The tight junction protein ZO-2 has several functional nuclear export signals

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The tight junction (TJ) protein ZO-2 exhibits dynamic subcellular localization, shifting between the TJ region in confluent cells and the nucleus in sparse cultures.
• The canine ZO-2 sequence contains four potential nuclear export signals (NES): two in the PDZ domain (NES-0, NES-1) and two in the GK region (NES-2, NES-3).

Purpose of the Study:

• To investigate the functionality of ZO-2's nuclear export signals (NES-0 and NES-3) and their role in subcellular localization.
• To determine the requirements for efficient nuclear export of ZO-2 segments and the full-length protein.

Main Methods:

• Nuclear export assays using peptides coupled to ovalbumin injected into MDCK cell nuclei.
• In vitro experiments with wild-type and mutated ZO-2-GST fusion proteins injected into cell nuclei.
• Transfection of MDCK cells with full-length ZO-2 constructs, including mutated NES sequences.

Main Results:

• NES-0 and NES-3 were confirmed as functional nuclear export signals, sensitive to leptomycin B.
• NES-1, previously considered non-functional, became active for nuclear export upon phosphorylation at Ser369.
• Efficient nuclear export of ZO-2 segments required specific combinations of NES (NES-0/NES-1 and NES-2/NES-3).
• Mutation of any single NES in full-length ZO-2 resulted in its nuclear accumulation.

Conclusions:

• Multiple functional nuclear export signals regulate the subcellular localization of ZO-2.
• The phosphorylation status of Ser369 is critical for the activity of NES-1.
• Disruption of any NES impairs the nuclear exit of ZO-2, highlighting the importance of these signals for its dynamic distribution.