Identification of the transactivation domain of the human FHL3.
Four and a half LIM domain protein 3 (FHL3) has the transactivation and repressor activity, and plays important roles in regulating the expression levels of various genes. In this study, FHL3 was proved to possess the auto-activation ability when constructed into the pGBKT7 plasmid (a GALA DNA-binding domain (BD) cloning vector of the yeast two-hybrid system) and transformed into yeast Y190 cells. To determine the transactivation domain of FHL3, five mutants were constructed by sequentially deleting each LIM domain of FHL3 and then inserting them into the pGBKT7 plasmid. After being transformed into yeast Y190 cells, expression levels of the mutants were identified by Western blot analysis. The beta-galactosidase assay showed that the mutant without the fourth LIM domain (LIM4) lost the auto-activation ability. Further investigations on the mutants with deleted first or second zinc finger of LIM4 confirmed that the second zinc finger motif in LIM4 was responsible for the auto-activation of FHL3.
Huang
X
X
Laboratory of Molecular Genetics, Beijing Institute of Basic Medical Sciences, 100850 Beijing, China.
Wang
J F
JF
Xia
W R
WR
Zou
M J
MJ
Cai
X
X
Xu
D G
DG
D020816:Amino Acid Motifs; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D060588:LIM Domain Proteins; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers
C117563:FHL3 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D015534:Trans-Activators