Serine Phosphorylation of L-Selectin Regulates ERM Binding, Clustering, and Monocyte Protrusion in Transendothelial Migration.

The migration of circulating leukocytes toward damaged tissue is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first cellular barrier that is breached in this process. Human CD14+ inflammatory monocytes express L-selectin, bestowing a non-canonical role in invasion during TEM. In vivo evidence supports a role for L-selectin in regulating TEM and chemotaxis, but the intracellular mechanism is poorly understood. The ezrin-radixin-moesin (ERM) proteins anchor transmembrane proteins to the cortical actin-based cytoskeleton and additionally act as signaling adaptors. During TEM, the L-selectin tail within transmigrating pseudopods interacts first with ezrin to transduce signals for protrusion, followed by moesin to drive ectodomain shedding of L-selectin to limit protrusion. Collectively, interaction of L-selectin with ezrin and moesin fine-tunes monocyte protrusive behavior in TEM. Using FLIM/FRET approaches, we show that ERM binding is absolutely required for outside-in L-selectin clustering. The cytoplasmic tail of human L-selectin contains two serine (S) residues at positions 364 and 367, and here we show that they play divergent roles in regulating ERM binding. Phospho-S364 blocks direct interaction with ERM, whereas molecular modeling suggests phospho-S367 likely drives desorption of the L-selectin tail from the inner leaflet of the plasma membrane to potentiate ERM binding. Serine-to-alanine mutagenesis of S367, but not S364, significantly reduced monocyte protrusive behavior in TEM under flow conditions. Our data propose a model whereby L-selectin tail desorption from the inner leaflet of the plasma membrane and ERM binding are two separable steps that collectively regulate protrusive behavior in TEM.

BCL9/9L-ß-catenin Signaling is Associated With Poor Outcome in Colorectal Cancer.

BCL9/9L proteins enhance the transcriptional output of the ß-catenin/TCF transcriptional complex and contribute critically to upholding the high WNT signaling level required for stemness maintenance in the intestinal epithelium. Here we show that a BCL9/9L-dependent gene signature derived from independent mouse colorectal cancer (CRC) models unprecedentedly separates patient subgroups with regard to progression free and overall survival. We found that this effect was by and large attributable to stemness related gene sets. Remarkably, this signature proved associated with recently described poor prognosis CRC subtypes exhibiting high stemness and/or epithelial-to-mesenchymal transition (EMT) traits. Consistent with the notion that high WNT signaling is required for stemness maintenance, ablating Bcl9/9l-ß-catenin in murine oncogenic intestinal organoids provoked their differentiation and completely abrogated their tumorigenicity, while not affecting their proliferation. Therapeutic strategies aimed at targeting WNT responses may be limited by intestinal toxicity. Our findings suggest that attenuating WNT signaling to an extent that affects stemness maintenance without disturbing intestinal renewal might be well tolerated and prove sufficient to reduce CRC recurrence and dramatically improve disease outcome.

Bcl9/Bcl9l are critical for Wnt-mediated regulation of stem cell traits in colon epithelium and adenocarcinomas.

Canonical Wnt signaling plays a critical role in stem cell maintenance in epithelial homeostasis and carcinogenesis. Here, we show that in the mouse this role is critically mediated by Bcl9/Bcl9l, the mammalian homologues of Legless, which in Drosophila is required for Armadillo/beta-catenin signaling. Conditional ablation of Bcl9/Bcl9l in the intestinal epithelium, where the essential role of Wnt signaling in epithelial homeostasis and stem cell maintenance is well documented, resulted in decreased expression of intestinal stem cell markers and impaired regeneration of ulcerated colon epithelium. Adenocarcinomas with aberrant Wnt signaling arose with similar incidence in wild-type and mutant mice. However, transcriptional profiles were vastly different: Whereas wild-type tumors displayed characteristics of epithelial-mesenchymal transition (EMT) and stem cell-like properties, these properties were largely abrogated in mutant tumors. These findings reveal an essential role for Bcl9/Bcl9l in regulating a subset of Wnt target genes involved in controlling EMT and stem cell-related features and suggest that targeting the Bcl9/Bcl9l arm of Wnt signaling in Wnt-activated cancers might attenuate these traits, which are associated with tumor invasion, metastasis, and resistance to therapy.

BCL9 is an essential component of canonical Wnt signaling that mediates the differentiation of myogenic progenitors during muscle regeneration.

Muscle stem cells and their progeny play a fundamental role in the regeneration of adult skeletal muscle. We have previously shown that activation of the canonical Wnt/beta-catenin signaling pathway in adult myogenic progenitors is required for their transition from rapidly dividing transient amplifying cells to more differentiated progenitors. Whereas Wnt signaling in Drosophila is dependent on the presence of the co-regulator Legless, previous studies of the mammalian ortholog of Legless, BCL9 (and its homolog, BCL9-2), have not revealed an essential role of these proteins in Wnt signaling in specific tissues during development. Using Cre-lox technology to delete BCL9 and BCL9-2 in the myogenic lineage in vivo and RNAi technology to knockdown the protein levels in vitro, we show that BCL9 is required for activation of the Wnt/beta-catenin cascade in adult mammalian myogenic progenitors. We observed that the nuclear localization of beta-catenin and downstream TCF/LEF-mediated transcription, which are normally observed in myogenic progenitors upon addition of exogenous Wnt and during muscle regeneration, were abrogated when BCL9/9-2 levels were reduced. Furthermore, reductions of BCL9/9-2 inhibited the promotion of myogenic differentiation by Wnt and the normal regenerative response of skeletal muscle. These results suggest a critical role of BCL9/9-2 in the Wnt-mediated regulation of adult, as opposed to embryonic, myogenic progenitors.

Mutagenesis of the ezrin-radixin-moesin binding domain of L-selectin tail affects shedding, microvillar positioning, and leukocyte tethering.

L-selectin is a cell adhesion molecule that mediates the initial capture (tethering) and subsequent rolling of leukocytes along ligands expressed on endothelial cells. We have previously identified ezrin and moesin as novel binding partners of the 17-amino acid L-selectin tail, but the biological role of this interaction is not known. Here we identify two basic amino acid residues within the L-selectin tail that are required for binding to ezrin-radixinmoesin (ERM) proteins: arginine 357 and lysine 362. L-selectin mutants defective for ERM binding show reduced localization to microvilli and decreased phorbol 12-myristate 13-acetate-induced shedding of the L-selectin ectodomain. Cells expressing these L-selectin mutants have reduced tethering to the L-selectin ligand P-selectin glycoprotein ligand-1, but rolling velocity on P-selectin glycoprotein ligand-1 is not affected. These results suggest that ERM proteins are required for microvillar positioning of L-selectin and that this is important both for leukocyte tethering and L-selectin shedding.

The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.