A dominant negative mutant of TLK1 causes chromosome missegregation and aneuploidy in normal breast epithelial cells.
BACKGROUND
In Arabidopsis thaliana, the gene Tousled encodes a protein kinase of unknown function, but mutations in the gene lead to flowering and leaf morphology defects. We have recently cloned a mammalian Tousled-Like Kinase (TLK1B) and found that it phosphorylates specifically histone H3, in vitro and in vivo. We now report the effects that overexpression of a kinase-dead mutant of TLK1B mediates in a normal diploid cell line.


RESULTS
Expression of a kinase-dead mutant resulted in reduction of phosphorylated histone H3, which could have consequences in mitotic segregation of chromosomes. When analyzed by FACS and microscopy, these cells displayed high chromosome number instability and aneuploidy. This phenomenon was accompanied by less condensed chromosomes at mitosis; failure of a number of chromosomes to align properly on the metaphase plate; failure of some chromosomes to attach to microtubules; and the occasional presentation of two bipolar spindles. We also used a different method (siRNA) to reduce the level of endogenous TLK1, but in this case, the main result was a strong block of cell cycle progression suggesting that TLK1 may also play a role in progression from G1. This block in S phase progression could also offer a different explanation of some of the later mitotic defects.


CONCLUSIONS
TLK1 has a function important for proper chromosome segregation and maintenance of diploid cells at mitosis in mammalian cells that could be mediated by reduced phosphorylation of histone H3 and condensation of chromosomes, although other explanations to the phenotype are possible.
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
Gulshan
G
Sunavala-Dossabhoy
Yuan
Y
Li
Briana
B
Williams
Arrigo
A
De Benedetti
D000782:Aneuploidy; D000818:Animals; D064127:Aurora Kinases; D001940:Breast / Q000166:cytology / Q000201:enzymology*; D002460:Cell Line; D043171:Chromosomal Instability; D020090:Chromosome Segregation; D002875:Chromosomes / Q000648:ultrastructure; D004847:Epithelial Cells / Q000201:enzymology / Q000528:radiation effects / Q000648:ultrastructure; D005260:Female; D016193:G1 Phase; D005720:Gamma Rays; D006657:Histones / Q000378:metabolism; D051379:Mice; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D006657:Histones; D034741:RNA, Small Interfering; C498929:Tlk1 protein, mouse; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases