The PIKKs are unusual protein kinases in that their sequence suggests common evolution from lipid kinases. The common evolution is apparent in the similarity of their regulation. While responding to different input signals, common themes of regulation include dynamic localization dependent on partner proteins and a protein or protein/nucleic acid activator ().
Figure 2 Common themes of PIKK regulation. Each PIKK is regulated through localization (to a nucleic acid or intracellular compartment), a partner protein that is required for both the recruitment and activation of the kinase, and a protein or protein/nucleic (more ...)
PIKK relocalization concentrates the kinases to where their activator is found. This concentration may be primarily responsible for activation since regulation of both ATR and ATM can by bypassed in experimental systems that increase the kinase/activator local concentrations. For example, overexpression of the TopBP1 ATR-activating domain induces ATR activation in the absence of DNA damage, producing pan-nuclear γH2AX staining and cellular senescence [18
]. Furthermore, concentrating the Mec1ATR
and 9-1-1 complexes together on chromatin is sufficient to activate ATR without a DNA lesion in S. cerevisiae
]. Therefore, RPA-coated ssDNA primarily serves as a scaffold for the assembly of proteins required for ATR activation, with the independent recruitment of checkpoint activating complexes providing a mechanism of regulation to ensure that ATR is not inappropriately activated. Similarly, immobilizing and concentrating ATM or key ATM regulators on chromatin also activates ATM signaling [64
Kinase signaling is further regulated by post-translational modifications on the PIKKs, accessory proteins, and activator proteins. These modifications may provide opportunities for fine-tuning or amplifying the signal. Finally, conformational changes underlie PIKK activation. We do not yet understand why the dimer to monomer ATM transition is important or how TopBP1 interaction with ATR-ATRIP allows greater substrate access. Further mutagenesis and domain mapping approaches will be useful, but ultimately high-resolution structures will be critical.
While the amount of information about how the PIKKs are activated has exploded in recent years, there remain many unanswered questions about this interesting family of kinases. Experimental results such as the lack of phenotype of the ATM S1981A mutant in mice, and relatively minor defects associated with mutations in the ATRIP-RPA binding surfaces, suggest that the simple activation models need further revision. New proteins like Aven must be incorporated into the models. Of course, the signaling downstream of these kinases has not been addressed in this review, but is equally complex.
Future research into the PIKKs will surely help us to understand not only how cells maintain genome integrity to prevent diseases such as cancer, but also may point to opportunities for drug development. mTOR has been targeted for decades with the natural product rapamycin – an antiproliferative antibiotic useful to cause immunosuppression. ATM and ATR pathway inhibitors are being developed as potential radio- and chemo-sensitizing agents for cancer therapy. Since cancer cells have defects in genome maintenance activities and have considerable genome integrity challenges due to their microenvironment and oncogene-induced replicative stress, targeting the DDR pathways may be useful as a monotherapy in cancer. However, successful targeting of these pathways requires that we understand their genome maintenance activities, how they are regulated, what genome maintenance activities are defective in cancer cells, which proteins might provide the best targets, and which patients might benefit most from this type of therapeutic approach. Furthermore, biomarkers for patient selection and drug evaluation will be needed. Thus, filling in the details of the PIKK signaling pathways is critical. Fortunately, our task is made easier by the significant similarities between the mechanisms regulating these kinases.