Mammalian homologues of the plant Tousled gene code for cell-cycle-regulated kinases with maximal activities linked to ongoing DNA replication

Abstract

The Tousled (TSL) gene of the plant Arabidopsis thaliana encodes a serine/threonine kinase that is essential for proper flower development. Here we report the cloning and characterization of two human putative homologues of the Arabidopsis TSL gene, termed TLK1 and TLK2 (Tousled-like kinase). At the protein level, the two human Tlks share 84% sequence similarity with each other and almost 50% with Arabidopsis Tsl. Furthermore, nuclear localization signals and predicted coiled-coil regions are conserved in the N-terminal domains of all three kinases. The mammalian Tlks share several functional properties with plant Tsl, including a broad expression, a propensity to dimerize and autophosphorylate, and a preference for similar substrates. Most interestingly, human Tlks are cellcycle-regulated enzymes, displaying maximal activities during S phase. Whereas protein levels are virtually constant throughout the cell cycle, both Tlks appear to be regulated by cell-cycle-dependent phosphorylation. Drug-induced inhibition of DNA replication causes a rapid loss of Tlk activity, indicating that Tlk function is tightly linked to ongoing DNA replication. These findings provide the first biochemical clues as to the possible molecular functions of Tlks, a highly conserved family of kinases implicated in the development of multicellular organisms.

The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1

“…Cells were washed with ice cold PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and resuspended in ice cold HEPES lysis buffer (50 mM HEPES pH7 .4, 150 mM NaCl, 0.5% Triton X-100) containing 30 mg/ml RNase A, 30 mg/ml DNase and protease and phosphatase inhibitors (Sillje et al, 1999). After 15 min on ice, cells were collected by scraping and centrifuged at 13 000 r.p.m.…”

Exposure to acute hypoxia induces a transient DNA damage response which includes Chk1 and TLK1

“…Mammalian TLK1 was identified in 1999 and was shown to be maximally active in S-phase leading to the hypothesis that TLK1 has a role in cell cycle progression. 34 More recently TLK1 has been reported to be involved in both the processing of newly replicated DNA and chromatin formation. The kinase activity of TLK1 decreases in response to genotoxic stress, such as ionising radiation or treatment with hydroxyurea.…”

Mitotic crisis: The unmasking of a novel role for RPA

“…Both Tousled-like kinases Tlk1 and Tlk2, 25 were detected, of interest because these ubiquitous kinases become activated during DNA replication in human cells and are rapidly inhibited in an ATM-and Chk1-dependent manner following DNA damage. [26][27][28] Significantly, Tlk1 also promotes repair of DSBs and UV-induced damage, perhaps by stimulating histone removal at the site of DNA damage. [29][30][31][32][33] That the Tlk1-Plk1 association is relevant is supported by the finding that Tlk1 is important for proper chromosome segregation during mitosis.…”