P-selectin glycoprotein ligand 1 (PSGL-1) is a leukocyte adhesion molecule involved in cell tether and rolling on activated endothelium. Our work shows that PSGL-1 associates with Syk. This association is mediated by the actin-linking proteins moesin and ezrin, which directly interact with Syk in an ITAM-dependent manner. PSGL-1 engagement induces tyrosine phosphorylation of Syk and SRE-dependent transcriptional activity. Treatment of cells with the Syk inhibitor piceatannol and overexpression of either a Syk dead kinase mutant or an ITAM-mutated moesin abrogated PSGL-1-induced transcriptional activation. These data unveil a new functional role for the ERMs (ezrin/radixin/moesin) as adaptor molecules in the interactions of adhesion receptors and intracellular tyrosine kinases and show that PSGL-1 is a signaling molecule in leukocytes.

During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane–cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, β-actin and α-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti–ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin–ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin–ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.

Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. ␣-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of ␣-tubulin with the C terminus of paxillin. Another member of the tubulin family, ␥-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both ␣-and ␥-tubulin in vivo was confirmed by coimmunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with ␣-and ␥-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.

A screen for natural products bearing pharmacological properties has yielded a secretion of the mollusk Cryptomphalus aspersa (SCA), which possesses skin-regenerative properties. In this report, we outline some of the cellular and molecular effects underlying this observation. First, we found that SCA contained antioxidant SOD and GST activities. In addition, SCA stimulated fibroblast proliferation and rearrangement of the actin cytoskeleton. Additional mechanisms involved in the regenerative effect of SCA included the stimulation of extracellular matrix assembly and the regulation of metalloproteinase activities. Together, these effects provide an array of molecular mechanisms underlying SCA-induced cellular regeneration and postulate its use in regeneration of wounded tissue.

P‐selectin glycoprotein ligand 1 (PSGL‐1) is an adhesion receptor localized on the tips of microvilli that is involved in the rolling of neutrophils on activated endothelium. We found that PSGL‐1 was concentrated at the uropod of chemokine‐stimulated lymphoid cells. Dynamic fluorescence videomicroscopy analyses of migrating lymphocytes demonstrated that PSGL‐1 and moesin redistributed towards the cellular uropod at the trailing edge of these cells, where activated ezrin/radixin/moesin (ERM) proteins were located. An eighteen amino acid sequence in the juxta‐membrane region of the PSGL‐1 cytoplasmic tail was found to be critical for uropod targeting and moesin binding. Substitution of S336, S348, and the basic cluster R337K338 by alanines within this region significantly impaired both moesin binding and PSGL‐1 polarization. These results underline the role of moesin in the subcellular redistribution of PSGL‐1 in lymphoid cells and make evident the importance of specific serine residues within the cytoplasmic tail of PSGL‐1 for this process.