Affinity purification mass spectrometry identifies MIB1 as a novel interaction partner with RYK. (A) Illustration depicts the RYK-associated protein interaction network discovered by affinity purification mass spectrometry (black lines). The STRING database was used to curate additional interactions among proteins found to interact with RYK (gray lines). The size of the node indicates the total number of peptides identified in eight independent purifications. (B) Schematic representation of RYK and MIB1 proteins depicting predicted domains. (C) Western blot for endogenous MIB1 demonstrates interaction with streptavidin affinity-purified glue-tagged RYK, RYKICD, or a membrane tethered form of the RYKICD (MYR-RYKICD). Untransfected, glue-TCF4, and glue-NCK2 cells served as negative controls. (D) Western blot for glue-RYKICD demonstrates interactions with flag affinity-purified full-length MIB1, the N terminus of MIB1, or a point mutant (C997S) of MIB1 that renders it catalytically impaired. N-terminally truncated MIB1 and negative controls do not copurify RYK. Blots represent n > 3.

MIB1 promotes RYK protein turnover. (A) Western blot of cell lysates showing that MIB1 cDNA overexpression results in a decrease in full-length RYK but not RYKICD or MYR-RYKICD. (B) Western blot of cell lysates showing that MIB1 cDNA overexpression results in a decrease in full-length mouse RYK and noncleavable RYK-RC but not mouse RYKICD. (C) Western blot of cell lysates showing that MIB1 cDNA overexpression results in a decrease in full-length mouse RYK in the presence of γ-secretase inhibitors. (D) Western blot of cell lysates showing that MIB1-dependent degradation of RYK can be reversed by inhibition of the proteasome with MG132 or inhibition of the lysosome with chloroquine. (E) Western blot of cell lysates demonstrating that the inhibition of the expression of TSG101, a member of the ESCRT pathway, reverses the ability of MIB1 to degrade RYK. (F) Western blot of cell lysates demonstrating that the turnover of full-length RYK in the presence of translation inhibition by cycloheximide can be reversed with proteasome or lysosome inhibition. (G) Western blot of cell lysates demonstrating that the turnover of full-length RYK in the presence of cycloheximide can be reversed by knockdown of MIB1 with siRNAs. Blots represent n = 3.

MIB1 quantitatively reduces RYK surface expression in the absence of protein degradation. (A, B, D, and E) Histograms of individual cell fluorescence intensities depicting the distribution of surface labeling of RYK in nonpermeabilized HEK293T cells treated for 2 h with chloroquine and MG132, labeled, and then analyzed by flow cytometry. (A) Histogram shows the background of anti-HA immunoreactivity in vector only as compared with glue-RYK–transfected cells. Background intensity was used to determine the RYK-positive gate in each experiment individually. (B) Histogram of surface RYK expression on cells transfected with glue-RYK and vector only or flag-MIB1 or flag-MIB1 mutants. (C) Graph represents the number of cells in the RYK-positive gate shown in B. Data demonstrate a significant decrease in surface RYK expression when cotransfected with MIB1 but not MIB1-C997S or MIB1-ΔRN3. Data are mean + SD (error bars). *, P < 0.05. (Student’s t test, n = 3). (D) Histogram of surface RYK expression on cells transfected with N-terminal glue-tagged RYKΔICD and with or without flag-MIB1. (E) Histogram of surface RYK expression on cells transfected with glue-RYK and control, MIB1, or RYK siRNAs. (F) Graph representing the number of cells in the RYK-positive gate shown in E. Data demonstrate that MIB1 loss of function significantly increases the level of RYK expression on the cell surface. Data are mean + SD (error bars). **, P < 0.005. (Student’s t test, n = 3).

Endogenous RYK and MIB1 are required for Wnt/CTNNB1 signaling at the level of receptor activation. (A) RYK and MIB1 siRNA transfection inhibits cDNA overexpression-mediated activation of BAR by Wnt-3A and Disheveled (DVL3) but not CTNNB1 or nondegradable CTNNB1* (S33A, S37A, T41A, and S45A). Data are mean + SD (error bars). (B) Western blot of cell lysates after a time course of treatment with Wnt-3A–conditioned media in cells transfected with control or MIB1 siRNAs. (C) Western blot after 1 h of treatment with Wnt-3A–conditioned media in HEK293T cells transfected with the indicated siRNAs. In rows labeled conA, cell lysates were stripped of membrane-associated proteins by overnight incubation with conA Sepharose resin. (D) Western blot after 1-h treatment with Wnt-3A–conditioned media in U2OS cells transfected with the indicated siRNAs. Blots represent n = 3.

C. elegans MIB1 RNAi genetically interacts with lin-18/RYK. (A) Phylogram representing the relationship of ceMIB to the top four hits from a BLASTp analysis of the human proteome. (B) Quantification of the bivulva phenotype from three independent RNAi feeding experiments. *, P < 0.0001; #, P < 0.05, Fisher’s exact test. (C–F) Images of L4 stage C. elegans larvae. ceMIB RNAi, mutation in lin-18/RYK, or the combination of both results in the bivulva phenotype characteristic of abnormal vulva cell fate specification. Anterior is to the left and dorsal is up. Arrowheads indicate vulval invaginations. Bar, 10 µm.

MIB1 promotes RYK ubiquitination. (A) Western blot analyzing metal-affinity purification of His-tagged ubiquitin under denaturing conditions. The blot shows that that MIB1 can promote the ubiquitination of the N-terminal glue-tagged RYK, RYKICD, and membrane-tethered MYR-RYKICD but not RYK lacking the ICD (RYKΔICD). Mutant MIB1, C997S, and truncated MIB1, ΔRN3, have little or no catalytic activity with respect to RYK. (B) Western blot analyzing streptavidin purification of glue-RYKICD. The blot shows the conjugation of endogenous ubiquitin promoted by MIB1 and by nonphosphorylatable MIB1-S807A, but not by MIB1-C997S. (C) Western blot analyzing metal affinity purification of His-tagged ubiquitin under denaturing conditions showing that MIB1 can promote the ubiquitination of C-terminal myc-tagged mouse RYK, RYKICD, and noncleavable RYK-RC. Blots represent n > 3. The asterisk indicates a nonspecific band.

MIB1 colocalizes with RYK on intracellular membranes. (A–D) Single-plane confocal images of immunofluorescent labeling of HEK293T cells transiently transfected with cDNA encoding full-length glue-tagged RYK and flag-tagged MIB1 or variants of MIB1. RYK is primarily localized to the plasma membrane when expressed alone (A). However, when overexpressed with MIB1 (B), RYK and MIB1 colocalize to intracellular membranous organelles that are also positive for Rab5. Catalytically impaired MIB1-C997S colocalizes with RYK in aggregates at or near the plasma-membrane (C). In contrast, overexpression of MIB1 lacking all three enzymatic domains MIB1-ΔRN3 (D) minimally affects RYK surface localization and colocalizes at the sites of cell protrusions. (E) Quantification of immunocytochemistry demonstrating that MIB1 and MIB1-C997S reduce the distribution of RYK along the plasma membrane. Data are mean + SEM (error bars). *, P < 0.005 (Student’s t test, n = 4). Bar, 10 µm.

Endogenous RYK and MIB1 are required for Wnt/CTNNB1 signaling. (A and C) Graphs showing results from a Wnt/CTNNB1-activated luciferase-based reporter (BAR). Data are mean + SD (error bars). (B and D–F) Quantitative RT-PCR data showing a requirement for RYK and MIB1 in the expression of Wnt target genes in multiple cell lines. Data are mean + SD (error bars). (A) HEK293T cells were transfected with siRNAs for 2 d and then treated overnight with control or Wnt-3A–conditioned media. Results are normalized to one for unstimulated cells. Multiple nonoverlapping siRNAs targeting either RYK or MIB1 significantly reduce Wnt-3A–mediated reporter activation. Data are representative of n > 5 experiments. (B) Increases in AXIN2 mRNA levels resulting from treatment with Wnt-3A–conditioned media are reduced in the context of MIB1 or RYK siRNA transfection in HEK293T cells. (C) Graph showing that MIB1 and RYK are required for Wnt-3A–mediated activation of BAR in U2OS cells treated as in A. n = 5. (D, E, and F) Graphs showing the results of quantitative RTPCR for AXIN2 (D and F) and NKD1 (E), demonstrating a requirement for MIB1 and RYK for the activation of endogenous Wnt-target genes by Wnt-3a in U2OS and HeLa cells. n = 3.

MIB1 requires endogenous RYK to activate Wnt/CTNNB1 signaling. (A) Graph demonstrating that MIB1 cDNA overexpression but not catalytically impaired MIB1-C997S can activate BAR in the presence or absence of Wnt-3A–conditioned media. Cells were transfected 6–8 h before treatment with conditioned media overnight. Data are mean + SD. n = 3. (B) Data generated from quantitative RT-PCR or HEK293T cells overexpressing MIB1 showing activation of the Wnt target gene, AXIN2. Data are mean + SD (error bars). *, P < 0.05 using a Student’s t test. n = 3. (C) Western blot of HEK293T cells overexpressing MIB1 showing that MIB1 can activate LRP6 phosphorylation and stimulate CTNNB1 stabilization. In rows labeled conA, cell lysates were stripped of membrane-associated proteins by overnight incubation with conA Sepharose resin. Blot represents n = 3. (D) Data showing the enhancement of Wnt-3A–induced BAR activity by the overexpression of MIB1 but not by mutants of MIB1 that lack the ability to bind RYK or lack enzymatic activity. The dotted line indicates the level of reporter activation by Wnt-3A–conditioned media alone. Data are mean + SD (error bars). n = 3. (E) Data demonstrating that MIB1-mediated activation of BAR is reversed by multiple independent siRNAs targeting either CTNNB1 or RYK. The dotted line indicates the level of reporter activation by MIB1 overexpression alone. Data are mean + SD (error bar). n = 3.