Differential-expression and tyrosine-phosphorylation profiles of caveolin isoforms in human T cell leukemia cell lines.
Caveolin, the essential structural component of caveolae, serves as a scaffolding protein onto which signaling molecules are assembled, and functions as a negative regulator for signal transduction. Caveolin-1 and -2 are expressed in most cell types, but are not expressed in normal blood cells and cell lines. We previously demonstrated that caveolin-1 is expressed in a panel of human leukemia cell lines that show an activated T cell phenotype. In that study, we detected two caveolin bands by Western blotting using a polyclonal antibody (pAb) reacting with caveolin-1, -2, and -3. We identified caveolin-1alpha by its large molecular weight, but did not discriminatively detect other caveolin families. Since anti-caveolin-1 monoclonal antibody (mAb) was reported not to detect caveolin-1 in some cases, here we developed a sensitive method for the discriminative detection of caveolin-1, -2, and -3 by modified Western blotting. Caveolins were solubilized using a two-step procedure and detected by immunoprecipitation with a pAb to caveolins followed by Western blotting with mAbs specific to each caveolin. Using this method we detected caveolin-1beta, -2alpha and -2beta, but not caveolin-3 in the leukemia cell lines. Caveolin-1alpha, which was identified by pAb, was not detected by this method. We show here that caveolin-1alpha and -2alpha, but not caveolin-1beta and -2beta, are tyrosine phosphorylated. This modification is likely to cause the lack of reactivity of caveolin-1alpha to the mAb, and suggests a possible close relationship to cell activation.
Tsuji
Yuichiro
Y
Department of Otolaryngology, Osaka Medical College, Daigakumachi, Takatsuki 569-8686, Japan.
Hatanaka
Michiyo
M
Maeda
Tamaki
T
Seya
Tsukasa
T
Takenaka
Hiroshi
H
Shimizu
Akira
A
D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D045744:Cell Line, Tumor; D006801:Humans; D015458:Leukemia, T-Cell / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D014443:Tyrosine