Translation of the radioresistance kinase TLK1B is induced by gamma-irradiation through activation of mTOR and phosphorylation of 4E-BP1.
BACKGROUND
The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila, or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and chromosome missegregation probably due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases their resistance to ionizing radiation, also likely through changes in chromatin remodeling. The TLK1B mRNA is translationally repressed by its 5'UTR and is regulated by the availability of eIF4E. We now report that radiation or doxorubicin result in an increase in the translation of TLK1B, and we have uncovered the likely mechanism for this effect.


RESULTS
Radiation causes a shift in the polysomal distribution of TLK1B mRNA, from the untranslated region and small polysomes to the large polysomes, concomitant with an increase in the expression of TLK1B protein. This change is preceded by an increase in phosphorylation of the eIF4E inhibitory protein 4E-BP1, which releases eIF4E when it is phosphorylated. The phosphorylation of 4E-BP1 depends on mTOR, since rapamycin blocked the increase in phosphorylation induced by radiation, and prevented the increase in TLK1B protein expression. The activation of mTOR was likely due to the rapid activation of Akt following radiation. The activation of Akt could be inhibited with wortmannin, an inhibitor of PI3 kinase, hence placing PI3 kinase upstream of Akt as a very early event following radiation. Wortmannin also inhibited translation of TLK1B mRNA following activation by IR. This was shown both by western blot and by measuring the initiation capacity of the mRNA, as indicated by its distribution on polysomes.


CONCLUSIONS
The translational upregulation of TLK1B elicited by DNA double strand breaks represents an interesting mechanism of translational regulation of a protein involved in radioprotection and highlights a novel mechanism of the stress response following radiation.
Sunavala-Dossabhoy
Gulshan
G
Department of Biochemistry and Molecular Biology and the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA. gsunav@lsuhsc.edu
Fowler
Marcie
M
De Benedetti
Arrigo
A
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D004249:DNA Damage; D039642:Eukaryotic Initiation Factors; D005720:Gamma Rays; D005786:Gene Expression Regulation; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011132:Polyribosomes / Q000235:genetics; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D011836:Radiation Tolerance; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; D039642:Eukaryotic Initiation Factors; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011494:Protein Kinases; C498929:Tlk1 protein, mouse; C546843:mTOR protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus; D000077191:Wortmannin