[LIM protein KyoT2 interacts with human tight junction protein ZO-2-i3].
It was reported that LIM protein KyoT2 negatively regulated transcription by association with the RBP-J DNA-binding protein. Using yeast two-hybrid system with LIM protein KyoT2 as a bait, we have isolated an alternatively spliced form of human tight junction protein 2--ZO-2-i3. Sequence analysis indicated that ZO-2-i3 is composed of 19 exons, and selected usage of exons led to an alteration in the region following the kinase domain as compared with the published sequence. To identify the interaction between LIM protein KyoT2 and ZO-2-i3, yeast two-hybrid system, purification of KyoT2 protein, and GST pull-down assay were performed in the experiments. After KyoT2 and ZO-2-i3 changed vectors, positive two-hybrid yeast was obtained. Using KyoT2 protein and antibody in GST pull-down assay positive result was also obtained. Therefore we conformed KyoT2 interacted ZO-2-i3 in vitro. Furthermore it was identified in yeast that KyoT2 associated with ZO-2-i3 through its LIM2 domain.
Huang
Hong-Yan
HY
Department of Physiology, Fourth Military Medical University, Xi'an 710032, China. hhongy@cmmail.com
Li
Rong
R
Sun
Qiang
Q
Wang
Jian
J
Zhou
Peng
P
Han
Hua
H
Zhang
Wan-Hui
WH
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005091:Exons; D005796:Genes / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007438:Introns; D060588:LIM Domain Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019108:Tight Junctions / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D062827:Zonula Occludens-2 Protein
D018076:DNA, Complementary; C101149:FHL1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; C569046:TJP2 protein, human; D014157:Transcription Factors; D062827:Zonula Occludens-2 Protein