[Search for interacting proteins of esophageal cancer related gene-1 encoded protein through the yeast two-hybrid system].
OBJECTIVE
To understand the role that esophageal cancer related gene-1 (ECRG-1) plays and to search for ECRG-1-interacting proteins.


METHODS
A DNA fragment encoding the carboxy-terminus of ECRG-1 (amino acids 40 - 418) was inserted into pGBKT7-DNA-BD vector and fused in-frame to the DNA-binding domain of GAL4. Then, it was used as a bait to screen the human fetal liver cDNA library by yeast two-hybrid, with the cDNA fragment inserted into pACT2 vector and fused in-frame to the Gal4 activation domain. If ECRG-1 interacted with a protein encoded by a cDNA fragmant in the yeast, the transcription of reporter Gene could be activated. With the false positive clonies eliminated, the inserts in the positive plasmids were sequenced and compared to those in the GenBank.


RESULTS
In approximately 3 x 10(6) independent tansformants screened, 23 clonies exhibited the expression of reporter gene. After eliminating the false positive clonies, two cDNA fragments were obtained. DNA sequencing revealed that one encoded Miz-1 (Myc-interacting Zn finger protein-1), and another encoded FLNA (actin-binding protein-280), Miz-1, being a Zn finger protein, could be bound to p15 promotor and activated the transcription. FLNA, being an actin-binding protein took part in the TGF-beta pathway via interaction with Smad.


CONCLUSIONS
ECRG-1 is able to be specifically bound to Miz-1 and FLNA in the yeast. It may play a role in the regulation of cell cycle via interaction with Miz-1 and FLNA.
Wang
Jianbo
J
Department of Chemical Etiology and Carcinogenesis, Cancer Institute (Hospital), Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China.
Fan
Yu
Y
Guo
Liping
L
Lu
Shixin
S
D003285:Contractile Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism* / Q000502:physiology; D064448:Filamins; D015723:Gene Library; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008099:Liver / Q000196:embryology / Q000502:physiology; D008565:Membrane Proteins; D008840:Microfilament Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000502:physiology; D057057:Serine Proteases; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D003285:Contractile Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C578661:FLNC protein, human; D064448:Filamins; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C446704:TMPRSS11A protein, human; D014157:Transcription Factors; C107961:ZBTB17 protein, human; D057057:Serine Proteases