Differential Mode of Regulation of the Checkpoint Kinases CHK1 and CHK2 by Their Regulatory Domains

Abstract

CHK1 and CHK2 are key mediators that link the machineries that monitor DNA integrity to components of the cell cycle engine. Despite the similarity and potential redundancy in their functions, CHK1 and CHK2 are unrelated protein kinases, each having a distinctive regulatory domain. Here we compare how the regulatory domains of human CHK1 and CHK2 modulate the respective kinase activities. Recombinant CHK1 has only low basal activity when expressed in cultured cells. Surprisingly, disruption of the C-terminal regulatory domain activates CHK1 even in the absence of stress. Unlike the full-length protein, C-terminally truncated CHK1 displays autophosphorylation, phosphorylates CDC25C on Ser 216 , and delays cell cycle progression. Intriguingly, enzymatic activity decreases when the entire regulatory domain is removed, suggesting that the regulatory domain contains both inhibitory and stimulatory elements. Conversely, the kinase domain suppresses Ser 345 phosphorylation, a major ATM/ATR phosphorylation site in the regulatory domain. In marked contrast, CHK2 expressed in either mammalian cells or in bacteria is already active as a kinase against itself and CDC25C and can delay cell cycle progression. Unlike CHK1, disruption of the regulatory domain of CHK2 abolishes its kinase activity. Moreover, the regulatory domain of CHK2, but not that of CHK1, can oligomerize. Finally, CHK1 but not CHK2 is phosphorylated during the spindle assembly checkpoint, which correlates with the inhibition of the kinase. The mitotic phosphorylation of CHK1 requires the regulatory domain, does not involve Ser 345 , and is independent on ATM. Collectively, these data reveal the very different mode of regulation between CHK1 and CHK2.

C-terminal domain of Chk1 regulates its subcellular location and kinase activity for DNA repair

“…Further truncations of the C-terminus were made to characterize the regulatory regions. C309, missing the 167 C-terminal amino acids (309-476), still displayed some autophosphorylation activity, while C284, which lacks the 192 C-terminal amino acids (284-476) containing the entire SQ/TQ domain, displayed no autophosphorylation [5]. We, therefore, hypothesize that the C-terminal regulatory domain plays an important role in modulating the kinase domain.…”

“…Ng et al [5] incubated full-length Chk1 and a truncated mutant, C390, which lacked the 86 C-terminal amino acids (390-476), with radioactive ATP and found that full length Chk1 was only weakly radiolabeled, while C390 was robustly autophosphorylated. To measure the kinase activity of Chk1 against external substrates, a GST-Cdc25C 195-259 fusion protein containing the Chk1 phosphorylation site (Ser216) was used.…”

“…Restriction enzymes were purchased from Promega (Promega, Madison, WI). Oligo(dT) 5 and Trizol RNA were purchased from Invitrogen (Invitrogen, Carlsbad, California). DMEM was purchased from GIBCO (GIBCO BRL, Invitrogen, USA).…”

“…In yeast and mammalian cells Chk1 is located both in the cytoplasm and nuclei before DNA damage, but it accumulates in nuclei after DNA damage, indicating a distinct change in subcellular location [22]. Recombinant Chk1, expressed in cultured cells, displayed only basal enzymatic activity [5]. Unlike the full-length protein, C-terminus-truncated Chk1 leads to autophosphorylation and phosphorylation of Cdc25C at Ser216.…”

“…Chk1 acts downstream of Atmlike proteins in yeasts and higher eukaryotes and regulates the checkpoint response that prevents chromosome segregation in the presence of DNA damage. Chk1 is a key mediator linking the mechanisms that monitor DNA integrity to components of the cell cycle engine [5]. Chk1 participates in the regulation of the cell cycle process, monitoring the integrity of genomic DNA and driving the repair of damaged genomic DNA.…”