Cell cycle checkpoints activated in response to genotoxic stress prevent replication of damaged DNA presumably to allow time for repair or apoptosis. As loss of these checkpoints results in cell transformation or death, checkpoints are vital in preserving genomic integrity and protecting cells from a variety of adverse environmental conditions. Here, we present evidence that hSMG-1 and ATM, but not ATR, control p53 phosphorylation and abundance (). Through the use of ATM
-deficient lymphoblasts and siRNA knockdown in epithelial cells, we found that hSMG-1 initiates p53 phosphorylation during hyperoxia while ATM helps to maintain phosphorylation over time. While p53 in turn stimulates p21 and executes the G1 checkpoint, we surprisingly discovered ATM, hSMG-1 and to a lesser extent ATR also control p21 stability. Thus, p21 abundance over time is regulated by PIKKs controlling both p53-dependent synthesis and p53-independent proteolysis of p21. P21 is a critical mediator of cellular responses to DNA damage as low levels are required to inhibit cell growth while higher levels are required to protect against hyperoxia-induced cell death (Vitiello et al., 2006
). As such, the dual functions of PIKKs to control both synthesis and proteolysis of p21 may allow levels of p21 and cell fate to be tightly coordinated with the extent of DNA damage.
Model of p21-dependent cell cycle arrest during oxidative stress
Using A549 cells and ATM
(+/+) and ATM
(−/−) lymphoblasts, we made the novel finding that hSMG-1 mediated phosphorylation of p53 on serine15 occurs prior to ATM-dependent phosphorylation. Moreover, early hSMG-1 signaling in hyperoxia was not necessary to activate ATM signaling at later times. hSMG-1 was previously shown to regulate p53 (Ser15) phosphorylation in response to ionizing radiation (Brumbaugh et al., 2004
). The current study extends the role of hSMG-1 by showing it to be a proximal mediator of checkpoint signaling in response to chronic oxidative stress. The rapid activation of hSMG-1 over ATM appears to be unique to hyperoxia as both hSMG-1 and ATM were required to phosphorylate p53 in response to low dose (19cGy) ionizing radiation (data not shown).
Sequential activation of hSMG-1 and ATM may be due to the chronic and progressive nature of hyperoxia as a damaging agent. Differences in spatiotemporal localization of substrates could account for the differences in hSMG-1 and ATM activation. For instance, ATM physically interacts with damaged DNA in a complex containing Mre11, Rad50 and Nbs1 (MRN complex) (Abraham & Tibbetts, 2005
; Lee & Paull, 2005
; Smith et al., 1999
). Thus, signals to recruit and phosphorylate p53 could take longer using a slow progressive model of damage like hyperoxia relative to a bolus of ionizing radiation (Abraham & Tibbetts, 2005
). The dynamics of hSMG-1 localization in response to damage have also not been elucidated, so it is possible that hSMG-1 could diffuse more readily to DNA lesions. Alternatively, hSMG-1 and ATM may be responding to different lesions that are sequentially produced during hyperoxia but simultaneously produced with IR. Consistent with this, hSMG-1 somehow recognizes and responds to aberrantly spliced mRNAs while ATM binds DNA double strand breaks. Precedent for PIKKs to sequentially respond to different lesions comes from studies showing that ATM rapidly responds to ionizing radiation while ATR responds later presumably due to replication fork stalling caused by damaged DNA (Bakkenist & Kastan, 2003
; Tibbetts et al., 1999
). Additional studies are required to clarify why hSMG-1 activation precedes that of ATM in hyperoxia.
This study also found that endogenous and conditionally expressed p21 are destabilized in response to hyperoxia. The loss of p21 stability was reversed by wortmannin, suggesting the involvement of PIKKs. Targeting of PIKK family members ATM and hSMG-1, and perhaps ATR to a lesser extent, restored p21 stability in hyperoxia indicating that p21 stability is regulated through DNA damage signaling. P21 destabilization could affect replication and/or repair through its interactions with PCNA, a component of both processes. Consistent with p21 destabilization affecting DNA repair, the loss of p21 stability in response to low doses of UV was shown to alleviate p21 inhibition of repair through effects on PCNA (Bendjennat et al., 2003
). Alternatively, decreased p21 stability could be important in regulating proliferation. We and others have shown that p21 expression alters the expression or stability of PCNA (Engel et al., 2003
; Gehen et al., 2007
). The enhanced degradation of the more abundant PCNA allows the less abundant p21 to bind and inhibit sliding-clamp functions of remaining PCNA more completely, thereby enhancing the G1 checkpoint. P21 has also been shown to block apoptosis at expression levels much higher than that needed to inhibit cell proliferation (Vitiello et al., 2006
). Hypothetically, extensive or prolonged PIKK signaling would enhance p21 proteolysis, thereby reducing levels below the protective threshold and allowing apoptosis to ensue. The ability of PIKKs to control both synthesis and destruction of p21 may therefore allow cells to finely tune their response to DNA damage.
It is likely that PIKKs control proteasome activity because p21 proteolysis was inhibited by wortmannin and with siRNAs targeting ATM or hSMG-1. In addition, p21 stability is known to be regulated by the proteasome (Blagosklonny et al., 1996
; Maki & Howley, 1997
). Although ubiquitination is a signal for protein degradation, surprisingly, the modification of all six lysines in p21 to arginine did not alter the proteasome-dependent nature of p21 degradation (Sheaff et al., 2000
). Instead, p21 was shown to physically interact with the C8 α subunit of the proteasome and over-expression of mutant forms of p21 unable to bind the proteasome showed greatly enhanced stability. Therefore, proteasomal degradation of p21 can proceed through unique mechanisms including direct binding to the proteasome (Coulombe et al., 2004
; Touitou et al., 2001
). Since PIKKs are activated when cells are damaged and the proteasome is responsible for degrading proteins, understanding how PIKKs control proteasome function may provide insight into how cells remove damaged proteins when injured.
Despite the clear capacity of hSMG-1 and ATM to phosphorylate p53 and regulate p21 stability during hyperoxia, the role of ATR remains less clear. Consistent with our previous study using U2OS cells with stable expression of dominant-negative ATR, siRNA knockdown of ATR failed to inhibit p53 (Ser15) phosphorylation during hyperoxia (Helt et al., 2005
). These findings however do not agree with another study showing that over-expression of dominant-negative ATR in HEK293 cells blocks hyperoxia-induced p53 (Ser15) phosphorylation (Das & Dashnamoorthy, 2004
). Since hyperoxia activates ATR-dependent phosphorylation of Chk1, unknown genetic differences between these cell lines might be affecting whether p53 is a substrate for ATR. Although ATR may not phosphorylate p53, this study shows that ATR modestly destabilizes p21 and affects the G1 checkpoint in A549 but not in H1299 cells ( versus ). While these differences might be attributed to the presence or absence of p53, it is clear that ATM regulates p21 stability in both cell lines. Likewise, hSMG-1 regulates p21 stability in H1299 cells while having a minimal effect on p21 levels in A549 cells. Evidence suggests that hSMG-1 may be the primary regulator of p21 synthesis since the knockdown of hSMG-1 had dramatic effects on p53 expression levels (). Therefore, in cells with wild-type p53, diminished p21 synthesis appears to balance with enhanced p21 stability upon knockdown of hSMG-1 resulting in little change in overall p21 expression. Thus, the role of hSMG-1 in regulating p21 stability was clearly revealed in p53-deficient H1299 cells conditionally over-expressing p21. Taken together, these findings reveal differential capacity of PIKKs to precisely control synthesis versus
destruction of the cyclin-dependent kinase inhibitor p21 with hSMG-1 and ATM being the primary regulators of p21 production during hyperoxia.
In summary, this study establishes hSMG-1 as a critical regulator of cell cycle checkpoint signaling under oxidative stress. Furthermore, the results presented support that the G1 checkpoint is carefully and precisely regulated by PIKK-dependent signaling to allow for the optimal response to DNA damage. By regulating both the synthesis and proteolysis of p21, PIKKs can titer p21 levels required to inhibit cell proliferation, affect DNA repair, and block apoptosis. This dual function of PIKKs may therefore allow cells to respond appropriately to the extent of DNA damage present.