Identification of ezrin as an 81-kDa tyrosine-phosphorylated protein in T cells

Abstract

We have used APT affinity purification to isolate tyrosine-phosphorylated proteins from MRL lpr/lpr (lpr) mouse T cells. One such protein is pp81 ezrin, previously identified as a tyrosine-phosphorylated protein in epidermal growth factor-stimulated A431 carcinoma cells. Biochemical analyses in A431 and gastric parietal cells have revealed ezrin to be a cytoskeleton-associated cytosolic protein. In Jurkat T cells, however, using similar methods we have shown ezrin to be a cytosolic protein with no measurable cytoskeletal association. We also observed no increases in ezrin tyrosine phosphorylation in TCR-stimulated Jurkat T cells, unless the cells were pretreated with protein tyrosine phosphatase inhibitors, suggesting that T cell ezrin tyrosine phosphorylation is tightly controlled by protein tyrosine phosphatases. The fraction of tyrosine phosphorylated ezrin in lpr T cells was 5 to 10 times that observed in Jurkat T cells, which along with constitutive TCR-zeta phosphorylation and pp60fyn overexpression, is a feature of the lpr defect.