A mechanism of nucleocytoplasmic trafficking for the homeodomain protein PRH.
Proline-rich homeodomain (PRH)/hematopoietically expressed homeodomain (Hex) is a homeodomain protein that plays an important role in early embryonic patterning and hematopoiesis. PRH can act as either a tumor suppressor or an oncogene and its expression is dysregulated in certain types of lymphoid and myeloid leukemias. Aberrant exclusion of PRH from the nuclei has been associated with thyroid and breast cancers and a subset of myeloid leukemias. Accordingly, nuclear localization of PRH was found to be necessary for the inhibition of eIF4E-dependent transformation. Since PRH's nuclear-cytoplasmic localization has been associated with neoplastic transformation we sought to better understand how PRH is transported to the nuclear compartment. Here, we report an essential element that controls the mechanism of PRH nucleocytoplasmic trafficking, namely that it is imported into the nuclei by Karyopherin/Importin 7. Kap7 was identified as a binding partner for PRH in a GST-pull down from a HeLa cell protein lysate, followed by mass-spectrometry. The Kap7-PRH complex is dissociated in the presence of RanGTP, as expected for a nuclear import complex. Kap7 can bind directly to PRH in a GST-pull down assay with purified proteins, as well as mediates the transport of PRH to the nuclear compartment in a digitonin permeabilized cells assay. Finally, in vivo depletion of Kap7 dramatically reduces accumulation of PRH in the nucleus. Our data open the way for investigations of the mechanism of perturbed PRH localization in tumors and possible therapeutic interventions.
Ploski
Jonathan E
JE
Department of Psychology, Yale University, New Haven, CT, USA.
Topisirovic
Ivan
I
Park
Kevin W
KW
Borden
Katherine L B
KL
Radu
Aurelian
A
D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D056945:Hep G2 Cells; D018398:Homeodomain Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
C078492:HHEX protein, human; D018398:Homeodomain Proteins; D028884:Karyopherins; D009687:Nuclear Proteins; D010446:Peptide Fragments; C120587:RAN protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; C091775:ran-binding protein 1; D020931:ran GTP-Binding Protein