Introduction
During the course of this study we discovered new aspects of Cdt1 function and regulation with respect to origin licensing. In particular, using the hypomorphic variants we showed the need for multiple Cdt1-MCM interactions for efficient origin licensing and
extrapolated these results to structural insights into human Cdt1-MCM interactions. Also, using the hypermorphic variant, we discovered a new mechanism of Cdt1 regulation involving Cyclin A which relies on restraining Cdt1 activity independently from the known phosphodegron- mediated regulation of Cdt1 protein stability. Although we made significant headway in
describing and understanding this new phenomena, many questions still remain. The remainder of this chapter focuses on questions in the field that need to be addressed based on the
discoveries presented here and in Cdt1 biology in general.
The unexpected Cyclin A-dependent regulation of Cdt1 in addition to generating a phosphodegron.
Research in our lab has shown that Cyclin A/Cdk1-dependent phosphorylation of the linker region of Cdt1 is important to inhibit origin licensing and thus DNA re-replication. How does phosphorylation of the linker region in Cdt1 act to negatively regulate Cdt1 function? Many proteins are regulated by PTMs, including phosphorylation, and this regulation can involve changes in overall protein conformation (Nussinov et al., 2012). In the case of Cdt1,
Cdt1-MCM interactions required for origin licensing. In line with this idea, our lab has
previously shown that phosphorylating Cdt1 in the linker region by p38 and JNK during a stress response resulted in inhibition of MCM loading as well as protection from ubiquitin-mediated proteolysis (Chandrasekaran et al., 2011). However, the residues targeted for phosphorylation and the CRL4Cdt2 recognition motif are in almost opposite extremes of the protein, suggesting the possibility of global conformational changes in Cdt1. This idea can be tested in vitro by
expressing Cdt1 protein versions with and without phosphorylated linkers, i.e. phosphomimetics and unphosphorylatable variants, and biochemically and biophysically asking whether these mutational alterations change Cdt1 structure. On the other hand, the phosphorylation events in the linker region of Cdt1 can cause not a global conformation change but rather propagate to regions of Cdt1 important for Cdt1-MCM interactions. In this scenario, the global protein
conformation could remain the same but domains required for origin licensing (the middle and/or C-terminal domain) are altered and/or positioned in such a way to inefficiently engage the MCM complex.
It is possible that decorating the linker region with PTMs serves to recruit other protein factors that collectively inhibit Cdt1-MCM interactions. It is well known that phosphorylation events act as priming events for either more PTMs or for the recruitment of other protein factors to a protein (Cesaro and Pinna, 2015; Miller and Turk, 2018). One can test this idea by using an IP-mass spec approach. Presumably the mutationally altered Cdt1 variant could interact with a different set of protein factors than WT Cdt1. These ideas are not mutually-exclusive as PTMs can alter protein conformation while at the same time exposing protein-protein interacting interfaces, thus potentially inhibiting and recruiting protein inhibitors to Cdt1 concurrently.
How does Geminin really influence Cdt1 activity in origin licensing?
Crystal structures of the middle domain of Cdt1 in complex with Geminin are available (Lee et al., 2004) and although our results (see Chapter 2) support a model where Geminin inhibits a newly defined Cdt1-MCM binding domain, other studies suggest Geminin is also needed for origin licensing (De Marco et al., 2009). That is, Geminin is both an “activator” of origin licensing in G1 and subsequently an inhibitor of origin licensing in S and G2/M phase (De Marco et al., 2009). De Marco et al. discovered a quaternary structure between Cdt1 and
Geminin and suggested that this complex can be permissive at low Geminin concentrations and inhibitory at higher Geminin concentrations (De Marco et al., 2009). Although this idea fits well with Geminin levels throughout the cell cycle where Geminin levels are low during G1
(permissive), increase at the G1/S boundary, and continually increase into G2/M (inhibitory), more investigation in needed, particularly since Geminin levels in G1 are virtually undetectable in recent live-cell imaging analyses conducted in our lab and others (Grant et al., 2018) (Sakaue- Sawano et al., 2017). How does the Cdt1:Geminin stoichiometry result in a molecular switch between a permissive vs inhibitory complex for origin licensing? One can address this question by first determining the physiological protein concentration of Geminin in G1 and then use established in vitro origin licensing reconstitution assays in Xenopus egg extract using various stoichiometric Cdt1:Geminin ratios.
During the course of this study, we found that the middle domain of Cdt1 is required for efficient Cdt1-MCM interactions (Pozo et al., 2018), and this domain is also the Cdt1-Geminin interaction region (Lee et al., 2004). Is Geminin exerting its origin licensing inhibitory role by blocking Cdt1-MCM interactions through Cdt1’s middle domain? A way to test the idea that
Geminin is inhibiting this new Cdt1-MCM binding domain, which is different from the previously defined MCM binding domain, can be to first express fragments of Cdt1
corresponding to the middle domain (new MCM binding domain) and the previously known MCM binding domain (C-terminus). Next, in vitro interaction assays can be carried out where the middle and C-terminal fragments are mixed with cell lysates in addition to exogenous Geminin. If indeed the middle domain of Cdt1 is where Geminin exerts its inhibitory function, then only the middle fragment of Cdt1 will show decreased interaction with MCM. On the other hand, the C-terminus fragment will have near normal interaction with MCM as compared to full length Cdt1. This result would yield mechanistic insight into the inhibitory role of Geminin in origin licensing and further support the existence of an additional Cdt1-MCM binding domain. How do perturbations in origin licensing lead to developmental disorders or cancer?
The precise control of origin licensing is essential for normal cell proliferation. In
humans, Cdt1 is the licensing factor that is subjected to the most regulation. It is not surprisingly then, that changes in Cdt1 regulation and activity result in developmental problems and
oncogenesis (Arentson et al., 2002). For instance, the naturally-occurring mutations used in this study are found in patients suffering from a form of dwarfism called Meier-Gorlin syndrome (Bicknell et al., 2011a; de Munnik et al., 2012). This developmental disorder begins in utero and continues throughout adolescence. From our results we infer that the hypomorphic mutations actually lengthen the cell cycle by slowing down G1 progression, while the hypermorphic mutation leads to genome instability. Both of these scenarios result in cell proliferation defects that over time can result in developmental disorders.
thus Cdt1 can re-license origins and induce a DNA re-replication phenomenon. This process in turn results in genome instability and potentially oncogenesis (Fujita, 2006; Liontos et al., 2007; Petropoulou et al., 2008).
How and why did Cdt1 evolve to have a secondary role in mitosis?
Many proteins have more than one function in cells and Cdt1 is no exception. A role of Cdt1 in mitosis was discovered in our lab. Cdt1 is required for robust kinetochore-microtubule attachment during chromosome segregation in mitosis (Varma et al., 2012). Depleting Cdt1 in cells specifically during G2/M and after its licensing role in G1 results in mitotic cells arresting at prometaphase (Varma et al., 2012). We currently know that Cdt1 interacts with the Hec1 loop of the Ndc80 complex at the kinetochore-microtubule interface, and that this interaction
maintains the Hec1 subunit in an extended conformation (Varma et al., 2012). In addition, a recent study showed that Cdt1 directly binds microtubules and mapped the microtubule-binding regions to the middle and C-terminal domains of Cdt1 (Agarwal et al., 2018). Furthermore, Agarwal et al. showed that the mitotic kinase Aurora B interacts with and phosphorylates Cdt1 to stabilize kinetochore-microtubule attachments (Agarwal et al., 2018). These studies have
increased our knowledge of Cdt1 function during mitosis. However, this novel role for Cdt1 remains largely unexplored and it would be interesting to dissect the molecular mechanism of why the PTMs on Cdt1 at the G2/M boundary are necessary for its mitotic function.
It is interesting to consider that Cdt1 interacts with both the MCM complex and the Hec1 loop providing the correct “brace” to either keep the MCM ring open or to maintain the Hec1 subunit in its extended conformation. It appears as though Cdt1 has evolved in cells to maintain protein complexes in the right conformation to achieve their function. Furthermore, because of
the same time preventing its licensing activity. It would be interesting to know what PTMs are found in Cdt1 during its mitosis role. One way to detangle the posttranslational modification required for Cdt1 function at each cell cycle phase can be to carry out an IP mass spec approach from synchronized cells. What function for Cdt1 came first, origin licensing or kinetochore- microtubule attachment remains unclear.
The cell cycle is a very complex biological process essential for all living things. By focusing on single key events of the cell cycle, we can begin to understand how the cell cycle machinery controls cell proliferation throughout normal development and also during disease states. In this work, we focused on the process of origin licensing with emphasis on the function and regulation of the Cdt1 protein. Using naturally-occurring and engineered mutations in Cdt1, we made progress towards having a deep understanding of the molecular mechanisms controlling origin licensing and how this event is regulated throughout the cell cycle. Ultimately, studying in detail the various events of the cell cycle will lead to a more well-rounded and precise
understanding of the molecular mechanism behind this fundamental process.
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