Canonical Wnt-signalling has been implicated in mouse and human embryonic stem cell (ESC) maintenance, however its requirement is controversial. β-catenin is the key component in this highly conserved Wnt pathway, acting as a transcriptional transactivator. Yet, β-catenin has additional roles at the plasma membrane regulating cell-cell adhesion, complicating the analyses of cells/tissues lacking β-catenin. We report here the generation of a β-catenin deficient mouse ESC (mESC) line and show that self-renewal is maintained in absence of β-catenin. Cell-adhesion is partially rescued by plakoglobin up-regulation, but fails to be maintained during differentiation. When differentiated as aggregates, wild-type mESCs form descendents of all three germ layers, while mesendodermal germ layer formation and neuronal differentiation are defective in β-catenin deficient mESCs. A Tcf/Lef-signalling defective β-catenin variant, which re-establishes cadherin-mediated cell-adhesion, rescues definitive endoderm and neuroepithelial formation, suggesting that β-catenin cell-adhesion function is more important than its signalling function for these processes.

Secreted proteins constitute a large and biologically important subset of proteins that are involved in cellular communication, adhesion and migration. Yet secretomes are understudied because of technical limitations in the detection of low-abundance proteins against a background of serum-containing media. Here we introduce a method that combines click chemistry and pulsed stable isotope labeling with amino acids in cell culture to selectively enrich and quantify secreted proteins. The combination of these two labeling approaches allows cells to be studied irrespective of the complexity of the background proteins. We provide an in-depth and differential secretome analysis of various cell lines and primary cells, quantifying secreted factors, including cytokines, chemokines and growth factors. In addition, we reveal that serum starvation has a marked effect on secretome composition. We also analyze the kinetics of protein secretion by macrophages in response to lipopolysaccharides.

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Murine double-minute clone 2 protein (MDM2) is an E3 ubiquitin ligase that regulates the turnover of several cellular factors including the p53 tumor suppressor protein. As part of the mechanism of p53 induction in response to DNA damage, a cluster of serine residues within the central acidic domain of MDM2 become hypophosphorylated, leading to attenuation of MDM2-mediated p53 destruction. In the present study, we identify the protein kinase CK1delta as a major cellular activity that phosphorylates MDM2. Amino acid substitution, coupled with phosphopeptide analyses, indicates that several serine residues in the acidic domain, including Ser-240, Ser-242, and Ser-246, as well as Ser-383 in the C-terminal region, are phosphorylated by CK1delta in vitro. We also show, through expression of a dominant negative mutant of CK1delta or treatment of cells with IC261, a CK1delta-selective inhibitor, that MDM2 is phosphorylated by CK1delta in cultured cells. These data establish the identity of a key signaling molecule that promotes the phosphorylation of a major regulatory region in MDM2 under normal growth conditions.