Mechanism of G1 Radiation Checkpoint Activation POS-LoRBPS780 analysis was used to verify siRNA performance. L) Statistical analysis. Student t-test for cell viability data shown in A–I, (Li/NT vs Targ/NT) probing for a significant effect of target knockdown in unperturbed cells, (Li/CHK1 vs Targ/CHK1) probing for a significant effect of target knockdown in CHK1-perturbed cells, Target/NT vs Target/CHK1 probing for a significant effect of CHK1 knockdown in target-perturbed cells. M, N) Treatment interaction. Assessment for evidence of interaction between radiation and target knockdown. Values represent the degree of net synergism between target knockdown and IR in NT (M) or CHK1-perturbed (N) cell background. doi:10.1371/journal.pone.0031627.g005 phosphorylate mammalian TP53 on S15 and S37 in vitro [57]. Phosphorylation of TP53 on these sites is known to result in increased TP53 transcriptional activity [58], supporting an assumption whereby PRPK plays a direct role in regulating signalling through the TP53-p21CIP1/WAF1 axis. A very recent report [59] identified PRPK in a screen for sensitizers to spindle assembly checkpoint activation showing that knockdown inhibited mitotic slippage with adaptation, consistent with a role of this gene product in G1 checkpoint activation. The second kinase, STK4, is a component of the MAPK ontology, and is not known to directly phosphorylate TP53 [60]. STK4 has been reported to act upstream of the c-Jun N-terminal kinases (JNK1-3/MAPK8-10) and p38/MAPK11-14 mitogen activated kinase family kinases [61,62]. Both JNK and p38 kinases are known to phosphorylate and activate TP53 [63,64]. Furthermore p38 family kinases are known to become activated in response to IR and as being required for IR-induced accumulation of p21CIP1/WAF1 [65], yet the signalling that leads to their activation is not know. Other recent work showed that STK4 activity promotes p21CIP1/WAF1 stability in a JNK-dependent manner [66] suggesting participation of this kinase in signalling resulting in transcriptional as well as posttranscriptional control of p21CIP1/WAF1. Our findings, in concert with the current literature, places STK4 as acting through the SAPK/JNK and p38 signalling MAP kinase cascades into DNA damage driven G1 checkpoint activation. While it is possible that the p21CIP1/WAF1 loss observed in cells with PRPK or STK4 loss is a consequence of checkpoint loss, rather than its cause, the critical requirement of p21CIP1/WAF1 accumulation documented by our work together with the known involvement of PRPK and STK4 in the regulation of this CDK inhibitor make such an interpretation less likely. The canonical route leading to TP53 activation in cells upon genotoxic insult involves ATM or ATR and their substrates CHK1 and CHK2, which in turn facilitate TP53 phosphorylation and activation [67,68,69]. As indicated in the results chapter, none of these genes scored in the screen nor did their pharmacological inhibition abolish G1 checkpoint activation, strongly supporting a view whereby signalling implicating these components is not involved in G1 checkpoint control. The implication of the TP53/ p21CIP1/WAF1 signalling hub in both S/G2 and G1 checkpoint control, along with the documented requirement of PRPK and STK4, suspected to affect this hub, in G1, proposes a model whereby TP53/p21CIP1/WAF1 facilitates execution of multiple checkpoints, but executor hub activation is controlled by unrelated yet convergent signalling ontology (see Figure 6B). STK4 and PRPK cluster with CDK4 as hits through their similar propensity to reduce p21CIP1/KIP1 positivity in irradiated cells. Identification of CDK4 in this screen is unexpected, as this kinase is known for its role in promoting RB1 phosphorylation and hence knockdown should lead to attenuation of the event [70]. Knockdown of the closely related and potentially redundant kinase CDK6 did not confer radiation-resistant RB1 phosphorylation but led to loss of RB1 phosphorylation in control and irradiated cells (not shown and Table S1), in line with the perceived role of CDK4/6 in driving RB1 inactivation and indicative of the critical role of this kinase-group in driving RB1 phoshorylation in the cells. It is possible that off-target activities of oligonucleotides led to identification of CDK4 and this cannot be fully excluded, albeit this target validated with two unrelated oligonucleotides. There is no prior published evidence whereby CDK4 is required for the induction or maintenance of p21Cip1/Kip1 expression. However, CDK4 in complex with D cyclins can bind p21Cip1/Kip1 and it is possible that this interaction stabilizes the CDK inhibitor. Reduction in CDK4 could free cyclin D to activate kinases other than CDK4, capable of phosphorylating RB1, an event that has been seen in cells with CDK4/6 knockout cells [71], and this could explain the radiation-resistant RB1 phosphorylation observed upon CDK4 knockdown. Several other gene products identified as hits in the screen did not significantly impact p21CIP1/Waf1 accumulation, suggesting that they support checkpoint control through mechanisms independent of TP53 activation and p21Cip1/Kip1 expression. They include HK1, PRKACG and the DYRK1A dual specificity kinase. There is some evidence that mechanisms other than p21CIP1/WAF1-mediated inhibition of the RB1 phosphorylating CDKs may play a role in the DNA damage-associated activation of RB1. For example there is published evidence for the activation of an RB1-directed phosphatase [72] and the phosphorylation- mediated degradation of cyclin D [73,74,75] in irradiated cells. It is conceivable that HK1, PRKACG and DYRK1A act through such alternative means. In common between HK1 and PRKACG is their involvement in driving oxidative glycolysis [76], with knockdown of either enzyme predicted to cause cessation of this process. Identification of HK1 and PRKACG in the screen could hence suggest that glycolytic activity is required for G1 checkpoint activation following genotoxic stress. Short-term treatment of cells with Lonidamine, a selective inhibitor of glycolysis has recently been associated with increased sensitivity to radiation [77], providing corroborating evidence for the interaction of this process with DNA damage response, and potentially checkpoint control. We note that CDK4, as well as LATS2, a kinase that can activate DYRK1A, was identified in a recently reported RNA interference screen [78] searching for functions which overcome proliferation inhibition following enforced expression of RB1 in RB1 phosphorylation incompetent SAOS2 cells. It is conceivable that knockdown overcame the RB1 phosphorylation incompe- tence of SAOS2 cells, which is believed to result from high expression of p16INK4A in combination with stabilization of the p21CIP1/WAF1 paralogue p27KIP1. The functional analysis by Tschoep leaves open whether LATS2 or CDK4 knockdown operates by reinstating RB1 phosphorylation and Tschoep et al. 2011 did not identify other targets that scored positive in the screen reported here. Radiation affects cell cycle progression at multiple points of the cell cycle. The mechanism underlying DNA damage related G2 and M phase checkpoint control has received considerable attention resulting in increasingly detailed models for the signalling involved [21,79]. The available knowledge as to signalling involved in damage-associated G1 checkpoint activation is comparatively scarce. Efforts towards delineation of signalling leading to G2/M checkpoint activation may have been fuelled by the recognition that this checkpoint is essential in cells with TP53 loss and thus the potential to uncover therapeutic opportunities in PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31627