And the debate goes on: Wnt regulation of the destruction complex
Development is an amazing process in which a single cell divides and multiplies to create a multicellular organism. During this process cells must reorganize spatially and temporally to determine their final cell fate. This reorganization requires cells to communicate with one another. There are a handful of essential cell signaling pathways that aid in cell fate and organization (Basson, 2012). One of these essential signaling pathways is Wnt signaling. The importance of this pathway in development is emphasized by its conservation throughout animals (Loh et al., 2016). Not only is Wnt signaling necessary for development, but it is also essential for tissue homeostasis. For example, Wnt signaling is responsible for maintaining the colon’s stem cell population. In fact, 90% of all colorectal cancers contain an activating mutation in this pathway (CGAN, 2012). Even though this pathway is ubiquitous and has been studied for over thirty years, the field is still debating the mechanisms of pathway is regulation.
The basics: Wnt signaling pathway simplified
The main function of Wnt signaling is to regulate the levels of β-catenin (βcat), a co-
activator of transcription. In the absence of Wnt signaling, the destruction complex comprised of
the tumor suppressor APC, the scaffold Axin, and the kinases CK1 and GSK3 keeps βcat levels
low. The destruction complex recruits βcat to be sequentially phosphorylated by CK1 and GSK3
(Liu et al., 2002). This modification of βcat allows it to be recognized by a Skp-Cullin-Fbox E3
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(Aberle et al., 1997; Kitagawa et al., 1999; Liu et al., 2002). Thus, in the absence of Wnt
signaling βcat levels are kept low, preventing Wnt induced transcription.
During Wnt signaling, a Wnt ligand interacts with the seven transmembrane receptor Frizzled and its co-receptor LDL-receptor-related protein (LRP) 5/6 (Arrow in Drosophila), creating a 3-protein receptor-ligand complex which promotes and stabilizes Dishvelled interaction with this complex (Angers and Moon, 2009; DeBruine et al., 2017; Tamai et al., 2000). Phosphorylation of LRP5/6 enhances recruitment of Axin to the membrane and destruction complex function is turned down (DeBruine et al., 2017). Throughout the years, many models have described how destruction complex function is decreased, ranging from complex disassembly, inhibition of GSK3, to degradation of Axin. These will be briefly described in the next section. However, it is agreed that Wnt signaling increases cytoplasmic levels of
βCat, and thus allowing it to enter the nucleus and works with the family of TCF and LEF
transcription factors to behave as a co-activator of transcription to express different Wnt target genes.
There is agreement as to the main positive and negative regulators of the Wnt signaling
pathway (ex: Frizzled, Wnt, LRP5/6, Dsh, APC, Axin, CK1, GSK3, and βCat), yet many key
questions of destruction complex regulation remain. For example: Axin polymerization is necessary for destruction complex function and the destruction complex contains 100s of Axin molecules (Faux et al., 2008; Fiedler et al., 2011; Kishida et al., 1999; Schaefer et al., 2018) - does the size of the complex matter? Also, what do APC and Axin physically do to regulate destruction complex function both in the presence and absence of Wnt signaling? Another gap
in the field is mechanism of βcat transfer from the destruction complex to the E3 ligase. Does the destruction complex and the E3 ligase physically interact with one another? The F-box
protein βTrCP (Slimb in Drosophila), which can directly bind to a phosphorylated βcat (Wu et al., 2003) can co-immunoprecipitate (co-IP) with Axin (Hart et al., 1999; Li et al., 2012; Liu et al., 1999b), suggesting a possible role of Axin to recruit the E3 to the destruction complex. Lastly,
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what is the mechanism of destruction complex turn down? Dishvelled is necessary to recruit Axin to the Wnt receptor complex to turn down destruction complex function (Cliffe et al., 2003; Fiedler et al., 2011; Schwarz-Romond et al., 2007a; Schwarz-Romond et al., 2007b), yet it is not fully understood what Dishevelled is doing to the complex. By understanding how these proteins interact with one another on a molecular level we can better understand how the system goes awry in cancer I will discuss each of these questions in more detail below.
Size matters: Why is polymerization necessary for complex function?
Most models of destruction complex function represent the complex as a simple tetramer of APC:Axin:CK1:GSK3 with a 1:1:1:1 ratio. Interestingly, Axin and APC self-association have been shown to be necessary for complex function (Fiedler et al., 2011; Kishida et al., 1999; Kunttas-Tatli et al., 2014; Mendoza-Topaz et al., 2011). Axin contains a DIX domain which allows it to self-polymerize and form puncta (Faux et al., 2008), and loss of polymerization decreases destruction complex function (Fiedler et al., 2011; Kishida et al., 1999; Pronobis et al., 2016). APC also contains a conserved self-association domain (ASAD). When this domain is deleted in cancer cells, Axin was less able to form robust puncta (Kunttas-Tatli et al., 2014), and function in Wnt signaling is lost (Kunttas-Tatli et al., 2014; Kunttas-Tatli et al., 2015; Roberts et al., 2012). In concert with this, in Drosophila embryos completely lacking APC2, (the main APC family member during Drosophila embryogenesis), no Axin puncta were seen (Mendoza- Topaz et al., 2011). These data suggest that puncta formation is necessary for complex function, but why would polymerization matter?
Condensates: the more the merrier
A re-emerging field in science is that of “phase separation” biochemistry, allowing the creation of a large non-membrane compartment, called a condensate (review in (Banani et al., 2017)). Within a cell there are 100s to 1000s of different chemical reactions occurring ranging from transcription to protein synthesis, and protein degradation. But how do reactants find one
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another? By concentrating reactants in a condensate, different chemical processes can be enhanced, while their separation can inhibit the same reactions. One example of this is the nucleolus - a non-membrane-bound compartment which contains many of the components necessary for ribosome biogenesis (Boisvert et al., 2007; Hyman et al., 2014).
In the Introduction chapter, we proposed that the destruction complex is another
example of a cellular condensate. I propose that the destruction complex forms condensates to
increase its avidity for βCat and the E3 ligase. Interestingly, Axin’s binding affinity for βCat has
been suggested to be quite low, so like adding more people to a tug of war, concentrating Axin
molecules into puncta could increase Axin’s avidity/pull for βCat (Salic et al., 2000). In addition,
APC’s ability to recruit βCat is quite variable and dependent on its phosphorylation (Easwaran et al., 1999; Rubinfeld et al., 1996; Xing et al., 2004). In the absence of phosphorylation, none of
APC’s 10 βCat binding sites (3x 15 amino acid repeats; 7x 20 amino acid repeats) are able to sequester βCat and prevent TCF binding (Spink et al., 2001; Xing et al., 2004). Once APC is
phosphorylated, its affinity for βCat is enhance by 3-500 fold, and can now block TCF binding to
βCat (Xing et al., 2004). Taken together, these results suggest that Axin and APC may need to
form condensates simply to gain the “strength” to recruit βCat. Even though APC can robustly recruit βCat, it must first be phosphorylated, yet when absent from the destruction complex APC
has not been shown to contain a binding site for any kinases. However, Axin can directly bind both APC and the kinase GSK3, and associates with CK1(Dajani et al., 2003; Liu et al., 2002; Spink et al., 2000), therefore Axin can bring a kinase within close proximity to APC, which can
then be phosphorylated to more robustly recruit βCat. In fact, earlier studies have shown that
APC phosphorylation is dependent on Axin (Rubinfeld et al., 2001).
APC has also been shown to stabilize Axin assembly into puncta and increase puncta volume compared to when Axin is expressed alone (Pronobis et al., 2015). Since Axin is the scaffold on which the complex is assembled, these data suggest that APC:Axin interaction could also stabilize other components of the destruction complex that bind Axin. My proposed model
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would thus suggest that regulation of destruction complex size regulates its ability to recruit βcat
and other essential components of the destruction complex. Regulation of destruction complex size could also be a means of destruction complex downregulation. In support of this, when a GFP-tagged Axin is expressed at near endogenous levels, the number of Axin molecules per punctum is decreased by half in cells receiving Wnt signaling (Schaefer et al., 2018). Therefore, in the Wnt-ON cells, cells in which the destruction complex is turned down, there is a decrease in the Axin numbers within the complex. This further suggests that at this lower number of the
scaffold protein, the destruction complex is less efficient at recruiting βCat to label it for
proteasomal degradation. To further test whether Wnt regulates complex size, it would be worth counting the number of Axin molecules in Drosophila embryos that either over- or under-
express Wnt signaling. If Wingless (Wg, Drosophila Wnt) regulates complex size, then the average number of Axin molecules per punctum in the Wg over-expression embryos would be
half that of wg mutant embryos. It would also be interesting to explore how many molecules of
APC are also in the complex, and whether this number also change in response to Wnt signaling.
It is interesting to note that in esophageal cancers, low levels of Axin correlate with poor patient prognosis (Li et al., 2009). This suggest that there may be a minimal level of total Axin
protein to form enough complexes to regulate βCat. Interestingly, early reports on protein levels
within Xenopus egg extracts have suggested core complex proteins were expressed at
dramatically different levels (ex: APC:Axin = 5000:1) (Lee et al., 2001; Lee et al., 2003). Based on these data, it was suggested that Axin is the limiting component of destruction complex function. However more recent studies have suggested that Axin and APC levels in humans and Drosophila are within the same order or magnitude (Kitazawa et al., 2017; Schaefer et al., 2018; Tan et al., 2012). I found that in Drosophila embryos in vivo, raising levels of APC or Axin via protein over-expression does not enhance baseline destruction complex function, suggesting
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sense that one of the proteins that post-translationally alter βCat, such as CK1, GSK3, or the E3
would be rate limiting, causing βCat destruction to slow down. Interestingly, addition of GSK3 to Xenopus egg extracts enhances βCat destruction (Salic et al., 2000).
Condensates: A way to bring multiple complexes together?
The main function of the destruction complex is to phosphorylate βCat so it can be
recognized by the Skp-Cullin-F-box (SCF) E3 ligase, which labels βCat for proteasomal
degradation. Even though βTrCP (Slimb in Drosophila) has been known to be the F-box protein
that recognizes βCat for 20 years (Jiang and Struhl, 1998), how βCat is transferred to the E3 has remained mysterious. I propose 2 models: 1) The destruction complex and the E3 ligase
are two separate complexes and therefore βcat needs to be actively transported from one
complex to another or 2) The destruction complex and the E3 are a part of the same
condensate, thus allowing phosphorylated βCat to be passed from one protein to the next.
Several IP experiments have suggested the latter model (Hart et al., 1999; Kitagawa et al.,
1999; Li et al., 2012; Liu et al., 1999a), with βTrCP co-IPing with APC or Axin, but colocalization
of these complexes has never been observed in vivo and the mechanism of this interaction has never been worked out.
Our preliminary work in SW480 cells (Chapter 3) has provided new insights in
destruction complex interactions. First our data support previous reports that Axin, and not APC recruits Slimb into the complex. Interestingly, we rarely saw any other of the components of the E3 (Cul1 or SkpA) associated with Axin puncta. These data suggest a hypothesis that Slimb
shuttles phosphorylated βCat from the destruction complex to the rest of the E3 for
ubiquitination. In my studies that examined the interaction between Axin and Slimb, robust recruitment of Slimb into Axin puncta occurred approximately 50% of the time, usually when Axin levels were high, suggesting this interaction may be weak. These data suggest that the more Axin present, the more readily Slimb is recruited to the destruction complex.
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As mentioned previously, APC promotes Axin is stabilization in puncta and puncta size, and there are more Axin molecules per punctum in Wnt-OFF cells than in Wnt-ON cells
(Pronobis et al., 2015; Schaefer et al., 2018). Based on these data, we could hypothesize that in Wnt-OFF cells, when complexes are at their largest size, the destruction complex is more
readily able to recruit βTrCP. Therefore, a decrease in complex size in Wnt-ON would therefore
cause a decrease in Slimb recruitment and slow the passing of βCat to the E3 thus resulting in increased βCat levels. These data are supported by IP data that shows in response to Wnt
signaling, Axin is no longer able to recruit βTrCP (Li et al., 2012). A simple binding assay could
test whether Axin concentration affects its interaction with βTrCP. To test whether βTrCP is
shuttled between the destruction complex and the rest of the E3 ligase, it would also be
interesting to restrict βTrCP movement and observe whether this has an effect on βcat destruction. If βcat remains stuck in the destruction complex, it could suggest that βTrCP is necessary to shuttle βcat between complexes. However, if βcat is not stuck in the complex then perhaps another protein that also interacts with βTrCP could be responsible for transferring βcat
to the E3. In vertebrates, WTX could be a candidate gene as it has been shown to enhance
βcat ubiquitination and can directly interact with Axin and βTrCP1/2 (Major et al., 2007). However, there is no known WTX gene in Drosophila. Interestingly, preliminary results
comparing co-localization between fly Axin and Slimb and human Axin and βTrCP, βTrCP was
more robustly recruited to Axin puncta.
Multiple mechanisms of turing down of the destruction complex
Since most colorectal cancers contain an activating mutation in the Wnt pathway, understanding how the destruction complex is inhibited could provide insights in cancer drug treatments. Could a drug be created to promote destruction complex function? As mentioned above, the exact mechanism of destruction complex inhibition is still a mystery. There are several different proposed models for destruction complex regulation. Here I focus on three: Axin degradation, destruction complex re-localization, and loss of interactants.
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Axin is the scaffold of the destruction complex, as it contains known or presumptive
binding sites for itself, APC, CK1, GSK3, βCat, Dvl, and LRP5/6 and can recruit core complex
proteins into cytoplasmic puncta (Dajani et al., 2003; Faux et al., 2008; Fiedler et al., 2011; Kishida et al., 1999; Liu et al., 2002; Spink et al., 2000). Early work in Xenopus egg extracts suggested that Axin was exceptionally rate-limiting, with levels as much as 5000-fold below those of other destruction complex proteins (Lee et al., 2003; Salic et al., 2000) Recent research revealed that Axin levels are not universally much lower than any other protein with in the
destruction complex-in fact Axin levels in both Drosophila and in cultured normal and colorectal cancer cells are in the same order of magnitude as those of APC (Kitazawa et al., 2017;
Schaefer et al., 2018; Tan et al., 2012). Interestingly, previous work in Drosophila examining the role of Wg on destruction complex function suggest very different modes of complex regulation. One group found that Axin levels were strongly reduced in response to Wg (Tolwinski et al., 2003). Another suggested that in response to Wg signaling, Axin was initially stabilized, causing an increase in both the membrane and cytoplasmic pools of Axin (Wang et al., 2016a; Wang et al., 2016b; Yang et al., 2016). Finally, a third group suggested that Wg had no effect in Axin levels, but instead Axin was recruited to the membrane in a Dsh dependent manner (Cliffe et al., 2003). In the following sections, I’ll explore these different models, and consider how the
destruction complex may be regulated by changes in localization, phosphorylation state, and protein levels.
Axin degradation or disassembly: control the scaffold, control the complex?
Several labs have suggested that activation of Wnt signaling initiates the degradation of Axin, though they differ in whether this is a rapid, primary response or a longer adaptation (Lee et al., 2003; Mao et al., 2001; Tolwinski et al., 2003; Wang et al., 2016a; Wang et al., 2016b; Yang et al., 2016). But how is Axin lost? One possible mechanism for Axin loss is via
modification by the poly-ADP-ribosylating (PARP) enzyme Tankyrase (Tnks) (Huang et al., 2009). PARsylation of Axin can lead to ubiquitination and then proteasomal degradation of Axin
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(Huang et al., 2009). In Drosophila and human cultured cells, after a 15-minute incubation with Wnt, Axin PARsylation is increased, and then 1-2 hours later Axin protein levels are decreased (Li et al., 2012; Wang et al., 2016b). Regulation of the destruction complex scaffold makes intuitive sense, since degradation of Axin would result in loss of complex formation. However, loss of Tnks has no effect on embryonic viability in Drosophila; in fact adult flies are grossly normal (Feng et al., 2014). These data suggest that TNKS may be a fine-tuning regulator of Wnt
signaling or that there is another mechanism to label Axin for degradation. Interestingly, in Tnks
mutant flies, Axin levels are still decreased 1-2 hour after Wnt signaling (Yang et al., 2016) If TNKS is unnecessary for Axin degradation, then how are Axin levels decreased after Wnt signaling? Are there two phases of Axin degradation? Axin degradation after long Wnt exposure could be mediated by the RING domain E3 ligase SIAH 1/2. Recently SIAH 1 and SIAH 2 have been identified as novel inhibitor of Axin levels in HEK293T cells (Ji et al., 2017). Once again, however, it is important to note that the single Drosophila Sina family member is adult viable without obvious defects in Wnt signaling (Carthew and Rubin, 1990).
Does a change in scenery result in a change in function?
Early work exploring how Wnt signaling effected Axin often utilized Axin-over-expression studies (Cliffe et al., 2003; Mendoza-Topaz et al., 2011; Tolwinski et al., 2003). However, over-
expression of Axin is known enhance degradation of βcat/Arm in several model systems
(Hamada et al., 1999; Nakamura et al., 1998). Therefore, is the observed regulation of Axin seen after over-expression similar to how endogenous Axin would behave? Recent papers looking at near endogenous levels of Axin have come to somewhat different conclusions about Axin localization in Wnt-OFF and Wnt ON cells (Schaefer et al., 2018; Wang et al., 2016a; Wang et al., 2016b; Yang et al., 2016).
One group expressed Axin at near endogenous levels during embryogenesis and visualized Axin by immuno-staining with antibodies to an epitope tag. Based on their Axin staining, in Wnt-OFF cells Axin was found throughout the cytoplasm at low levels, and no
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cytoplasmic puncta were apparent. In Wnt-ON cells Axin levels were initially elevated both in the cytoplasm and at the membrane (visualized as an increase in staining), but dropped Axin levels several hours later (visualized as a decrease in staining). Interestingly, when we expressed a