Tyrosine phosphorylation of a c-Src-like protein is increased in membranes of CD4- CD8- T lymphocytes from lpr/lpr mice.
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. p56lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56lck to migrate as pp60lck; however, pp60lck 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 approximately 6.3 to 6.5. Although on two-dimensional gels pp60c-src had a pI (6.4 to 6.8) within a similar region, p60c-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 pp60c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59fyn, 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 lpr/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.
Katagiri
T
T
Cell Biology and Immunology Program, University of North Carolina School of Medicine, Chapel Hill 27599.
Ting
J P
JP
Dy
R
R
Prokop
C
C
Cohen
P
P
Earp
H S
HS
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005808:Genes, Recessive; D006863:Hydrogen-Ion Concentration; D051379:Mice; D010641:Phenotype; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D013601:T-Lymphocytes / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D010770:Phosphotransferases; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn