Stress-Stimulated Mitogen-Activated Protein Kinases Control the Stability and Activity of the Cdt1 DNA Replication Licensing Factor

Abstract

DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin licensing protein whose activity must be restricted to G 1 phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G 2 phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitinmediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin licensing. Two other CRL4 Cdt2 targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G 2 promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.Precise and complete genome duplication presents a unique challenge during the cell division cycle. To permit efficient replication, DNA synthesis initiates at many chromosomal sites, known as origins of DNA replication. During G 1 phase, origins are loaded with an inactive form of the DNA helicase core, the minichromosome maintenance (MCM) complex. Origins with loaded MCM complexes are "licensed" because they are competent for replication initiation in the subsequent S phase. MCM loading is accomplished through recruitment of MCM complexes from the nucleoplasm by the Cdt1 protein to an origin-bound assembly of the origin recognition complex (ORC) and the Cdc6 protein. ORC and Cdc6 then load MCM onto DNA (48,57,58).Failure to properly control MCM loading can lead to replication errors and genome instability if insufficient origin licensing occurs in G 1 or if inappropriate origin relicensing occurs after the onset of S phase. For example, high levels of Cdt1 or Cdc6 activity in S or G 2 phase can promote origin relicensing, which leads to extensive rereplication and cell death; modest deregulation of either Cdc6 or Cdt1 promotes genome instability and tumorigenesis (5,28,44). Thus far, the best-understood mechanisms restricting origin licensing to G 1 phase are cell cycle-regulated accumulation and degradation of licensing proteins and inhibition of several licensing proteins after Sphase onset through phosphorylation by cyclin-dependent kinases (CDKs) (7,23,34).Given the critical need to maintain tight control and coordination of origin licensing, it is likely that additional important regulatory mechanisms have yet to be uncovered. Cell cycle progression is arrested in response to a variety of cellular stresses, including exposure to inflammatory cytokines, bacterial toxins,...

Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

“…It has been shown previously that Cdt1 levels increase after S phase and that Cdt1 protein is stabilized during G2 phase (4,9). However, the levels of a protein are not necessarily related to its turnover rate.…”

“…In many cells G2 is very short. Three mechanisms contribute to Cdt1 accumulation in G2: the inactivation of the ubiquitin ligase activity associated with Cul4-DDB1-Cdt2 (7), the partial stabilization of Cdt1 caused by phosphorylation by the MAP kinases p38 and JNK (9), and the regulation by Geminin. These mechanisms are particularly important because they must counteract the decrease of Cdt1 RNA during G2 phase (Fig.…”

“…Cdt1 activity is limited to G1 through the control of its synthesis, degradation, and activity. The low level in S phase is thought to result from targeted degradation (6)(7)(8), whereas its higher level in G2 is thought to result from its stabilization (9). However, the increase of Cdt1 in G2 poses a potential risk in allowing rereplication, which could occur if there were residual activity of the DNA-replicating enzymes in G2.…”

CDK1-dependent Inhibition of the E3 Ubiquitin Ligase CRL4CDT2 Ensures Robust Transition from S Phase to Mitosis

“…Nevertheless, replication can extend close to the time of mitosis, and this late replication has been implicated in the genome instability associated with common fragile sites (38). Because PCNA DNA triggers CRL4 CDT2 -mediated degradation, persistence of PCNA DNA so late in S phase was not fully consistent with observations by us and others that CRL4 CDT2 substrates reaccumulate well in advance of mitosis (5,11,19,39). We therefore considered the possibility that substrates may be actively stabilized during the transition from S phase to mitosis.…”

“…To test that possibility, we employed pharmacological inhibition of candidate kinases: cyclin-dependent kinases (CDKs) and the stress-activated mitogen-activated protein kinases (MAPK) p38 and JNK, which we previously showed could stabilize CRL4 CDT2 substrates (39). We briefly treated prometaphase cells with kinase inhibitors prior to UV irradiation and then tested for reversal of mitotic stability by immunoblotting (Fig.…”