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 lprllpr 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. ps6 kk phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused ps6 kk to migrate as pp6wk; however, pp6oIck could be clearly distinguished from the pp6O in lpr cells by two-dimensional gel electrophoresis. The pp6O from lpr cells exhibited several isoforms at pH -6.3 to 6.5. Although on two-dimensional gels pp60crc had a pl (6.4 to 6.8) within a similar region, p60'-`mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp6O isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp69c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p5915", the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59#' 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 pp6O from lpr cells. We conclude that p59'v"' phosphorylation is increased in membranes from lprllpr 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.The massively enlarged lymphoid tissue in lprllpr mice is populated by abnormal lymphocytes expressing an array of markers associated with lymphocyte activation (3). Since these cells lack L3T4 and Lyt-2 (CD4-CD8-) (6,41,58), it is the presence of Thy-1 and the productive rearrangement of the T-cell receptor genes (16, 43) that suggest a T-cell origin. These abnormal CD4-CD8-or double-negative T (DNT) cells accumulate in great numbers in the peripheral lymphoid organs, coincident with development of a lupus erythematosus-like syndrome (26). Although the identity of the recessive lpr gene that causes this disorder is unknown, it is expressed intrinsically in T lymphocytes because the lpr phenotype is expressed only in cells of lpr origin when mixtures of lpr and normal bone marrow are given to irradiated hosts (30). DNT cells from lpr mice are known to express c-myb constitutively at u...

Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa

“…An alternative approach commonly used to overcome this problem is to probe with a antibody that specifically recognizes tyrosine phosphorylated at position 416 (Y416) of SRC (anti-pY416) (Bagrodia et al, 1993;Cartwright et al, 1989;Katagiri et al, 1989;Kralisz and Cierniewski, 2000). When mouse SRC becomes activated autophosphorylation of Y416 occurs (Boerner et al, 1996), such that phosphorylation of this residue correlates well with enzyme activity (Amini et al, 1986;Bagrodia et al, 1993;Cartwright et al, 1989;Katagiri et al, 1989;Kralisz and Cierniewski, 2000). To investigate whether SRC activity increased as cells transited from a noncapacitated to a capacitated state, 2D western blot analyses were performed.…”

Hyperactive mTOR pathway promotes lymphoproliferation and abnormal differentiation in autoimmune lymphoproliferative syndrome

“…Indeed, DNT cells of lpr mice exhibit elevated inositol phospholipid turnover, constitutive tyrosine phosphorylation, and aberrant expression of CD45. 42,43 These alterations are expected to induce a state of T-cell activation likely associated with mTOR activation. Because KLRG1 inhibits mTOR signaling, 44 lack of KLRG1 on ALPS DNT cells could further enhance this pathway.…”

Regulation of the Neuronal Nicotinic Acetylcholine Receptor by Src Family Tyrosine Kinases

“…Antibodies-The following antibodies were used in this study: mouse monoclonal antibody (mAb) 337 specific for the ␤4 cytoplasmic domain of human AChR (30); mAb 268 specific for the ␣5 subunit of human AChR (J. Lindstrom); rat mAb 35, which immunoprecipitates ␣1, ␣3, and ␣5 AChR subunits (48,49); B3B9 anti-ERK-2 mouse mAb (a kind gift from M. Weber (50)); mouse mAb EC10 specific for residues [17][18][19][20][21][22][23][24][25][26][27] in the Unique domain of chicken c-Src (51); Q0, a pan-Src family rabbit polyclonal antibody raised against the C-terminal peptide (residues 522-533) of c-Src and reactive with c-Src, Fyn, c-Yes, and other SFKs (24,52); mouse mAb 2-17, reactive with residues 2-17 in the Unique domain of c-Src from all species (Quality Biotech, Camden, NJ); rabbit polyclonal anti-Fyn antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA); mouse mAb anti-c-Yes (Wako Industries, Japan); mouse mAb specific for human transferrin receptor (Zymed Laboratory Inc., South San Francisco, CA); and mouse mAb PY99 specific for phosphotyrosine (Santa Cruz). ChromPure mouse, rabbit, or rat IgGs (Jackson Laboratory) were used as species-matched, negative antibody controls.…”