The checkpoint kinase Chk2 is an important component of the DNA damage machinery, mediating cell cycle arrests at multiple checkpoints and apoptosis (8
). After DNA damage, ATM phosphorylates Chk2 on T68 (39
), promoting Chk2 oligomerization through the interaction of the amino-terminal SCD of one molecule with the FHA domain of another molecule (3
) as well as the autophosphorylation at T383 and T387 in the activation loop (5
), for full activation of Chk2. Phosphorylation of T68, a necessary but not sufficient step for Chk2 activation, is not required for Chk2 autophosphorylation (47
) (Fig. ), and amino acid substitutions at T68 do not abolish the kinase activity (56
) or the DNA damage-induced association with chromatin (34
We have identified three serine phosphoresidues (S19, S33, and S35) located in the amino-terminal SQ/TQ-rich region of Chk2 that provides a consensus motif for phosphorylation by ATM. In vivo phosphorylation of these serine residues in normal cells was rapidly induced in response to IR doses of >1 Gy (generating >19 DSBs/cell), in contrast to the phosphorylation of T68 occurring in response to much lower doses of IR (~0.25 Gy, estimated to generate <8 DSBs/cell). Interestingly, Chk2 autophosphorylation at T387 occurred at a later time point (45 to 90 min), when phosphorylation levels of T68, S19, and S33/S35 decreased markedly, suggesting that these residues serve as a trigger for Chk2 activation. Given that the proteasome inhibitor ALLN did not prevent the decline in T68, S19, and S33/S35 phosphorylation levels, our results are consistent with dephosphorylation rather than proteolytic degradation to account for this phenomenon. In addition, the SCD appears to have a dual function, i.e., to initially promote Chk2 activation through SCD phosphorylation and to limit the amplification of the number of activated Chk2 molecules through its dephosphorylation.
Phosphorylation of S19 and S33/S35 was normal in an T68A mutant, indicating the independence of these events. Conversely, phosphorylation of S19 and S33/S35 was markedly attenuated in Chk2-KD, possibly reflecting a hindering effect played by Chk2 oligomerization rather than catalytic loss, as demonstrated by the efficient phosphorylation of these residues in Chk2-KD-FHAΔ
, a mutant form of Chk2 that is unable to oligomerize because of the deletion of the FHA domain (5
The rapidity of S19 and S33/S35 phosphorylation is consistent with a direct activity of ATM on these residues. Indeed, the absence of S19 and S33/S35 phosphorylation in two NBS cell lines lacking full-length Nbs1 protein expression, together with the impaired activity of ATM towards its substrates (14
), further supports this contention. The partial phosphorylation of T68 in NBS cells might thus be explained by the differential affinity of ATM, much greater for T68 than for S19 and S33/S35 (39
). Therefore, the ability of Nbs1 to increase ATM affinity for its substrate (43
) should be dispensable for T68 phosphorylation and indispensable for S19 and S33/S35 phosphorylation. Such a model is also supported by the lack of S19 and S33/S35 phosphorylation in response to IR doses of <1 Gy which nevertheless vigorously activate ATM (7
). The role of ATM in S19 and S33/S35 phosphorylation was also highlighted by the fact that neither 4-NQO nor HU treatment affected these residues. Conversely, T68 was phosphorylated by 4-NQO and HU, although in an ATM-independent and ATR-dependent manner, again differentiating between these phosphorylation events. Our results with an ATR-defective Seckel cell line and ATR-silenced cells excluded an involvement of ATR in the IR-induced phosphorylation of S19 and S33/S35.
We evaluated the molecular roles of S19 and S33/S35 in MCF7 cells, characterized by a normal ATM-Chk2- and p53-dependent DNA damage response, and in HCT15 cells, which lack endogenous Chk2, stably transfected with single (S19A), double (S33A S35A) or triple (S19A S33A S35A) Chk2 mutants. In these cells, the overall nuclear distribution of Chk2 and relocalization of phospho-T68 in IR-induced foci was unaffected by Ser-to-Ala mutations in S19/S33/S35 (see Fig. S4 in the supplemental material). In vitro kinase assays revealed deficient autophosphorylation and trans
phosphorylation catalytic activities by Chk2S19A
, and Chk2S3A
, thus implicating these serine residues in the activation of Chk2. Size fractionation analysis of cell extracts from transiently transfected HCT15 cells revealed a marked decrease in the amount of mutant Chk2 in the fractions corresponding to 161 to 238 kDa, suggesting that the Ser-to-Ala substitutions impair Chk2 dimerizations. This observation was confirmed by a direct analysis of the dimerization ability of Chk2, obtained by using two different tagged forms of the protein, and is in accordance with the model proposed for its activation recently supported by crystallographic analysis (42
Mdmx, an important regulator of p53, was recently shown to be phosphorylated by Chk2 on Ser367 after DNA damage, causing ubiquitination and degradation of Mdmx and activation of p53 (17
). We have shown that in cells with stably silenced endogenous Chk2, ectopic Chk2wt
restored the IR-induced degradation of Hdmx, whereas Chk2S3A
had a partial effect, suggesting a role for S19 and S33/S35 in the Chk2/Hdmx pathway, leading to p53 accumulation and apoptosis in response to a high level of DNA damage.
Analysis of Chk2 in relation to the cell cycle revealed that in response to radiation, T68 became phosphorylated in G1
, S, and G2
/M, whereas S19 and S33/S35, and to a lower extent T387, became phosphorylated in G1
phase only. This event was observed not only in lymphoblastoid cells but also in hTERT-immortalized epithelial cells. One mechanism to account for this cell cycle-restricted phosphorylation might depend on the activity of a kinase that specifically targets (in an ATM-dependent manner) these residues in the G1
phase, as in the case of other target substrates of phosphatidylinositol 3-kinase in yeast (49
) and mammalian cells (20
). Alternatively, a phosphatase(s) might selectively dephosphorylate S19 and S33/S35 in S and G2
/M, assuming that DNA damage induces phosphorylation of these residues in all phases of the cell cycle. However, preliminary results with phosphatase inhibitors, e.g., calyculin A or orthovanadate (data not shown), would argue against the latter possibility.
Whatever the underlying mechanism, the biological significance of the G1
phase-restricted phosphorylation of S19 and S33/S35 remains unclear, given that the radiation-induced Chk2 kinase activity, though maximal in G1
phase cells (4.3-fold), was also seen in G2
/M phase cells (2.6-fold) and to a lower extent in S-phase (2.1-fold) cells. This would be compatible with Chk2 playing a major role in G1
/S checkpoint arrest in the presence of a high level of DNA damage and with the restoration of the G1
/S checkpoint in Chk2 null cells (Fig. ) (23
) by the wild-type Chk2, but not by the S3A mutant Chk2. It is possible that this phosphorylated form of Chk2, occurring in response to >18 DSBs/cell, might provide a more effective and sustained G1
arrest to prevent replication of cells with damaged DNA that reenter G1
after escaping mitotic checkpoint arrest.
Finally, we showed that ectopic expression of wild-type Chk2 in the absence of de novo DNA damage has an antiproliferative effect, possibly reflecting a constitutive trans
autophosphorylation and activation of Chk2 when overexpressed (47
) and, in turn, an increased apoptotic cell death and senescence, as recently reported (16
). Conversely, the Chk2S3A
mutant lacked the antiproliferative activity of Chk2wt
, underscoring the biological role of S19 and S33/S35 phosphoresidues in Chk2 activity.
Together, our data shed light on several aspects of the intricate and tightly coordinated phosphorylation events leading to the ATM-dependent functional activation of Chk2 kinase. Finally, the differential phosphorylation of Chk2 at multiple residues as a function of the IR dose (T68 is responsive to even 0.25 Gy, estimated to generate <8 DSBs/cell, whereas S19 and S33/S35 are responsive to >1 Gy, estimated to generate >19 DSBs/cell [12
]) might represent a mechanism by which ATM fine-tunes Chk2's pleiotropic responses to increasing numbers of genotoxic lesions.