Menin localizes to chromatin through an ATR-CHK1 mediated pathway after UV-induced DNA damage.
BACKGROUND
Menin is the tumor suppressor protein product of the gene identified in MEN1 syndrome. Evidence suggests menin binds DNA and interacts with proteins implicated in DNA damage pathways. The canonical cellular response to UV-induced DNA damage involves activation of the ataxia-telangiectasia-mutated and Rad3-related (ATR) kinase pathway.


METHODS
HEK293 cells were irradiated in a UV chamber. Menin's cellular location before and after UV irradiation was investigated by extracting four separate cellular components--a soluble, two chromatin and a nuclear matrix. To block the ATR pathway, we treated with 5 microM of caffeine for 1 h before irradiation. The ATR pathway was further investigated by transiently transfecting HEK293 cells with two mammalian CHK1 expression constructs--full length CHK1 and truncated active CHK1.


RESULTS
A 24-h post UV-irradiation time course was studied and demonstrated menin concentration in the chromatin peaked at 4 h. At 4 h post-irradiation, menin concentration in the chromatin increased in a dose dependent manner and demonstrated a 2.8-fold maximal increase. HEK293 cells were pretreated with caffeine, an inhibitor of the ATR. Caffeine decreased menin localization to the chromatin after UV. Constitutively active CHK1 (1-365) transfection increased chromatin-bound menin, mimicking UV irradiation.


CONCLUSIONS
Menin localizes to the chromatin after UV irradiation. Caffeine blocks menin localization to the chromatin after UV-irradiation. Over expressing active CHK1 (1-365) increased chromatin-bound menin, similar to UV. The data suggest menin localization to chromatin after UV irradiation is the result of an ATR-CHK1 dependent pathway.
Farley
Steven M
SM
Department of Surgery, Greater Los Angeles Healthcare System, Wadsworth VA, West Los Angeles, California, USA. sfarley@mednet.ucla.edu
Chen
Gao
G
Guo
Sydney
S
Wang
Min
M
A
Jingbo
J
Lee
Felix
F
Lee
Frank
F
Sawicki
Mark
M
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology*; D006801:Humans; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015398:Signal Transduction; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D002843:Chromatin; C105373:MEN1 protein, human; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases