Prolyl isomerization

*Correspondence to: Alessandro A Sartori; Email: [email protected] Submitted: 06/18/13; Accepted: 06/20/13 http://dx.doi.org/10.4161/cc.26077 Comment on: Steger M, et al. Mol Cell 2013; 50:333-43; PMID:23623683; http://dx.doi.org/10.1016/j.molcel.2013.03.023 The repair of DNA double-strand breaks (DSBs) is highly complex. To maintain genome integrity and avoid tumorigenesis, cells choreograph the 2 major DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), both spatially and temporally through post-translational modifications. DSBs activate ATM kinase, which triggers a signaling cascade, leading to activation of cell cycle checkpoints and DNA repair. There is also growing evidence implicating cyclin-dependent kinases (CDKs) in the regulation of DSB repair. CDKs, first discovered for their role in cell cycle regulation, belong to a large superfamily of proline-directed kinases that exclusively phosphorylate serine or threonine residues preceding a proline (S/T-P motifs). The unique stereochemistry of proline allows prolyl-peptide bonds to adopt cis and trans conformations. The intrinsically slow interconversion between these isomers can be greatly accelerated by peptidyl-prolyl isomerases (PPIases). PPIases include 3 major subfamilies (cyclophilins, FKBPs, and parvulins) that act in general protein folding, but only one enzyme, Pin1, can isomerize phosphorylated S/T-P motifs. By inducing conformational changes in a subset of phosphorylated proteins, Pin1 was shown to act as a molecular switch in multiple cellular processes. Most recently, we showed that Pin1 also has a profound impact on the regulation of DSB repair. In order to identify novel Pin1interacting proteins, we performed GST pull-down assays using Pin1 as bait and analyzed the recovered proteins by mass spectrometry. Interestingly, the list of >600 specific interactors included the key DSB response factors MDC1, 53BP1, BRCA1, BARD1, PTIP, and CtIP. This prompted us to test the involvement of Pin1 in GFP-based HR and NHEJ reporter assays. We found that depletion of Pin1 caused a significant decrease in NHEJ frequency, while Pin1 overexpression led to a strong reduction in HR. Since DNA-end resection constitutes a critical point in DSB repair pathway choice, we reasoned that Pin1 may restrict HR and promote NHEJ by actively suppressing the formation of single-stranded DNA. This hypothesis was substantiated by increased hyperphosphorylation of RPA2 and a concomitant defect in NHEJ upon DSB induction in Pin1 MEFs. Moreover, the observed hyper-resection phenotype of Pin1-depleted cells was strictly dependent on CtIP, which plays a key role in the initiation of DNA-end resection. Confirming our proteomics screen data, we could show that Pin1 binds to CtIP in a phosphorylation-dependent manner and identified 2 conserved S/T-P motifs in CtIP (S276 and T315) to be required for Pin1 interaction. We could show that CtIP-pT315 serves as the major Pin1 binding site, but that CtIP isomerization takes place exclusively at the pS276-P277 site. In order to identify the kinase(s) responsible for phosphorylating CtIP at S276 and T315, we raised individual phospho-specific antibodies and used them in combination with various kinase inhibitors. Treatment of cells with roscovitine, a pan-CDK inhibitor, strongly reduced both Pin1CtIP interaction and T315 (but not S276) phosphorylation. Consistently, overexpression of dominant-negative (dn) forms of CDKs resulted in a strong (CDK2-dn) or moderate (CDK1-dn) reduction of Pin1-CtIP interaction. We also monitored CtIP phosphorylation levels during the cell cycle, and found that pT315 was upregulated during S phase and peaked in late S/G 2 , whereas pS276 was largely undetectable. In fact, we noticed that the region encompassing S276 is highly conserved in mammals and rather matches the consensus sequence for mitogen-activated protein kinases (MAPKs), with some family members known to be activated in response to DNA damaging agents. Interestingly, we observed a slight increase in Pin1CtIP complex formation in presence of DSBs. These observations are consistent with a fascinating kinase convergence mechanism, in which CDK1/2mediated T315 phosphorylation is a prerequisite for Pin1 recognition, while S276 phosphorylation by another DNA damage-inducible proline-directed kinase is crucial for CtIP isomerization. To investigate the role of CtIP isomerization at the molecular and cellular level, we generated cell lines stably expressing siRNA-resistant GFP-tagged wild type or mutant CtIP, in which both S276 and T315 were changed to nonphosphorylatable residues (CtIP-2A). Importantly, we observed increased resection of DSBs and reduced NHEJ Prolyl isomerization A new PIN code for DSB repair