Carboxypeptidase E (CPE) Inhibits the Secretion and Activity of Wnt3a

Oncogene (2016) 35, 6416–6428 © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE Carboxypeptidase E (CPE) inhibits the secretion and activity of Wnt3a N Skalka1,4, M Caspi1,4, L Lahav-Ariel1, YP Loh2, K Hirschberg3 and R Rosin-Arbesfeld1 The Wnt pathway has essential roles in cell proliferation, cell fate determination and tumorigenesis by regulating the expression of a wide range of target genes. As a core signaling cascade, the canonical Wnt pathway is regulated at different levels by numerous proteins. We have previously shown that carboxypeptidase E (CPE) is a novel regulator of the canonical Wnt signaling pathway. Here, we show that CPE and the Wnt3a ligand are co-secreted from cells. We show that although the C'-terminal Lys residue of Wnt3a is critical for its activity and is important for the effect of CPE on the Wnt pathway, CPE does not execute its effect by removing this Wnt3a residue. Interestingly, CPE through its N'-terminal sequence, forms aggregates with Wnt3a and possible endoplasmic reticulum (ER) stress leading to its loss of function. Together, our current results provide a mechanistic insight into the way CPE regulates the canonical Wnt signaling pathway. Oncogene (2016) 35, 6416–6428; doi:10.1038/onc.2016.173; published online 4 July 2016 INTRODUCTION induced tumorigenicity, and may serve as a potential tool in the The Wnt/β-catenin signaling pathway has key roles in regulation design of novel colorectal cancer therapeutic strategies. of embryogenesis and adult homeostasis. However, aberrant constitutive activation of this pathway leads to uncontrolled cell proliferation, growth and survival, promoting the progression of RESULTS 1–3 various types of human cancers, especially colorectal cancer. In A novel Wnt3a fusion protein is secreted and active β the absence of a Wnt signal, the levels of -catenin, the key We have previously shown that full-length CPE downregulates the effector of the Wnt signaling pathway, are kept low by the β ‘β ’ Wnt/ -catenin signaling pathway and interacts with the Wnt3a -catenin destruction complex that contains, among others, the ligand both intracellularly and extracellularly.6 To further investi- tumor suppressor protein adenomatous polyposis coli (APC) and gate the relationship between CPE and Wnt3a, we have Axin. This complex promotes the phosphorylation of β-catenin at constructed two vectors encoding mWnt3a fused to the Cherry its N-terminal Ser/Thr residues by casein kinase 1a and glycogen protein either in its C' or N'-terminus (Cherry-Wnt3a and Wnt3a- synthase kinase 3β, marking it for ubiquitylation and subsequent Cherry, respectively). As shown in Figure 1a, Wnt3a-Cherry has a proteasomal degradation. Activation of the pathway by the Wnt ligand or by mutations in APC, Axin or β-catenin disrupts the similar expression pattern to that of the untagged Wnt3a degradation of the latter, resulting in cytoplasmic accumulation of expressed in L-Wnt3a cells. The reciprocal construct (Cherry- β-catenin that translocates into the nucleus where it associates Wnt3a) exhibited a more diffused pattern which resembles the with TCF transcription factors and upregulates the transcription of cherry protein cellular distribution (not shown), and western blot Wnt target genes. The Wnt pathway is tightly regulated, and analysis suggests that the cherry tag is cleaved off during protein uncontrolled changes in Wnt-induced transcription are thought to synthesis (Figure 1b). Both the Wnt3a-Cherry and the Wnt3a lie in the basis of tumorigenesis.1 Previously, we have found expressed from the Cherry-Wnt3a construct were secreted as seen that carboxypeptidase E (CPE), a pro-hormone processing exo- in Figure 1b (lower panel). To ensure that the different plasmids peptidase that cleaves its substrates’ C'-terminal Lysine or Arginine do not induce cell death and that total amount of proteins was residues,4,5 interacts with Wnt3a and decreases Wnt signaling loaded, the conditioned medium (CM) containing the secreted levels.6 In this study, we utilized N'-terminal and C'-terminal proteins was subjected to western blot analysis using an anti-RPL6 tagged Wnt3a fusion proteins and show that although the C'- antibody. In addition, to verify that the changes in constructs terminal Lys residue is crucial for Wnt3a's activity, CPE inhibits Wnt expression are not influenced by the position of the Cherry tag we signaling independently of its enzymatic activity and its ability to have reacted the same lysates to both anti-Wnt3a and anti-Cherry cleave the C'-terminal residue of Wnt3a. Our results further show antibodies (Supplementary Figure S1). Finally, we show that both that CPE reduces Wnt3a protein levels probably through overexpressed Wnt3a ligands are active although the Cherry- autophagy and that the N'-terminal region of CPE triggers the Wnt3a displays higher activity (Figure 1c). This finding could result aggregation of Wnt3a into insoluble cellular fractions. Taken from the Cherry tag being cleaved off as part of the signal peptide together, these results suggest that CPE has novel roles in Wnt- processing along the secretion route. In this scenario, the Wnt3a 1Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 2Section on Cellular Neurobiology, Program on Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA and 3Department of Pathology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. Correspondence: Dr R Rosin-Arbesfeld, Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel. E-mail: [email protected] 4These authors contributed equally to this work. Received 26 November 2015; revised 26 February 2016; accepted 8 April 2016; published online 4 July 2016 Wnt signaling and carboxypeptidase E (CPE) N Skalka et al 6417 Figure 1. Wnt3a-Cherry is both secreted and active. (a) L-Wnt3a cells were transfected with Wnt3a-Cherry. Twenty-four hours later, the cells were fixed, permeabilized and stained with anti-Wnt3a antibodies. Cells were visualized by a confocal microscope. (b) HEK293T cells were transfected with the indicated vectors. Forty-eight hours later, medium was collected and cells were harvested. Lysates and cell media were subjected to SDS–PAGE analysis using anti-Wnt3a antibodies. Anti-actin (lysate) or anti-LRP6 (CM) was used as loading controls. Asterisks mark a degradation product. (c) HEK293T cells were co-transfected with the indicated vectors along with the TOPFLASH/FOPFLASH constructs. Forty-eight hours post transfection, cells were harvested and luciferase values were measured. Values are shown as mean ± s.d. from at least three independent experiments performed in triplicates. Asterisks denote statistical significance of cells transfected with the different Wnt expressing constructs as compared with control samples in an unpaired Student's t-test. *P ⩽ 0.05. **P ⩽ 0.008. C, cherry. ligand reverts to its native form and may be more active co-transfecting HEK293T cells. Figure 2c depicts the interaction compared to the tagged Wnt3a protein. between Wnt3a-Cherry and the full-length CPE protein. As expected, the truncated CPE (ΔN-CPE) did not bind Wnt3a 6 CPE binds and colocalizes with Wnt3a to the Golgi and vesicle-like (Figure 2c). The interaction between CPE and Wnt3a was also 6 structures shown in β-cells that abundantly express the CPE protein. The CPE is known to localize to the trans-Golgi network and secretory cells were transfected with Wnt3a-HA and subjected to immuno- vesicles.7,8 Thus, we examined the subcellular distribution of the precipitation (IP) using an anti-HA antibody (Figure 2d). Wnt3a-Cherry and the CPE-GFP proteins using both live-cell imaging and immunofluorescence. Our results show that the two The C'-terminal lysine of Wnt3a is critical for its signaling activity proteins are expressed in the Golgi apparatus and seem to CPE like other members of the CP family cleaves C'-terminal basic colocalize in secretory vesicles when overexpressed in COS-7 cells residues (Lysine or Arginine) in a broad spectrum of substrates.10 (Figure 2a and Supplementary Movie S1). Immunofluorescence Interestingly, it has been shown that carboxypeptidase Z (CPZ) experiments revealed that CPE and Wnt3a colocalize with the modulates Wnt signaling by removing the terminal arginine of the Golgi marker—Golgin-97 (Figure 2b). As β-catenin was also Wnt4 protein.11,12 As Wnt3a contains a Lysine residue at the reported to localize to the trans-Golgi network,9 we tested C'-terminal position, we decided to test whether this amino acid is whether CPE can direct β-catenin to the Golgi apparatus. important for the function of Wnt3a. Site-directed mutagenesis was However, our results show that the two proteins do not interact performed to construct a Wnt3a that either lacks its last lysine (Supplementary Figure S2A and not shown), and CPE does not (ΔK351) or contains a glutamic acid residue at this position (K351E). seem to sequester β-catenin toward a trans-Golgi network The mutated Wnt3a proteins show a similar expression pattern as localization (Supplementary Figure S2B). Next, we tested the the wild-type Wnt3a-Cherry, when expressed in COS-7 cells ability of Wnt3a-Cherry protein to interact with full-length CPE by (Figure 3a) and retain their ability to interact with full-length CPE © 2016 Macmillan Publishers Limited, part of Springer Nature. Oncogene (2016) 6416 – 6428 Wnt signaling and carboxypeptidase E (CPE) N Skalka et al 6418 Figure 2. CPE and Wnt3a colocalize and interact in vesicle-like structures. (a) COS-7 cells were co-transfected with CPE-GFP and Wnt3a-Cherry. Twenty-four hours later, the cells were fixed and images were taken using a confocal microscope. Enlargement shows vesicles containing Wnt3a and CPE proteins.

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