PubMed · 15669262
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Takahiro Arakawa. 2004. [Intracellular localization, modification].. https://pubmed.ncbi.nlm.nih.gov/15669262/
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Many receptor tyrosine kinases (RTKs) can be detected in the cell nucleus, such as EGFR, HER-2, HER-3, HER-4, and fibroblast growth factor receptor. EGFR, HER-2 and HER-4 contain transactivational activity and function as transcription co-factors to activate gene promoters. High EGFR in tumor nuclei correlates with increased tumor proliferation and poor survival in cancer patients. However, the mechanism by which cell-surface EGFR translocates into the cell nucleus remains largely unknown. Here, we found that EGFR co-localizes and interacts with importins alpha1/beta1, carriers that are critical for macromolecules nuclear import. EGFR variant mutated at the nuclear localization signal (NLS) is defective in associating with importins and in entering the nuclei indicating that EGFR's NLS is critical for EGFR/importins interaction and EGFR nuclear import. Moreover, disruption of receptor internalization process using chemicals and forced expression of dominant-negative Dynamin II mutant suppressed nuclear entry of EGFR. Additional evidences suggest an involvement of endosomal sorting machinery in EGFR nuclear translocalization. Finally, we found that nuclear export of EGFR may involve CRM1 exportin as we detected EGFR/CRM1 interaction and markedly increased nuclear EGFR following exposure to leptomycin B, a CRM1 inhibitor. Collectively, these data suggest the importance of receptor endocytosis, endosomal sorting machinery, interaction with importins alpha1/beta1, and exportin CRM1 in EGFR nuclear-cytoplasmic trafficking. Together, our work sheds light into the nature and regulation of the nuclear EGFR pathway and provides a plausible mechanism by which cells shuttle cell-surface EGFR and potentially other RTKs through the nuclear pore complex and into the nuclear compartment.
Using manual and automated high throughput microscopy (HTM), ligand-dependent trafficking of green fluorescent protein-androgen receptor (GFP-AR) was analyzed in fixed and living cells to determine its spatial distribution, solubility, mobility, and co-activator interactions. Within minutes, addition of the agonist R1881 resulted translocation of GFP-AR from the cytoplasm to the nucleus, where it displayed a hyperspeckled pattern and extraction resistance in low expressing cells. AR antagonists (Casodex, hydroxyflutamide) also caused nuclear translocation, however, the antagonist-bound GFP-AR had a more diffuse nuclear distribution, distinct from the agonist-bound GFP-AR, and was completely soluble; overexpressed GFP-AR in treated cells was extraction resistant, independent of ligand type. To more dramatically show the different effects of ligand on AR distribution, we utilized an AR with a mutation in the DNA binding domain (ARC619Y) that forms distinct foci upon exposure to agonists but retains a diffuse nuclear distribution in the presence of antagonists. Live-cell imaging of this mutant demonstrated that cytoplasmic foci formation occurs immediately upon agonist but not antagonist addition. Fluorescence recovery after photobleaching (FRAP) revealed that agonist-bound GFP-AR exhibited reduced mobility relative to unliganded or antagonist-bound GFP-AR. Importantly, agonist-bound GFP-AR mobility was strongly affected by protein expression levels in transiently transfected cells, and displayed reduced mobility even in slightly overexpressing cells. Cyan fluorescent protein-AR (CFP-AR) and yellow fluorescent protein-CREB binding protein (YFP-CBP) in the presence of agonists and antagonists were used to demonstrate that CFP-AR specifically co-localizes with YFP-CBP in an agonist dependent manner. Dual FRAP experiments demonstrated that CBP mobility mirrored AR mobility only in the presence of agonist. HTM enabled simultaneous studies of the sub-cellular distribution of GFP-AR and ARC619Y in response to a range of concentrations of agonists and antagonists (ranging from 10(-12) to 10(-5)) in thousands of cells. These results further support the notion that ligand specific interactions rapidly affect receptor and co-factor organization, solubility, and molecular dynamics, and each can be aberrantly affected by mutation and overexpression.
It is known that Fas death domain-associated protein (Daxx) possesses both putative nuclear and cytoplasmic functions. However, the nuclear transport mechanism is largely unknown. This study examined the nuclear location signal (NLS) of Daxx and whether the nuclear transport of Daxx was mediated by small ubiquitin-related modifier (SUMO). Two NLS motifs of Daxx, leucine (L)-rich nuclear export signal (NES)-like motif (188IXXLXXLLXL197) and C-terminal lysine (K) rich NLS2 (amino acids 627-634) motif, were identified and the K630 and K631 on the NLS2 motif were characterized as the major sumoylation sites of Daxx by in vitro sumoylation analysis. Proteins of inactive SUMO (SUMO-delta), a sumoylation-incompetent mutant, and Daxx NLS mutants (Daxx-NES(mut) and Daxx NLS2(mut)) were dispersed in cytoplasm. The cytoplasmic dispersed Daxx mutants could be relocalized to nucleus by cotransfection with active SUMO, but not with inactive SUMO-delta, demonstrating the role of SUMO on regulating the cytoplasmonuclear transport of Daxx. However, inactive SUMO-delta could also be relocalized to nucleus during cotransfection with wild-type Daxx, suggesting that SUMO regulation of the cytoplasmonuclear transport of its target protein Daxx does not need covalent modification. This study shows that cytoplasmic SUMO has a biological role in enhancing the cytoplasmonuclear transport of its target protein Daxx and it may be done through the non-sumoylation interactions.