The mitogen-activated protein kinase (MAPK) Kss1 has a dual role in regulating filamentous (invasive) growth of the yeast Saccharomyces cerevisiae. The stimulatory function of Kss1 requires both its catalytic activity and its activation by the MAPK/ERK kinase (MEK) Ste7; in contrast, the inhibitory function of Kss1 requires neither. This study examines the mechanism by which Kss1 inhibits invasive growth, and how Ste7 action overcomes this inhibition. We found that unphosphorylated Kss1 binds directly to the transcription factor Ste12, that this binding is necessary for Kss1-mediated repression of Ste12, and that Ste7-mediated phosphorylation of Kss1 weakens Kss1-Ste12 interaction and relieves Kss1-mediated repression. Relative to Kss1, the MAPK Fus3 binds less strongly to Ste12 and is correspondingly a weaker inhibitor of invasive growth. Analysis of Kss1 mutants indicated that the activation loop of Kss1 controls binding to Ste12. Potent repression of a transcription factor by its physical interaction with the unactivated isoform of a protein kinase, and relief of this repression by activation of the kinase, is a novel mechanism for signal-dependent regulation of gene expression.[Key Words: Protein-protein interaction; signal transduction; gene regulation; developmental control; invasive growth]Received June 8, 1998; revised version accepted July 24, 1998.Commitment of a cell to a developmental fate entails a change in the pattern of gene expression. In many instances, this gene regulation is controlled by signaltransduction pathways that affect the phosphorylation of transcription factors (or their associated proteins) by protein kinases (for review, see Karin 1994;Hill and Treisman 1995;Treisman 1996). Phosphorylation can control the localization or sequestration of many transcription factors, as shown for yeast Swi5, Drosophila dorsal and yan, SV40 large T antigen, and vertebrate NF-B, NFAT, STATs, and SMADs (for review, see Hill and Treisman 1995;Vandromme et al. 1996;Heldin et al. 1997). Phosphorylation by protein kinases can also influence the DNA-binding activity of transcription factors. For example, DNA-binding of Elk-1 is stimulated by the Erk and Jnk mitogen-activated protein kinases (MAPKs), whereas DNA-binding of c-Jun is inhibited by CKII (for review, see Treisman 1996). The transactivation potential of transcription factors can also be regulated independently of DNA binding. For example, protein kinase A (PKA)-mediated phosphorylation of CREB, as well as Jnk-mediated phosphorylation of c-Jun, stimulates the binding of the coactivators CREB-binding protein (CBP) and P300 (for review, see Goldman et al. 1997), and Cdkmediated phosphorylation of Rb leads to dissociation of this repressor from E2F (for review, see DePinho 1998). Finally, protein kinases can regulate transcription factor stability (Clevers and van de Wetering 1997). In all of these cases, the catalytic (phosphotransferase) activity of the protein kinase involved is an indispensable component of its regulatory action.Recent studies of...

Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in the absence of any additional yeast proteins. These complexes were specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7. Neither the kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste7 and Kss1 were necessary for complex formation. Ste7-Kss1 (and Ste7-Fus3) complexes were present in yeast cell extracts and were undiminished in extracts prepared from a ste5delta-ste11delta double mutant strain. In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or pheromone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissociation of the high-affinity complex was shown to be required for either phosphorylation event. Deletions of Ste7 in the region required for its stable association with Kss1 and Fus3 in vitro significantly decreased (but did not eliminate) signaling in vivo. These findings suggest that the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ensure fidelity in signal transmission and to insulate one signaling pathway from another.

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC Cdh1 ) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC Cdh1 ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage. INTRODUCTIONDuplication of large mammalian genomes requires that DNA replication initiate at thousands of chromosomal origins. In order for an origin to be competent for replication, it must first be bound by a multiprotein complex, the prereplication complex (preRC). PreRCs are constructed in a stepwise process through the chromatin binding of the origin recognition complex (ORC), which then recruits both the Cdc6 ATPase and Cdt1, two proteins that are required for the stable loading of the minichromosome maintenance complex (MCM). The Cdc6 and Cdt1-dependent loading of MCM complexes at origins licenses them for replication during the G1 phase of the cell cycle. Sufficient preRCs must be assembled during G1 to promote complete replication, but new preRCs must not assemble after S phase begins because relicensing of previously fired origins leads to rereplication and genome instability (Vaziri et al., 2003;Melixetian et al., 2004;Zhu et al., 2004;Archambault et al., 2005). For these reasons, preRC assembly is one of the most highly regulated events in the control of DNA replication. Cells restrict preRC assembly to the G1 period through a combination of overlapping mechanisms that regulate individual preRC components (reviewed in Bell and Dutta, 2002;Blow and Hodgson, 2002;Nishitani and Lygerou, 2002;Diffley, 2004;Machida et al., 2005;Fujita, 2006).Cdc6 is not only an essential factor for preRC construction, but it has also been implicated in the activation of the cell cycle checkpoint that prevents entry into mitosis while DNA replication is incomplete (Clay-Farrace et al., 2003;Oehlmann et al., 2004;Lau et al., 2006). These observations suggest that Cdc6 functions not only during G1, but also in later cell cycle stages. Moreover, Cdc6 plays a role in setting the threshold for...

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment.

Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs). Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression. Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain. Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs. However, maintenance of repression at PREs is more dependent on Dig1 and͞or Dig2 and less dependent on Kss1 than repression at FREs. In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs. Differential utilization of two types of MAPK-mediated regulation (bindingimposed repression and phosphorylation-dependent activation), in combination with distinct Ste12-containing complexes, contributes to the mechanisms by which separate extracellular stimuli that use the same MAPK cascade can elicit two different transcriptional responses.

Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create "PIP-FUCCI". The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions. ARTICLE HISTORY

Cell proliferation is a fundamental requirement for organismal development and homeostasis. The mammalian cell division cycle is tightly-controlled to ensure complete and precise genome duplication and segregation of replicated chromosomes to daughter cells. The onset of DNA replication marks an irreversible commitment to cell division, and the accumulated efforts of many decades of molecular and cellular studies have probed this cellular decision, commonly called the restriction point. Despite a long-standing conceptual framework of the restriction point for progression through G1 phase into S phase or exit from G1 phase to quiescence (G0), recent technical advances in quantitative single cell analysis of mammalian cells have provided new insights. Significant intercellular heterogeneity revealed by single cell studies and the discovery of discrete subpopulations in proliferating cultures suggests the need for an even more nuanced understanding of cell proliferation decisions. In this review, we describe some of the recent developments in the cell cycle field made possible by quantitative single cell experimental approaches.

Although molecular mechanisms that prompt cell cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S-phase progression. In sum, we show that a cell’s response to DNA damage depends on its exact cell cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none.