8801431     Medline TA:  Mol Endocrinol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  333-45     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Blotting, Western
CCAAT-Enhancer-Binding Protein-alpha / genetics,  metabolism,  physiology*
Cell Nucleus / physiology
Chromatin / physiology
DNA-Binding Proteins / genetics,  metabolism,  physiology*
Electrophoretic Mobility Shift Assay
Enhancer Elements, Genetic / genetics,  physiology
Fluorescence Recovery After Photobleaching
Fluorescence Resonance Energy Transfer / methods
HeLa Cells
Homeodomain Proteins / physiology
Humans
Mutation
Pituitary Gland / metabolism,  physiology*
Transcription Factor Pit-1
Transcription Factors / genetics,  metabolism,  physiology*
Transcription, Genetic / physiology
Grant Support
ID/Acronym/Agency:
DK-47301/DK/NIDDK NIH HHS; F32 DK-60315-01/DK/NIDDK NIH HHS; F32 DK-GM10093/DK/NIDDK NIH HHS; R01 DK043701/DK/NIDDK NIH HHS; R01 DK043701-09/DK/NIDDK NIH HHS; R01 DK054345-04A1/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/CCAAT-Enhancer-Binding Protein-alpha; 0/Chromatin; 0/DNA-Binding Proteins; 0/Homeodomain Proteins; 0/POU1F1 protein, human; 0/Transcription Factor Pit-1; 0/Transcription Factors
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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