Tyrosine Phosphorylation of a c-Src-Like Protein Is Increased in Membranes of CD4^- CD8^- T Lymphocytes from lpr/lpr Mice

Abstract

Mice homozygous for the autosomal recessive lpr gene have a disorder that results in autoimmunity and massive accumulation of T lymphocytes lacking CD4 and CD8 surface markers. These abnormal T cells exhibit constitutive tyrosine phosphorylation of a component of the CD3-T-cell receptor complex. We compared membrane tyrosine phosphorylation in lpr/lpr CD4^- CD8^- T cells and control T cells. lpr membranes exhibited a 7.3-fold increase (n = 16) in tyrosine phosphorylation of a 60-kilodalton protein. The increase was correlated with the Lpr but not the CD4^- CD8^- phenotype in that p60 phosphorylation was not increased in membranes from normal CD4^- CD8^- thymocytes. To identify the p60 in lpr cells, we examined the activity of several T-cell tyrosine-specific protein kinases. p56^lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56^lck to migrate as pp60^lck; however, pp60^lck could be clearly distinguished from the pp60 in lpr cells by two-dimensional gel electrophoresis. The pp60 from lpr cells exhibited several isoforms at pH ~6.3 to 6.5. Although on two-dimensional gels pp60^c-src had a pI (6.4 to 6.8) within a similar region, p60^c-src mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp60 isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp60^c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59^fyn, the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59^fyn was selectively immunoprecipitated from T-cell membranes with specific antisera, its molecular weight, proteolytic cleavage pattern, and behavior on two-dimensional gels were identical to those of the pp60 from lpr cells. We conclude that p59^fyn phosphorylation is increased in membranes from Ipr/lpr CD4^- CD8^- T cells and that the increase is correlated with constitutive tyrosine phosphorylation and perhaps with the expansion of this unusual T-cell population.