Identifying The Interaction Between Lim Protein Kyot2 And Human Tight Tight Juncion Protein Zo-2

Notch proteins are a family of evolutionarily conserved transmembrane receptors which are involved in cell fate determination of many cell lineages. Notch ligand Delta is also transmembrane protein with characteristic cysteine-rich N-terminal domain responsible for Notch binding. The activation of Notch by binding with Notch ligand can lead to inhibition of cell differentiation. The intracellular region of Notchl transactivates genes by interacting with a DNA binding protein RBP-J, which plays an important role in Notch signaling.Using yeast two-hybrid system Honjo's group in Kyoto University isolated a novel protein, KyoT, which physically interacts with RBP-J. Differential splicing gives rise to three transcripts of the KyoT gene, KyoTl, KyoT2 and KyoTS. KyoT2, but not KyoTl negatively regulates transcription by association with RBP-J.Using yeast two-hybrid system with LIM domain protein KyoTl as a bait, we have isolated analternatively spliced form of human tight junction protein 2------ZO-2-i3. The tight junction (orzonula occludens) is the most apical junction between neighbouring epithelial cells or endothelial cells. In vertebrate organisms, the tight junction is the cellular structure, which serves to prevent the?<sub><sub><sub><sub><sub><sub><sub><sub><sub>y?EK.X?l*籺<sub><sub><sub><sub><sub>2002. 5_<sub><sub><sub><sub><sub><sub><sub><sub> 5free passage of molecules and cells through the paracellular pathway. The second function of the tight junction is to maintain different protein and lipid composition between the apical and basolateral plasma membrane domains of polarized epithelial cells or endothelial cells.ZO-2 is localized in the cytoplasmic plaque domain of tight junction. ZO-2 interacts with the integral membrane proteins via its first PDZ domain or GUK. domain. At the same time ZO-2 directly or indirectly binds to actin, a cytoskeleton protein. Therefore ZO-2 established a linker between tight junction and cytoskeleton. ZO-2 may regulate the formation or function of tight junction and contact between tight junction and cytoskeleton.As for the alternatively spliced form of human tight junction protein 2梈O-2i3, sequence analysis indicated that ZO-213 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 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-2i3 exchanged their vectors, yeast two-hybrid test revealed physical binding of the two proteins. Using KyoT2 protein and antibody in GST pull-down assay similar result was also obtained. Therefore we confirmed KyoT2 interacted ZO-2-i3 in vitro. Furthermore it was identified in yeast that KyoT2, through its LIM2 domain, associated with ZO-2-i3.