Karyopherins in nuclear transport of homeodomain proteins during development

Abstract

Homeodomain proteins are crucial transcription factors for cell differentiation, cell proliferation and organ development. Interestingly, their homeodomain signature structure is important for both their DNA-binding and their nucleocytoplasmic trafficking. The accurate nucleocytoplasmic distribution of these proteins is essential for their functions. We summarize information on (a) the roles of karyopherins for import and export of homeoproteins, (b) the regulation of their nuclear transport during development, and (c) the corresponding complexity of homeoprotein nucleocytoplasmic transport signals. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Figure 1. Cartoon of the DNA binding architecture of homeodomains

Helix III of the homeodomain binds the major groove of DNA, with helix I and II lying outside the double helix. Helix III, as a recognition helix, contains the C-terminal basic cluster that contacts both the phosphate backbone and specific bases. The N-terminal arm containing the N-terminal basic cluster lies in the minor groove and makes additional contacts.

Figure 2. Sequences of homeodomains

Selected homeoproteins were aligned by the Clustal W program [137] embedded in MegAlign (DNASTAR, Inc). The colors of the top panel represent the frequency of given residues among sequences shown. The red column represents perfectly conserved residues for which there are no exceptions. The yellow and green columns indicate conserved residues with exceptions (frequency around 70%-90%). The grey and blue columns indicate residues that are much less well conserved among the homeoproteins (from high to low: red>yellow>green>grey>blue). Four amino acid residues in helix III and two amino acid residues in helix I indicated by ^‘*’ are conserved among all homeoproteins [32, 34, 49]. Note that two basic clusters are located at both ends of the homeodomain. The shaded region between helix II and helix III can form an extended loop for specific types of homeodomains.

Figure 3A. Sequences of the N-terminal basic clusters

Selected homeoproteins were aligned by the Clustal W program. Note that amino acid R5 in this cluster is highly conserved. Mutation of this amino acid in HNF1α causes it to be sequestered in the cytoplasm [49].

Figure 3B. Sequences of the C-terminal basic cluster

Selected homeoproteins were aligned by the Clustal W program. Note that the shaded region represents identified core residues for NLS function. The boxed region indicates a hydrophobic group of amino acids in the Prospro homeodomain which functions as a NES.

Figure 4. Model of nuclear import of homeoproteins mediated by the homeodomain

There are two basic amino-acid clusters (BC1 and BC2) at the ends of the homeodomain of homeoproteins. There are three forms of NLS found in the homeodomains: both BC1 and BC2 can function as an NLS independently (NLS1 or NLS2). BC1 can function as an NLS (NLS3) in conjunction with BC2. NLS1 and NLS2 can be recognized by either importin αs or karyopherin βs directly but NLS3 is only targeted by karyopherin βs. Therefore, nuclear import of homeoproteins can be mediated by both the classical and the nonclassical pathways. The homeodomain could use only a single NLS in certain conditions. As shown in the top left panel, if BC2 is structurally concealed, BC1/NLS1 could be functional. Alternatively as shown in the top right panel, if BC1 is structurally concealed, its BC2/NLS2 could function. When the homeodomain shows the NLS3 conformation (lower panel), only importin βs interact with NLS3 and nuclear import is mediated by a nonclassical pathway. The causes of possible interconversions among the three different NLS conformations are largely unknown (indicated by question marks).