OVERVIEW:
Wnt signaling is an essential cell-cell signaling pathway which regulates development and is often perturbed in cancer. Wnt signaling regulated the levels of beta-catenin, a co-
activator of transcription. In the absence of Wnt signaling, the destruction complex comprised of the tumor suppressor APC, the scaffold protein Axin, and the kinases CK1 and GSK3 recruit beta-catenin and phosphorylate it and thus labeling beta-catenin for proteasomal degradation. Once beta-catenin is phosphorylated, it can now be recognized by the Skp-Cullin-F-box
containing E3 Ligase. Even though it has been known for over 20 years that beta-TrCP is the F- box protein that recognizes phosphorylated beta-catenin, the mechanism of beta-catenin transfer from the destruction complex to the E3 ligase is unknown. We utilized cell culture to visualize that Slimb, the fly homolog of beta-TrCP, is recruited to the destruction complex by Axin. Co-immunoprecipitation and co-localization assays in which we sequentially deleted different domains of Axin reveal that the RGS domain of Axin is necessary for Axin and Slimb to interact. Our data suggests that Slimb shuttles phosphorylated beta-catenin from the destruction complex to the E3 ligase to be ubiquitinated and then passed to the proteasome for
degradation. INTRODUCTION:
During development, Wnt signaling is essential for setting up the body plan and determining cell fate in all animals. Either loss of or mis-regulation of this pathway leads to embryonic lethality. Wnt signaling is also essential for tissue homeostasis. For example, Wnt
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signaling maintains the stem cells in the crypts of the colon, which divide to replace cells lost in the villi. Over 90% of colon cancers contain a mutation inappropriately activating Wnt signaling. The protein most often altered in colon cancer is the tumor suppressor Adenomatous polyposis coli (APC). In fact, more than 80% of all colon cancers express only a truncated form of APC.
APC is a core component of the destruction complex (DC), a key negative regulator of
the Wnt pathway. Βeta-catenin (βcat), main effector of Wnt signaling, forms a complex with TCF/LEF family transcription factors to co-activate of transcription of Wnt target genes. The core components of the DC are APC, the scaffold protein Axin, and the kinases CK1 and GSK3. These proteins form large supermolecular-complexes containing 10s-100s of Axin molecules
(Schaefer et al., 2018), in which APC and Axinwork together to recruit βcat and target it for
destruction. To regulate levels of βcat it must be post-translationally modified, via addition of
ubiquitin. Once βcat has been poly-ubiquitinated it can now be recognized by the proteasome for protein degradation. The process of βcat destruction occurs in four steps: 1-Recruitment, 2- Phosphorylation, 3-E3-ligase ubiquitination, and 4-Degradation by the proteasome.
1. Recruitment
The βcat:DC interaction is mediated through APC and/or Axin. Both contain βcat binding motifs that bind to the Armadillo (Arm) repeats of βcat (Xu and Kimelman, 2007). In Drosophila, APC2 is the main APC family member that is expressed during embryonic development. APC2
contains 8 βcat binding motifs: three 15 amino acid repeats (15R), and five 20 amino acid
repeats (20R). Despite having multiple βcat binding domains, a single APC2 molecule’s ability
to recruit βcat is quite low (Spink et al., 2001; Xing et al., 2004), however phosphorylation of the
20Rs increases APC2´s affinity for βcat (Xing et al., 2004). Interestingly studies in cancer cell
lines and in developing Drosophila embryo suggest many of APC2’s βcat binding motifs are
dispensable for destruction complex function (Pronobis et al., 2016; Yamulla et al., 2014).
Axin contains a single βcat binding motif which is essential for DC function (Pronobis et
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efficiently recruit βcat? As noted above, the DC assembles into a large supra-molecular
complex containing 10s to 100s of molecules of Axin (Schaefer et al., 2018). So perhaps by
concentrating APC2 and Axin molecules into spots or puncta, the DC’s avidity for βcat is
increased. Axin, can self-polymerize via is C-terminal DIX domain, which can polymerize in a head to tail fashion and forming the base scaffold of the DC (Fiedler et al., 2011; Schwarz- Romond et al., 2007a; Schwarz-Romond et al., 2007b). If Axin loses this ability to self-
polymerize, then the DC is no longer to target βcat for destruction (Fiedler et al., 2011). Thus, in
order for βcat to be recruited to the DC, Axin and APC2 need to form higher-order oligomers to increase their avidity for βcat.
2.Phosphorylation
The βcat binding pocket in the E3 ligase can only recognize a phosphorylated βcat (Wu
et al., 2003). Phosphorylation of βcat occurs in 2 steps, via 2 different kinases, CK1 and GSK3.
These kinases are recruited to the DC via interaction with Axin (Dajani et al., 2003; Liu et al.,
2002). The first phosphorylation step is carried out by CK1α. The N-terminal end of βcat, before
the start of its Arm repeat domains, carries a series of conserved serine and threonine residues
spaced four amino acids apart. CK1 phosphorylates βcat at serine 45 (Liu et al., 2002). This
phosphorylation primes βcat to now be bound and then phosphorylated by GSK3β at serine 33,
37, and threonine 41 (Liu et al., 2002). This series of sequential phosphorylation events are
necessary for βcatto now be recognized by the E3 ligase. 3. Ubiquitination
Many substrates are post-translationally modified by attachment of the small protein ubiquitin (Ub). Ubiquitination regulates several cell processes, including degradation by the proteasome, cell cycle progression, regulation of transcription, DNA repair, and signal
transduction (Berndsen and Wolberger, 2014). Ubiquitination of βcat labels it for recognition and
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Wnt target genes. Ubiquitination requires a coordinated dance between 3 different enzymes, E1, E2, and E3. The E1 catalyzes the activation of Ub in an ATP-dependent reaction (Berndsen
and Wolberger, 2014). Ub is then transferred to an E2 enzyme. The E3 ligase theninteracts
with both the substrate and the E2-ubiquitin to promote attachment of Ub to the substrate (Berndsen and Wolberger, 2014). Humans only have a few E1s, approximately 40 E2s, and
more than 500 E3 ligases (Wenzel et al., 2011). The E3 responsible for βcat ubiquitination is
SCFβTrCP. This complex is comprised of: Cullin1 (Cul1), Skp1, the F-box protein βTrCP, and
Ring box (RBX) subunits, which work together to bind to phosphorylated βcat and attach
multiple Ub subunits. Cul1 is the scaffold of the complex, at one end bringing together the Rbx/E2-Ub proteins and at the other end binds Skp1. The small protein Skp1(SkpA in
Drosophila) is the linker between Cul1 and βTrCP. The F-box protein βTrCP (Slimb in
Drosophila) contains the substrate recognition domain of the E3 ligase. The βcat recognition site
spans WD40 repeats on the c-terminal end of βTrCP (Wu et al., 2003). This domain forms a
propeller structure with a pocket that binds only to phosphorylated proteins. βTrCPcan to bind
to several phospho-proteins and thus regulate diverse cell signaling pathways (ex: NFκB and
Hedgehog (Fuchs et al., 2004; Jiang and Struhl, 1998)) however we will focus on its role in
regulating βcat levels. After βTrCP-βcat binding βcat is poly-ubiquitinated and can now be recognized by the proteasome.
4.Degradation
Protein degradation in Eukaryotes cells can occurs via 2 major ways: lysosomal or autosomal proteolysis or the ubiquitin proteasome system. The ubiquitin proteasome system is the major protein degradation pathway utilized in Eukaryotes (Kisselev et al., 2000). The proteasome is a large multi-protein complex composed of 2 subcomplexes: the 20S catalytic core and 1-2 19S regulatory particle(s). These subcomplexes get their name from the size of the proteins after run on a separation column. The 20S core complex is barrel shaped and is
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recognizes poly-ubiquitinated proteins and is believed to aid in transferring the substrate into the catalytic core. Within the barrel shaped 20S protein hydrolysis occurs to create 3-15 aa long peptides. This process is ATP-dependent (Tanaka, 2009). Thus, in the case of Wnt signaling,
the 19S subcomplex of the proteasome recognizes a poly-ubiquitinated βcat and passes it to
the 20S subcomplex to degrade βcat, keeping cytoplasmic levels of βcat low.
While regulation of βcat levels via protein degradation is a key function of Wnt signaling
our understanding of howβcat is transferred from the DC to the E3 is minimal. Based on recent
research from our lab we propose that the DC is a phase separated machine (see Ch1 for more detail). The E3 ligase is a well-studied complex, yet the number of molecules that form an active complex is unknown. If the E3 is also a supra-molecular machine, similar to the DC, we
hypothesize 2 different mechanisms for βcat transfer to the E3. 1) Since the E3 is able to bind and Ub diverse phospho-proteins, it might make sense for the E3 to form a complex separate
from the DC. In this scenario, the DC and the E3 form two separate complexes, requiring βcat to
be shuttled between the complexes. 2) The E3 is a part of the phase separated DC. In this
model once βcat is phosphorylated it is directly transferred to the E3. This would prevent
dephosphorylation of βcat by cellular phosphatases during transit. Immuno-precipitation (IP)
experiments in animalsand in cell culture suggest that at least βTrCP interacts with Axin (Hart
et al., 1999; Kitagawa et al., 1999; Li et al., 2012; Liu et al., 1999). However, none of these
studies looked colocalization between Axin and βTrCP, or other components of the E3, leaving
both models an option, especially if βTrCP was the shuttling protein.
We thus set out to examine these two models, using immuno-fluorescence, IPs, and super-resolution microscopy. Our data support the first mechanism. We found that the
Drosophila βTrcP homolog Slimb is recruited into Axin puncta, independent of Axin’s ability to interact with βcat or APC2. However, we do not detect other components of the SCF complex. Our data also suggest that Slimb either directly or indirectly interacts with Axin’s RGS domain, a
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known APC2 direct interaction domain, perhaps suggesting competitive interaction between APC2-Axin-Slimb.
RESULTS
A system to examine whether the destruction complex and the E3 ligase co-localize
A key step in Wnt signaling is the transfer of phosphorylated βcat from the DC to the E3
ligase complex to begin βcat degradation. One key question in the field involves the mechanism
by which βcat is transferred from one complex to another. Do these complexes form separate
structures within the cytoplasm of the cells, thus requiring some form of protein shuttle to move
βcat? Or is the destruction complex more like a factory for βcat destruction, containing the machinery to first phosphorylate βcat, and then directly pass it down the assembly line to the E3 ligase? Previous work is more consistent with the latter model, as Axin can co-
immunoprecipitate (coIP) with mammalian βTrcP (Hart et al., 1999; Li et al., 2012) and one role
of APC is to protect βcat from dephosphorylation before it is ubiquitinated (Su et al., 2008). To further address this issue, we utilized a colon cancer cell line (SW480) to transfect components of the destruction complex and the E3 ligase to visualize whether they co-localize.
To visualize the destruction complex, we tagged both Drosophila Axin and APC2 with GFP, RFP, or Flg epitope tags (Pronobis et al., 2015; Roberts et al., 2011). For our studies we
utilized the Drosophila proteins, which can rescue βcat destruction in this colorectal cell line (Pronobis et al., 2015; Roberts et al., 2011). Drosophila APC2 is also half the size of human APC1 and therefore easier to transfect and express in cells (Kreiss et al., 1999). When GFP- tagged APC2 (GFP:APC2) is transfected in cells alone, APC2 is found throughout the cytoplasm (Fig 3.1A). When Axin with an RFP tag is transfected alone (Axin:RFP) it forms cytoplasmic puncta, due to Axin’s ability to self-polymerize via its DIX domain (Fig 3.1B)
(Kishida et al., 1999).When GFP:APC2 is expressed along with Axin:RFP, GFP:APC2 is
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interaction leads to larger stabilized destruction complexes (Kunttas-Tatli et al., 2014; Pronobis et al., 2015).
We next examined whether the E3 ligase had any specific localization pattern on its own. To accomplish this, we tagged Drosophila Cul1, SkpA, and Slimb with either GFP, RFP or Flg tags. When each E3 component was expressed alone or when they were expressed in combination, each was diffusely localized in both the cytoplasm and nucleus, without obvious enrichment in any subcellular structure (Fig 3.1D, Fig 3.2A-B and data not shown). In a few cells, there was slight enrichment of proteins in puncta near the nucleus, which may be due to the E3’s known role in regulating centrosome duplication (Wojcik et al., 2000).
Axin and not APC2 can recruit Slimb into the destruction complex
Previous studies have shown that Axin and βTrcPco-IP with one another (Hart et al.,
1999; Kitagawa et al., 1999; Li et al., 2012; Liu et al., 1999).To test Axin and/or APC were
responsible for this co-recruitment we expressed co-expressed Flg:Axin and GFP:APC2 with a
RFP-tagged Slimb. We found that the βTrcP homolog Slimb was robustly recruited to Axin/APC
puncta (Fig 3.3A). However, this did not isolate whether Axin or APC recruited Slimb.
When APC was first discovered, it was believed to be the scaffold of the DC, as it can bind βcat
and coIP with the kinase GSK3 (Polakis, 1997). However, subsequent work revealed that Axin is the actual scaffold of the DC, mediating complex assembly by directly binding all of the core
destruction complex components: APC, GSK3, CK1, and βcat (Dajani et al., 2003; Liu et al., 2002; Spink et al., 2000). To define which whether APC or Axin and recruit Slimb, we expressed either co expressed Axin and Slimb or APC2 and Slimb. The ability of Axin to form puncta made
examining Slimb recruitment straightforward.Co-expression of an RFP-tagged Axin (Axin:RFP)
with a GFP-tagged Slimb (Slimb:GFP) revealed that Axin can recruit Slimb:GFP into
cytoplasmic puncta (Fig 3.1E-F). Interestingly, we observed that robust Slimb:GFP recruitment into Axin:RFP puncta was depentdent on the amount of Axin:RFP protein in the cell Fig 3.1E-F).
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Since APC2 has no specific localization pattern when expressed on its own, it is difficult to tell whether APC2 is able to recruit other proteins. We therefore utilized an APC2 construct containing a mitochondrial localization signal (mito:APC2,(Roberts et al., 2012)). As shown previously, even though mito:APC2 is recruited to the mitochondria, it remains functional, as
evidenced by reduction of βcat levels inSW480 cells and the ability to rescue Drosophila APC2
mutants (Fig 3.3B(Roberts et al., 2012)). Mito:APC2 can still recruit Axin (Fig 3.3C, (Roberts et
al., 2012)). We therefore expressed mito:APC2 with Slimb:GFP to test whether APC2 was capable of E3 recruitment. GFP-tagged mito:APC2 was expressed with RFP- tagged Slimb and colocalization between these proteins was observed. Mito:APC2 was unable to recruit any Slimb (Fig 3.3D), suggesting APC2 is does not directly interact with the E3.
We next wanted to begin to test they hypothesis of whether the whole E3 is recruited by Axin, or just Slimb. To test this we co-expressed Axin:RFP with either GPP-tagged Cul1 (GFP:Cul1) or GFP-tagged SkpA (GFP:SkpA). When expressed alone, both SkpA and Cul1 were found throughout the cytoplasm and nucleus (Fig 3.2A-B). We were surprised to find that while Slimb was robustly recruited to the Axin puncta, Cul1 and SkpA were not (Fig 3.2C-D).
Consistent with previous work with the mammalian homologs, we could coIP Axin and Slimb,
but did not detect co-IP of with either Cul1 or SkpA(Fig 3.2E-F). These data suggest that Axin is
able to recruit Slimb to the DC and not the other components of the E3. The RGS domain of Axin is required for efficient Slimb recruitment
Both Axin and Slimb directly bind to βcat, but at different locations on βcat (Xu and Kimelman, 2007). Therefore previous studies have suggested that the Axin:Slimb interaction
might not be direct, but instead a result of bridging by βcat (Liu et al., 1999). APC2 is also able
to directly bind to βcat, in a similar location as Axin (Xu and Kimelman, 2007). If Axin is only
recruiting Slimb via a βcat linker, then APC2 should also be able to recruit Slimb, something not
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Figure 3.1
Figure 3.1: Axin recruits Slimb into cytoplasmic puncta. A-F) SW480 cells transfected with
the labelled constructs and stained for βcat (except C). A) RFP-tagged wild type Drosophila
Axin self-polymerizes into cytoplasmic puncta and reduces βcat levels. B) When expressed in
cells, GFP:APC2 is diffuse throughout the cell and dramatically reduces βcat levels. C) When
co-expressed, GFP:APC2 is recruited into Axin:RFP puncta. D) GFP tagged Slimb is expressed through out the cytoplasm and nucleus. Slimb expressed alone is unable to significantly reduce
βcat levels. E-F) Co-expression of Axin:RFP and Slimb:GFP. Axin can recruit Slimb:GFP into cytoplasmic puncta and robust recruitment appears to be dependent on Axin concentration.
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Slimb via a βcat linker, we removed the βcat bindingsite from Axin (AxinΔβcat:RFP) (Fig 3.4A)
and co-expressed it with Slimb. If βcat was acting as a bridge between Slimb and Axin, then we
would expect to no longer see Slimb recruitment into Axin puncta. In contrast, if Axin and Slimb interact by another means, then Slimb should still be recruited into the puncta. When
AxinΔβcat:RFP was expressed alone, it was still able to form cytoplasmic puncta since it still
contains its self-polymerization domain (Fig 3.4A & C). When co-expressed with Slimb:GFP,
AxinΔβcat:RFP was still able to recruit Slimb:GFP into puncta (Fig 3.4F), suggesting that the
Slimb-Axin interaction is not solely a result of both proteins binding to βcat. It is interesting to
note that cells expressing AxinΔβcat:RFP were unable to decrease βcat levels (Fig 3.4F’’’),
however we think this is due to the essential role of Axin’s βcat binding motif in DC function
(Pronobis et al., 2016).
To further investigate which domain of Axin was required for Slimb recruitment, we generated
mutants of Axin deleting different domains or regions (Fig 3.4A).Each RFP-tagged Axin mutant
was co-expressed with Flg-tagged Slimb. We then pulled down for RFP (Axin mutant) and
assessed if Slimb was co-IPed. We created four different deletion mutants: 1) AxinΔRGS:RFP-
removed the RGS domain - which allows Axin and APC to directly interact (Spink et al., 2000). 2) AxinΔβcat:RFP, which deleted Axin’s βcat binding motif. 3) AxinΔDIX:RFP - this removed Axin’s polymerization domain and last 4) StAftRGS:RFP - (Start After RGS) this constructe deleted the first third of Axin (Fig 3.4A) We were surprised to see that the two Axin mutants lacking the RGS domain were unable to pull down Flg:Slimb (Fig 3.4B). These data suggest that