We recently reported a screen for potential Wnt and/or GSK3 regulated protein degradation substrates using in vitro expression cloning technique and biochemical reconstitution in a Xenopus egg cytoplasmic extract.1
Using LiCl as a general GSK3 inhibitor or GID (the GSK3 interacting domain of Axin) protein as specific inhibitor of GSK3 activity in the Wnt signaling pathway, we screened pools of cDNAs encoding ~10,000 polypeptides and identified 35 potential GSK3 regulated proteolytic substrates that responded to LiCl in the screen. 12 of the 35 candidates responded to both LiCl and GID, and to other destruction complex inhibitors such as Dsh (dishevelled) and AxinΔRGS (axin that misses the APC-interacting RGS domain), and are thus strong candidates for Wnt regulation. Wnt regulation of selected candidates was verified by expression in vivo in Xenopus embryos. The other 23 candidates responded to LiCl but not GID in the screen, and are thus potential GSK3 protein substrates but not Wnt regulated ( and ref. 1
Figure 1 Potential GSK3 phosphorylation dependent protein degradation substrates identified in biochemical screen (modified from Fig. 3 in ref. 1). 35 novel proteolytic targets of Wnt and/or GSK3 identified in the screen are grouped according to their cellular (more ...)
This study provided evidence that Wnt signaling regulates the stability of multiple proteins, and therefore various cellular processes, in additional to β-catenin mediated gene expression. Many of the identified substrates interact with each other or have similar cellular functions, indicating Wnt and/or GSK3 regulation of those cellular functions. One example includes MRLC (myosin regulatory light chain) and TMEM4 (or MASP for MIR-interacting saposin-like protein). MRLC is a main regulator of myosin contractility and cell motility and its activation has been known to be regulated by multiple signaling pathways.17–19
Wnt signaling has also been known to regulate MRLC activity and cell motility, although through the noncanonical Wnt pathways.20
Little is known about TMEM4, but it has been reported to interact with MRLC and positively regulate its activity and stability.21,22
Finding MRLC and TMEM4 in the screen indicates that the canonical Wnt signaling pathway may also control cell motility in some cell contexts through the destruction complex mediated regulation of MRLC and TMEM4 stability. Interestingly, components of the destruction complex, GSK3 and APC, have been implicated in regulation of cell polarity and cell migration through their regulation of microtubule polarity and dynamics.23,24
Another group of interesting targets identified in the screen include the RNA binding proteins TIAR (T-cell restricted intracellular antigen-related protein) and Sam68, a STAR (signal transduction and activation of RNA) family protein. TIAR has been shown to bind to U-rich sequences near 5' splice sites of pre-mRNAs and modulates alternative splicing.25,26
Indeed, one bioinformatics study estimates that ~15% of alternative cassette exons in the genome are regulated by TIA1/TIAR, suggesting a widespread role of TIAR in the regulation of alternative splicing.27
Sam68 has been reported to regulate the splicing of genes such as CD44 and Bcl-x, and its activity is regulated by cellular signaling and phosphorylation.28,29
Identifying TIAR and Sam68 as potential Wnt signaling regulated proteolysis substrates therefore raise the exciting possibility that Wnt signaling may regulate mRNA splicing and processing through regulation of the stability of these proteins.
Some proteins identified as Wnt regulated destruction complex targets in the screen also play roles in the Wnt signaling pathway. Two of the LiCl and GID positive targets, Trim29 and TACSTD1, have recently been reported to be positive regulators of β-catenin signaling. TACSTD1, also called EpCAM, is a membrane protein that is overexpressed in cancer cells and was one of the first identified cancer antigens.30–32
It undergoes proteolytic cleavage upon extracellular domain homophilic binding and releases the intracellular domain (EpICD). EpICD then enters the nucleus and binds to transcription factors including β-catenin and Lef-1, and activates downstream gene expression.33
Trim29, also known as ATDC (ataxiatelangiectasia group D complementing gene), was found to positively regulate Dishevelled levels and inhibit the destruction complex, thus stabilizing β-catenin.34
Potential Wnt regulation of the stability of EpCAM and ATDC therefore suggests that there exists feedback control of the canonical Wnt/β-catenin signaling pathway, probably resulting in fine control of the signaling strength and length in various cellular contexts.