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PAI‐RBP is a Negative Regulator of DNA‐Repair Proteins
Author(s) -
Zang Heesuk,
Heaton Joanne,
Howlett Niall,
Wittekindt Owen,
Gelehrter Thomas
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a911-d
Subject(s) - aphidicolin , biology , microbiology and biotechnology , transfection , staurosporine , phosphorylation , gene isoform , cell cycle , protein kinase a , apoptosis , gene , biochemistry
PAI‐RBP1 was originally identified as an RNA‐binding protein that interacts with the 3′UTR of the PAI‐1 mRNA. The protein has four isoforms. We found that HeLa cells stably transfected with one of the PAI‐RBP isoforms, PAI‐RBP4, showed increase in both spontaneous and aphidicolin‐induced chromosomal gaps and breaks. Western blot revealed that the overexpression of PAI‐RBP4 led to the down‐regulation of ATM and Chk1. Interestingly, mutation of PAI‐RBP4 on Ser74, a predicted target of PKA phosphorylation, abolished its ability to down‐regulate both kinases. Overexpression of another isoform, PAI‐RBP1, however, resulted in the down‐regulation of p53, ATR, and Chk2, which were not affected by PAI‐RBP4 expression. PAI‐RBP4 is a cytoplasmic protein. However, in response to mitomycin C, hydroxyurea, aphidicolin and UV irradiation, the PAI‐RBP4 protein translocates to the nucleus. Furthermore, aphidicolin could induce increased phosphorylation of RPA2 in HeLa cells stably transfected with PAI‐RBP4. In vitro DNA binding experiments indicated that PAI‐RBP4 could also bind to single‐stranded DNA in a sequence‐independent manner. We also examined cell growth, apoptosis, and the overall rate of translation. Cells expressing high level of PAI‐RBP4 showed decreased translation rate, decreased cell number at confluence and increased cell death, as examined by TUNEL assay. We found that PAI‐RBP4 reduced the level of eIF4E. In contrast to PAI‐RBP4's effect on eIF4E, PAI‐RBP1 increased the level of eIF4E. We have also found that PAI‐RBP4 down‐regulated 4E‐BP1, but not mTOR, or the S6 kinase. Taken together, these results suggest that PAI‐RBP isoforms may exhibit splicing variant‐specific effects on enzymes involved in the DNA‐damage responses as well as the eIF4E‐dependent translation machinery.