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Cellular proteins are degraded in either proteasomes or lysosomes depending on the types of ubiquitin chains that covalently modify them. It is not known whether the choice between these two pathways is physiologically regulated. The Lys48-polyubiquitin chain is the major signal directing proteins for degradation in proteasomes. Here, we report the unexpected finding that canonical Wnt signaling translocates some K48-linked polyubiquitinated proteins to the endolysosomal pathway. Proteasomal target proteins, such as b-catenin, Smad1, and Smad4, were targeted into endolysosomes in a process dependent on GSK3 activity. Relocalization was also dependent on Axin1 and the multivesicular body (MVB) proteins HRS/Vps27 and Vps4. The Wnt-induced accumulation of K48-linked polyubiquitinated proteins in endolysosomal organelles was accompanied by a transient decrease in cellular levels of free mono-ubiquitin, which may contribute to Wnt-regulated stabilization of proteins (Wnt/ STOP). We conclude that Wnt redirects Lys48-polyubiquitinated proteins that are normally degraded in proteasomes to endolysosomes.

Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer.

Sustained canonical Wnt signaling requires the inhibition of glycogen synthase kinase 3 (GSK3) activity by sequestration of GSK3 inside multivesicular endosomes (MVEs). Here, we show that Wnt signaling is increased by the lysosomal inhibitor chloroquine, which causes accumulation of MVEs. A similar MVE expansion and increased Wnt responsiveness was found in cells deficient in presenilin, a protein associated with Alzheimer's disease. The Wnt-enhancing effects were entirely dependent on the functional endosomal sorting complex required for transport (ESCRT), which is needed for the formation of intraluminal vesicles in MVEs. We suggest that accumulation of late endosomal structures leads to enhanced canonical Wnt signaling through increased Wnt-receptor/GSK3 sequestration. The decrease in GSK3 cytosolic activity stabilized cytoplasmic GSK3 substrates such as β-catenin, the microtubule-associated protein Tau, and other proteins. These results underscore the importance of the endosomal pathway in canonical Wnt signaling and reveal a mechanism for regulation of Wnt signaling by presenilin deficiency., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Arginine methylation has emerged as a widespread and reversible protein modification with the potential to regulate a multitude of cellular processes, but its function is poorly understood. Endolysosomes play an important role in Wnt signaling, in which glycogen synthase kinase 3 (GSK3) becomes sequestered inside multivesicular bodies (MVBs) by the process known as microautophagy, causing the stabilization of many proteins. Up to 20% of cellular proteins contain three or more consecutive putative GSK3 sites, and of these 33% also contain methylarginine (meArg) modifications. Intriguingly, a cytoskeletal protein was previously known to have meArg modifications that enhanced subsequent phosphorylations by GSK3. Here, we report the unexpected finding that protein arginine methyltransferase 1 (PRMT1) is required for canonical Wnt signaling. Treatment of cultured cells for 5-30 min with Wnt3a induced a large increase in total endocytic vesicles which were also positive for asymmetric dimethylarginine modifications. Protease protection studies, both biochemical and in situ in cultured cells, showed that many meArg-modified cytosolic proteins became rapidly translocated into MVBs together with GSK3 and Lys48-polyubiquitinated proteins by ESCRT-driven microautophagy. In the case of the transcription factor Smad4, we showed that a unique arginine methylation site was required for GSK3 phosphorylation and Wnt regulation. The enzyme PRMT1 was found to be essential for Wnt-stimulated arginine methylation, GSK3 sequestration, and canonical Wnt signaling. The results reveal a cell biological role for PRMT1 arginine methylation at the crossroads of growth factor signaling, protein phosphorylation, membrane trafficking, cytosolic proteolysis, and Wnt-regulated microautophagy.

The earliest event in Xenopus development is the dorsal accumulation of nuclear β-catenin under the influence of cytoplasmic determinants displaced by fertilization. In this study, a genome-wide approach was used to examine transcription of the 43,673 genes annotated in the Xenopus laevis genome under a variety of conditions that inhibit or promote formation of the Spemann organizer signaling center. Loss of function of β-catenin with antisense morpholinos reproducibly reduced the expression of 247 mRNAs at gastrula stage. Interestingly, only 123 β-catenin targets were enriched on the dorsal side and defined an early dorsal β-catenin gene signature. These genes included several previously unrecognized Spemann organizer components. Surprisingly, only 3 of these 123 genes overlapped with the late Wnt signature recently defined by two other groups using inhibition by Dkk1 mRNA or Wnt8 morpholinos, which indicates that the effects of β-catenin/Wnt signaling in early development are exquisitely regulated by stage-dependent mechanisms. We analyzed transcriptome responses to a number of treatments in a total of 46 RNA-seq libraries. These treatments included, in addition to β-catenin depletion, regenerating dorsal and ventral half-embryos, lithium chloride treatment, and the overexpression of Wnt8, Siamois, and Cerberus mRNAs. Only some of the early dorsal β-catenin signature genes were activated at blastula whereas others required the induction of endomesoderm, as indicated by their inhibition by Cerberus overexpression. These comprehensive data provide a rich resource for analyzing how the dorsal and ventral regions of the embryo communicate with each other in a self-organizing vertebrate model embryo.

Canonical Wnt signaling influences cellular fate and proliferation through inhibition of Glycogen Synthase Kinase (GSK3) and the subsequent stabilization of its many substrates, most notably β-Catenin, a transcriptional co-activator. MITF, a melanoma oncogene member of the microphthalmia family of transcription factors (MiT), was recently found to contain novel GSK3 phosphorylation sites and to be stabilized by Wnt. Other MiT members, TFEB and TFE3, are known to play important roles in cellular clearance pathways by transcriptionally regulating the biogenesis of lysosomes and autophagosomes via activation of CLEAR elements in gene promoters of target genes. Recent studies suggest that MITF can also upregulate many lysosomal genes. MiT family members are dysregulated in cancer and are considered oncogenes, but the underlying oncogenic mechanisms remain unclear. Here we review the role of MiT members, including MITF, in lysosomal biogenesis, and how cancers overexpressing MITF, TFEB or TFE3 could rewire the lysosomal pathway, inhibit cellular senescence, and activate Wnt signaling by increasing sequestration of negative regulators of Wnt signaling in multivesicular bodies (MVBs). Microarray studies suggest that MITF expression inhibits macroautophagy. In melanoma the MITF-driven increase in MVBs generates a positive feedback loop between MITF, Wnt, and MVBs. [ABSTRACT FROM AUTHOR]

Inhibition of GSK3 by Wnt signaling stabilizes many cellular proteins, but proof that this effect is independent of β-catenin-mediated transcription is lacking. Koch, Acebron, and colleagues (2015) now demonstrate that transcriptionally silent mammalian sperm require Wnt signaling via exosomes to prevent protein degradation during their lengthy travels through the epididymis. [ABSTRACT FROM AUTHOR]