Chk2 is a critical mediator of damage-dependent ATM/ATR signaling, and the T-loop exchange region within the kinase domain plays an essential role in Chk2 activation (2
). DNA damage-induced activation of Chk2 initiates an intramolecular cascade of phosphorylation events. The initial signaling events are phosphorylation of residues in the SQ/TQ domain that appear to facilitate dimerization of two Chk2 molecules. The dimerization is required for trans-autophosphorylation of Thr383
within the activation T-loop exchange region, an event needed for full activation of kinase function. Chk2 thus activated in turn phosphorylates and activates a variety of target proteins involved in DNA repair and cell cycle regulation. Although specific phosphatases have been identified that dephosphorylate Chk2 at Thr68
), the independence of Thr383
phosphorylation from Thr68
phosphorylation status suggests that other mechanisms exist to fine tune the kinase activation response (22
The dimerization of Chk2 is mediated by direct interaction of the SQ/TQ domain of one molecule with the FHA domain of an adjacent molecule. This arrangement facilitates the trans-autophosphorylation of Thr383
on one molecule pair and Thr387
on the second molecule pair. The crystal structure of the Chk2 kinase domain in an activated state was recently solved (23
) and revealed some physical detail of the T-loop exchange region. The crystal structure established that the T-loop consists of two α helices bordering a structured loop. Notably, residue Thr383
is within the “left-hand” helix (residues 377–386), whereas residue Thr387
lies within the more relaxed loop segment (residues 387–392). The positioning of Thr383
in structurally diverse regions suggests that the physical constraints upon Thr383
are different from those of Thr387
with the later being more flexible. Our study showed that T > D mimetic substitution of Thr387
could assume a structure recognized by a phospho-Thr387
-specific Chk2 antibody, whereas T > D substitution at Thr383
failed to do so. In addition, the T387D mutant displayed kinase activity, whereas the T383D mutant was inactive. The entire loop segment, at least in the activated state, projects into a hydrophobic pouch formed by the second Chk2 molecule. Thus, our observation of a functional dependence upon phosphorylation at Ser389
is consistent with the predicted steric impact of introduction of a phosphate group into a hydrophobic pocket.
The interdependence of Thr383
is assumed in fulfillment of an autophosphorylation model and has been demonstrated experimentally (6
). In our analysis we could confirm the dependence of phosphorylation at Thr387
on co-phosphorylation at Thr383
. Interestingly, although we were not able to detect any instance of a single phosphorylation at Thr387
, we did detect peptides with single phosphorylation at Thr383
. This would suggest that phosphorylation at Thr383
precedes phosphorylation at Thr387
. In fact our data showed evidence of singular phosphorylation events at all examined T-loop region sites (Thr372
, and Tyr390
) except for Thr387
, suggesting a unique structural prerequisite for Thr387
Unique among all the residues examined in our study, Thr389 was the only phosphorylation event associated with a reduction in activity. Specifically, inhibition of phosphorylation at Thr389 via T > A substitution resulted in a dramatic increase in the kinase activity of Chk2. On the other hand introduction of a phosphomimetic substitution T > D at Thr389 reduced measureable kinase activity to levels equal to wild-type protein. Our findings also support a model in which phosphorylation of Thr387 is dependent upon phosphorylation at Thr389, because the T > A substitution at Thr389 abolished recognition of Chk2 by a phospho-Thr387-specific antibody. Conversely, the T > D mimetic substitution at Thr389 restored recognition of phospho-Thr387 Chk2. Thus, phosphorylation at Thr389 appears to be a prerequisite to phosphorylation at Thr387 but associated with reduced kinase function (albeit at wild-type protein levels).
One unique aspect to our study is the ability to acquire quantitative information on the relative abundance of phosphorylation at specific sites in response to exposure to IR. In addition, we could simultaneously obtain insight into the relative distribution of specific phosphorylation events between nuclear and chromatin compartments. This allows us to relate phosphorylation events to protein localization. We observed relative changes in abundance indicating IR-induced de novo phosphorylation and IR-induced loss of phosphorylation. Likewise we determined relative changes in the localization of Chk2 species consistent with chromatin targeting and chromatin egress. These results suggest a complex interrelationship between phosphorylation status of multiple T-loop sites and chk2 localization.
We could document a de novo increase in phosphorylation at Ser379, Thr389, and Thr383/Thr389 following exposure to IR. We also observed a modest de novo increase in single phosphorylation species at Thr383 following IR. There was a notable increase in Thr383 single phosphorylation species in the chromatin fraction following IR, but this was compensated for by a loss in the nuclear abundance of the same species. We failed to detect the existence of an independent phosphorylation at Thr387 under any conditions. These results suggest that phosphorylation at Thr387 is dependent upon prior phosphorylation. Our findings also suggest that Thr389 phosphorylation is an IR-induced de novo event and a prerequisite for Thr387 phosphorylation. It may be that the Thr383/Thr389 double phosphorylation species represents the initiating IR-dependent event that precedes phosphorylation at Thr387. Phosphorylation at Thr378, which had no impact upon kinase function, was dramatically reduced in the chromatin fraction following IR treatment. The tyrosine phosphorylation at Tyr390 was associated with IR-induced loss in Thr383/Tyr390, Thr387/Tyr390, and Thr389/Tyr390. We conclude that Thr378 and Tyr390 might be involved in an IR-induced dephosphorylation event or serve as potential signals for recovery of activated Chk2 into a naïve state. Further studies are required to evaluate these two hypotheses.
Chromatin targeting events post-IR were presumed to be phosphorylation changes associated with recruitment of Chk2 to sites of damage as a result of DNA damage response initiation. We observed phosphorylation at Thr372, Ser379, Thr383, and Thr389 to be associated with chromatin targeting after IR. We also observed double phosphorylation at Thr378/Ser379, Thr383/Thr387, and Thr383/Thr389 to be associated with chromatin targeting. In some cases the IR-induced increases in chromatin presence of these phosphorylated forms of Chk2 were reciprocal with the same fold decrease in the nucleus. This result is supportive of an IR-induced exchange and was noted for phosphorylation at Thr372, Thr383, and Thr383/Thr387. In other cases the increase in chromatin-localized Chk2 was associated with a de novo increase in specific phosphorylated forms, suggesting an IR-induced phosphorylation. This was observed for phosphorylation at Ser379, Thr389, Thr378/Ser379, and Thr383/Thr389. These results imply that de novo phosphorylation at these sites is a requisite to chromatin targeting.
Chromatin egress is an observed localization change in Chk2 associated with normal DNA damage response (24
). It is speculated that egress may regulate downstream signaling, and the movement of Chk2 from sites of damage to the nucleoplasm is consistent with cellular target activation by Chk2. We defined chromatin egress as IR-induced reduction in the chromatin/nucleus ratio in the absence of reduced nuclear abundance of the same species. We observed chromatin egress of Chk2 to be associated with phosphorylation at Tyr390
, and Thr383
. A reciprocal exchange was noted for Tyr390
. Overall it seems that hyperphosphorylation of the 383–406 peptide was associated with chromatin egress. For some phosphorylation species, such as Thr387
, and Thr387
, the relative distribution and abundance did not change in response to IR. Thus, the functional significance of these events in IR-mediated signaling could not be established.
The critical role of ubiquitylation in regulating the molecular events that comprise cellular DNA damage response has recently come to light (25
). The observation that Chk2 function is dependent upon ubiquitylation suggests a possible role for this modification in regulating subcellular localization and/or protein turnover. We confirmed the requirement of phosphorylation at Ser379
and also showed that phosphorylation at Thr383
is required for Chk2 ubiquitylation. Because phosphorylation at Thr383
is required for kinase activation the dependence of ubiquitylation upon Thr383
phosphorylation may link this modification to kinase activity. Interestingly, we observed that the kinase inactive mutant Y390F was ubiquitylated. However, in contrast to either Ser379
, phosphorylation at Tyr390
was almost always reduced in response to IR. Thus, ubiquitylation in response to IR can be maintained in the presence of altered kinase activity. The full implication of the relationship between phosphorylation, ubiquitylation, and chromatin targeting/egress needs further study.
Our data suggests that IR induces de novo phosphorylation at Ser379, Thr389, and Thr383/Thr389. Of these events we suggest that the Thr383/Thr389 would be a prerequisite for phosphorylation at Thr387 and signal formation of oligomeric activated Chk2. The formation of the Chk2 species phosphorylated at Thr383/Thr387/Thr389 then signals chromatin egress of activated Chk2. The interplay between these three residues provides a mechanism for activation and egress of protein. Later events such as phosphorylation at Tyr390 may signal recycling of Chk2 from active to naïve state (). We confirm the interdependence of Thr383 and Thr387 and underscore the dependence of Thr387 phosphorylation on prior phosphorylation at both Thr383 and Thr389. These results support a model in which multiple interdependent phosphorylation events within the Chk2 T-loop region regulate chromatin targeting, kinase activation, chromatin egress, and recycling.
FIGURE 10. Interdependent phosphorylation within the activation loop mediates damage-induced activation and subcellular redistribution of Chk2. Our model suggests that initial phosphorylation at Thr383 in response to IR results in localization of Chk2 to chromatin. (more ...)