A conformational change in PBX1A is necessary for its nuclear localization

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• The extradenticle (EXD) homeodomain (HD) protein in flies requires HOMOTHORAX (HTH) for nuclear localization.
• Mammalian homologs of EXD, like PBX1A, interact with HTH homologs (MEIS1, 2, 3) and PREP1.

Purpose of the Study:

• To investigate the mechanism of PBX1A nuclear localization and its dependence on MEIS/PREP proteins.
• To elucidate the role of the N-terminal region of PBX1A in regulating its interaction with MEIS/PREP and nuclear entry.

Main Methods:

• Deletion mapping and green fluorescent protein (GFP) fusions to identify nuclear localization signals (NLSs).
• DNA-binding assays and pull-down experiments to study protein interactions and conformation.
• Site-directed mutagenesis to investigate the role of specific residues in PBX1A.

Main Results:

• PBX1A requires MEIS/PREP proteins for nuclear localization, similar to EXD/HTH.
• Removal of the N-terminal region of PBX1A abolishes MEIS/PREP interaction but permits nuclear accumulation.
• Two cooperative NLSs were mapped within the PBX HD, masked by the N-terminus.
• A mutation disrupting the N-terminus-HD interaction leads to constitutive nuclear localization and increased protease sensitivity.
• MEIS proteins induce a conformational change in PBX1A, unmasking NLSs and enhancing DNA-binding activity.

Conclusions:

• The N-terminus of PBX1A masks its nuclear localization signals.
• MEIS family proteins induce a conformational change in PBX1A, leading to nuclear localization and enhanced DNA-binding.
• PBX1 nuclear localization is spatially regulated by Meis1 expression in mouse development.