p27Kip1 localizes to detergent-insoluble microdomains within lymphocyte membranes

Background: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is re lated strictly to p27Kip1-mediated cell cycle inhibition or to other, possi bly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have b een shown to possess sphingolipid and cholesterol-rich microdomains that ar e insoluble in non-ionic detergents. These "rafts" provide a scaffold for s ignaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. Methods: Localization of p27Kip1 was studied by electron and confocal micro scopy. Association of p27Kip1 with membrane microdomains in unstimulated an d stimulated lymphocytes was determined using Western blots analysis of iso lated membranes variably treated with detergents. Results: We demonstrated that p27Kip1 was present in clusters associated wi th the plasma membrane in normal lymphocytes. The solubility profile of p27 Kip1 in isolated membranes indicated that it was localized to raft structur es. When lymphocytes were stimulated, however, p27Kip1 was excluded from ag gregated raft complexes. Conclusions: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27 Kip1 may play a functional role in modulating membrane signaling.