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-1α 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-1β, -2α and -2β, but not caveolin-3 in the leukemia cell lines. Caveolin-1α, which was identified by pAb, was not detected by this method. We show here that caveolin-1α and -2α, but not caveolin-1β and -2β, are tyrosine phosphorylated. This modification is likely to cause the lack of reactivity of caveolin-1α to the mAb, and suggests a possible close relationship to cell activation.