Protein complexes associated with ß-catenin differentially influence the differentiation profile of neonatal and adult CD8+ T cells.

The canonical Wnt signaling pathway is a master cell regulator involved in CD8+ T cell proliferation and differentiation. In human CD8+ T cells, this pathway induces differentiation into memory cells or a "stem cell memory like" population, which is preferentially present in cord blood. To better understand the role of canonical Wnt signals in neonatal or adult blood, we compared the proteins associated with ß-catenin, in nonstimulated and Wnt3a-stimulated human neonatal and adult naive CD8+ T cells. Differentially recruited proteins established different complexes in adult and neonatal cells. In the former, ß-catenin-associated proteins were linked to cell signaling and immunological functions, whereas those of neonates were linked to proliferation and metabolism. Wnt3a stimulation led to the recruitment and overexpression of Wnt11 in adult cells and Wnt5a in neonatal cells, suggesting a differential connexion with planar polarity and Wnt/Ca2+ noncanonical pathways, respectively. The chromatin immunoprecipitation polymerase chain reaction ß-catenin was recruited to a higher level on the promoters of cell renewal genes in neonatal cells and of differentiation genes in those of adults. We found a preferential association of ß-catenin with CBP in neonatal cells and with p300 in the adult samples, which could be involved in a higher self-renewal capacity of the neonatal cells and memory commitment in those of adults. Altogether, our results show that different proteins associated with ß-catenin during Wnt3a activation mediate a differential response of neonatal and adult human CD8+ T cells.

Separation of membrane proteins according to their hydropathy by serial phase partitioning with Triton X-114.

The detergent Triton X-114, because of its convenient cloud point temperature (22 degrees C), has been used extensively to extract membrane proteins and to separate them in two phases according to their hydropathy. The upper detergent-poor phase contains mostly hydrophilic proteins, whereas hydrophobic ones are found mainly in the lower detergent-rich phase. In this work, we developed a method to fractionate membrane proteins and estimate their hydropathy based on a series of cloud point partitions with Triton X-114. With this method, beetroot plasma membrane proteins were separated in different fractions according to their hydropathy, following the binomial distribution law as expected. This method revealed the presence of both hydrophilic and hydrophobic Ca(2+)-dependent protein kinases in those membranes. At least five distinct Ca(2+)-dependent kinases were observed in in-gel kinase activity assays. This separation procedure was also used as the first step in the purification of a hydrophobic 60-kDa kinase.