Cells have evolved intricate networks of proteins or signal transduction pathways that allow them to integrate internal or external signals in order to respond to specific cues during development and during stress. Molecules that originate from neighboring cells, tissues, and pathogens, can act over short and long distances to elicit a physiological response. Signaling pathways can also be triggered within the cell, as is the case when cells are placed under alert following DNA damage or when there is a problem with DNA replication. Such information is transmitted via protein-protein interactions and protein modifications and leads to the alteration of gene expression and other events that regulate cell division, DNA repair or cell death. Our laboratory focuses on checkpoint signaling events triggered by DNA damage or replication interference. We are taking genetic, biochemical and cell biological approaches to study these signaling pathways. We are interested in dissecting signaling complexes involved in the response to lesions that are caused by DNA damaging agents or stalled replication forks and determining how specific protein-protein interactions and cellular location are regulated to transmit this information. We are also interested in the interactions between the signaling molecules and the enzymes that function in DNA replication and repair.
The pathways that we study are involved in both the etiology and treatment of cancer. Loss-of-function mutations in mammalian checkpoint genes compromise the response to DNA damage at the cellular level and at the level of the organism lead to a predisposition to cancer. In addition, cancer therapies frequently rely on drugs or agents that trigger genomic instability by taking advantage of the fact that cancer cells have defects in the response to DNA damage. We are addressing these questions by embarking on the following projects:
I. Cross-talk between the checkpoint and other stress responsive pathways.
Many pathways integrate signals into common effectors in order to regulate progression through mitosis, which allows cells to coordinate cell division with nutrient availability and spindle and genomic integrity. Yeast cAMP-dependent protein kinase (PKA) signaling is regulated by glucose-sensing mechanisms and has been shown to have a role in the regulation of cell cycle progression in response to nutrient availability. Genomic integrity is safeguarded in part by checkpoints, which provide cells with a mechanism to detect DNA damage or ongoing DNA replication and respond by arresting the cell cycle to allow the completion of DNA replication and/or DNA repair. Our work has recently identified the mitotic regulator Cdc20 as a common effector for the PKA and DNA damage checkpoint pathways operating at the metaphase to anaphase (M-A) transition. In the past 6 years, a picture of how kinases signal cell cycle arrest in response to different DNA lesions has emerged. However, little is known regarding the mechanism of cross-talk between checkpoint and other stress and nutrient-responsive pathways.
Studies in budding yeast have identified a role for PKA signaling in the checkpoints activated by DNA damage. Checkpoint kinases and PKA cooperate to stop cell division in response to DNA damage. The figure shows budding yeast that have arrested (top), or have failed in the DNA damage checkpoint response and formed microcolonies, indicative of uncontrolled cell growth
The central hypothesis for how the PKA and DNA damage checkpoint pathways interact is that nutrient sensing and DNA damage regulate PKA to control progression through mitosis via the same or similar mechanism. Using the genetically amenable budding yeast model system, we can dissect the regulatory switches coupling mitotic progression to specific signals caused by nutrient availability or cellular and environmental genotoxic agents. Future studies will test whether these molecular switches are conserved in mammalian cells and whether they play important roles in coupling nutrient sensing pathways to genomic stability.
II. Checkpoints that monitor DNA replication as therapeutic targets for cancer
Most cancer cells are partially compromised in their ability to respond to DNA damage or replication blocks, which includes defects in checkpoints and apoptotic pathways and limits the effectiveness of current cancer treatments that involve the use of DNA-damaging agents such as radiation. Therefore, our understanding of how the checkpoint pathways are wired will allow us to design more effective therapeutic regimes for the treatment of cancer.
We have shown that the checkpoint kinase Chk1 interacts with chromatin where it is phosphorylated in the absence of exogenous DNA damage indicating that Chk1 has a role in an intrinsic checkpoint at every cell cycle. The model for this line of work is that Chk1 is present in complexes with proteins involved in DNA metabolism and plays a role in these processes. To test this model, we are exploring checkpoint-signaling through biochemical identification of Chk1-interacting proteins (Chips).
In invertebrates, Chk1 is essential for embryonic development,
the response to replication blocks, as well as the recovery from
The checkpoint kinases have made attractive drug targets as therapeutic enhancers of genotoxic cancer drugs. However, the first generation of kinase inhibitors is mostly comprised of molecules that compete for binding to catalytic sites, which by their chemical nature are non-specific. Our work with the Chips will map the domains involved in checkpoint signaling in order to identify regions that may be targeted for development of a different class of peptide or chemical inhibitors that act by blocking specific protein-protein interactions. This new class of inhibitors could be used to disrupt checkpoint signaling in vivo to enhance the effect of drugs that cause damage to DNA.
III. FUNCTIONAL SCREEN FOR SNPs IN CHECKPOINT GENES AND GENERATION OF MOUSE MODELS WITH ATTENUATED CHECKPOINT RESPONSE
The hypotheses that we are addressing here are (1) attenuated alleles of checkpoint genes confer an increased risk to individuals following exposure to agents that damage DNA and (2) retention of the attenuated allele following loss of the functional allele in the animal will increase the risk of developing cancer.
Studies addressing the roles of vertebrate Chk1 in the S-phase checkpoint in vivo have been difficult, due to the fact that Chk1 is essential for vertebrate development. We developed a new assay in the yeast model system, which is amenable to genetic manipulation that can be utilized to study the roles of Chk1p kinases in both the S-phase-checkpoint pathways and the recovery from stalled or collapsed replication forks. We have generated several hypomorphic and separation-of-function alleles by targeted changes to the amino acids that are invariant between the non-catalytic domains of the yeast and murine proteins. We have also used this assay to determine the structural requirements of Chk1 for these responses and, more importantly, this assay will be key in examining the functional consequence of variants in the human population that lead to changes in conserved residues (see below).
Our strategy is to use the yeast as a genetic tool to determine the functional significance of single nucleotide polymorphisms (SNPs) that result in amino acid changes in checkpoint proteins prior to generating the mouse model with the SNP. Preliminary studies have shown that substitution of invariant residues in human and yeast proteins resulted in the same phenotype both in yeast and human cells underscoring the conservation of the checkpoint kinases and the utility of this system.
Our approach to generate a mouse model will make use of systems that allow us to not only study the impact of carrying the variant allele in a homozygote and heterozygote state, but also allow for removal of the wild-type allele in different tissues of the adult mouse. This will result in mice that have retained the SNP allele mimicking a loss of the heterozygosity event. This approach will allow us to the study the consequences of carrying the variant in a homozygous state as a germ line mutation and also as a late somatic event in heterozygotes by conditional expression of the mutant allele in particular cell types in the adult mouse.
IV: SPECIFICITY OF CHECKPOINT SIGNALING BY CELLULAR ADDRESS, FROM CHROMATIN TO CYTOPLASM
One line of studies derived from the identification of Chk1-interacting proteins (Chips) is based on the hypothesis that signal transduction proteins are regulated at the level of sub-cellular localization, and that this level of regulation contributes to specificity of signaling in response to a variety of cues. Our studies have uncovered not only Chips that are potential targets of Chk1 (proteins involved in DNA metabolism and transcription factors) but also proteins that perform more general regulatory interactions that we would have predicted to be part of a signal transduction pathway. One such protein,14-3-3, falls in the adaptor/scaffold category. 14-3-3 recognition sites surround phospho-serine residues, and in many cases regulate proteins by changing their sub-cellular localization. We found that phosphorylation of Ser345 of Chk1 could serve as a docking site for 14-3-3 proteins that would lead to the interference with a Nuclear Export Signal present in a region surrounding Ser345. We used an immunoprecipitation approach coupled with proteomic analyses to identify additional proteins that associated with Flag-Chk1 and identified additional proteins involved in nucleo-cytoplasmic shuttling of proteins, as Chk1-associated proteins. In the future, we will identify all the proteins involved in this process and test our hypotheses that a role of the nuclear export system is to transmit the checkpoint signal from the nucleus to the cytoplasm where cross-talk with other stress-responsive pathways would occur.
The genetic and biochemical approaches used to study signaling pathways have the same shortcoming: the measured response is usually outside of the cellular context. In order to understand the changes that are triggered in the cell or tissue following either activation of receptors, stress, or genotoxic insults, we need to integrate approaches that will allow us to analyze signaling events in living cells. One of the long-term goals of this work is to establish in vivo biosensors that interpret signals, such as the fluorescence resonance energy transfer (FRET) system described for PKA and calcium signaling, or that measure changes in molecules, such as glucose and cAMP, in living cells during the response to nutrient or DNA-damage signals. These approaches will also lend themselves, in the future, to screens for drugs that interfere with or enhance these signaling events.
Funding for the Sanchez Lab has been provided by: