The role of the CD45 phosphotyrosine phosphatase in coupling the T cell antigen receptor complex (TCR) to intracellular signals was investigated. CD45‐ HPB‐ALL T cells were transfected with cDNA encoding the CD45RA+B+C‐ isoform. The tyrosine kinase activity of p59fyn was found to be 65% less in CD45‐ cells than in CD45+ cells, whereas p56lck kinase activity was comparable in both sub‐clones. In CD45‐ cells the TCR was uncoupled from protein tyrosine phosphorylation, phospholipase C gamma 1 regulation, inositol phosphate production, calcium signals, diacylglycerol production and protein kinase C activation. Restoration of TCR coupling to all these pathways correlated with the increased p59fyn activity observed in CD45‐transfected cells. Co‐aggregation of CD4‐ or CD8‐p56lck kinase with the TCR in CD45‐ cells restored TCR‐induced protein tyrosine phosphorylation, phospholipase C gamma 1 regulation and calcium signals. Receptor‐mediated calcium signals were largely due (60–90%) to Ca2+ influx, and only a minor component (10–40%) was caused by Ca2+ release from intracellular stores. Maximal CD3‐mediated Ca2+ influx occurred at CD3 mAb concentrations at which inositol phosphate production was non‐detectable. These results indicate that CD45‐regulated p59fyn plays a critical role in coupling the TCR to specific intracellular signalling pathways and that CD4‐ or CD8‐p56lck can only restore signal transduction coupling in CD45‐ cells when brought into close association with the TCR.

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The homogenates of amyloid-laden spleens prepared from CBA mice were analysed by SDS-PAGE and immunoblotting employing rat anti-murine monoclonal antibody, MSA 4-26. The results showed that the precursor of amyloid A protein (AA), serum amyloid A protein 2 (SAA2), and SAA intermediates with molecular weights of 10,000, 9000, and 8000 were contained in amyloid-laden tissues. The experiment using sonicated spleen cells and acute phase murine sera showed a delay in the degradation rate of SAA2 on cell fragments and the remains of SAA1 in supernatants. This result can explain disappearance of SAA2 from the murine serum during amyloidogenesis in vivo.

We have recently shown that glucocorticoids dramatically increase the number of interleukin 1 (IL 1) receptors on human peripheral blood mononuclear cells (PBMC) and that IL 1 selectively induces the phosphorylation of a cytosolic 65-kilodalton (kDa) protein (pp 65) in glucocorticoid-pretreated PBMC. We describe here the purification and biochemical characteristics of pp 65. 32P-Labeled pp 65 was purified to homogeneity from the cytosol fraction of IL 1 stimulated [32P]orthophosphate-labeled PBMC by sequential chromatography on Sephacryl S-200, high-performance liquid chromatography (HPLC) anion exchange, and hydroxyapatite HPLC. The purified pp 65 was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The unphosphorylated 65-kDa protein (p 65) was also purified to homogeneity in a similar way. About 40 micrograms of purified 65-kDa protein was recovered from 5 x 10(8) PBMC. Analysis of the amino-terminal sequence of the purified pp 65 revealed the amino terminus of pp 65 to be blocked. Amino acid sequence analysis of a cyanogen bromide cleaved peptide showed pp 65 to be a unique protein whose protein sequence has not yet been reported. Studies of the distribution of p(p) 65 based on Western blotting using specific polyclonal rabbit antibody to p(p) 65 showed that p(p) 65 exists in a variety of cells such as neutrophils, monocytes, B lymphocytes, and myeloid cells. It could not be detected in the T cell leukemia cell line (MOLT), melanoma cells, and fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

We have isolated from KB cells stimulated with interleukin-1 (IL-1) a protein kinase that phosphorylates a peptide (T669) based on the sequence around T669 of the epidermal growth factor (EGF) receptor. The enzyme, which had an apparent molecular mass of 45 kDa on gel-filtration chromatography, was purified 170,000-fold from cytosolic extracts by sequential chromatography on Mono Q, Mono S, phenyl-Sepharose, Superose 12, ATP-Sepharose and Mono Q. The enzyme activity co-chromatographed at the last step with a 45 kDa protein band that stained for phosphotyrosine. This peak fraction also contained some actin and a 60 kDa protein that stained weakly for phosphotyrosine. The T669 peptide is a substrate for mitogen-activated protein (MAP) kinase. Amounts of IL-1-induced T669 kinase and activated recombinant p42 MAP kinase having equal activity on T669 peptide were compared on commonly used MAP kinase substrates. T669 kinase was two or three orders of magnitude less active on myelin basic protein or microtubule-associated protein-2 than was MAP kinase. The IL-1-induced T669 kinase did not react with antiserum to p42/p44 MAP kinase. It was inactivated by treatment with protein phosphatase 2A or protein phosphotyrosine phosphatase 1B, so it may be regulated by dual phosphorylation in similar fashion to MAP kinase. The dephosphorylated enzyme was not re-activated by MAP kinase kinase. This novel enzyme could lie on a kinase cascade induced by IL-1. It may be responsible for phosphorylating T669 of the EGF receptor.