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 related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes.


METHODS
Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents.


RESULTS
We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated 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, p27Kip1 may play a functional role in modulating membrane signaling.
Yaroslavskiy
B B
BB
Department of Medicine, University of Pittsburgh School of Medicine, Pennsylvania 15213, USA.
Stolz
D B
DB
Watkins
S C
SC
Alber
S M
SM
Bradbury
N A
NA
Steinman
R A
RA
D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D018957:CD59 Antigens / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D037181:Lectins, C-Type; D008213:Lymphocyte Activation; D017830:Octoxynol / Q000494:pharmacology; D012995:Solubility; D013601:T-Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018957:CD59 Antigens; D018797:Cell Cycle Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003902:Detergents; D037181:Lectins, C-Type; D017830:Octoxynol; D025521:Tumor Suppressor Proteins; D000099192:CD69 Antigens