Structural study of the O-linked sugar chains of human leukocyte tyrosine phosphatase CD45.

The O-linked sugar chains of the human leukocyte cell surface glycoprotein CD45 were released as tritium-labeled oligosaccharides by beta-elimination in the presence of NaB3H4. Mono Q column chromatography revealed that they comprise neutral (64%) and acidic (36%) oligosaccharides, the latter of which were converted to neutral ones by Arthrobacter ureafaciens sialidase treatment. Structural studies of each oligosaccharide fractionated on a Bio-Gel P-4 column by sequential exoglycosidase digestion and by methylation analysis revealed that human leukocyte CD45 contains mainly core 1 and core 2 oligosaccharides, 15% of which are modified with poly (N-acetyllactosamine) chains in different extensions. CD45 consists of several isoforms which were isolated after cell surface sialic acid residues were labeled by periodate/NaB3H4 treatment. Bio-Gel P-6 column chromatography of a mixture of the tritium-labeled glycopeptide/oligosaccharides obtained by pronase-digestion followed by mild alkaline borohydride treatment showed that distribution of the sialylated core 2 oligosaccharides is different among CD45 isoforms.

Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.

Structural study of the sugar chains of human leukocyte common antigen CD45.

The leukocyte cell surface glycoprotein CD45 is a protein tyrosine phosphatase and is involved in signal transduction mediated by the T cell antigen receptor. The asparagine-linked sugar chains were released as oligosaccharides from purified CD45 by hydrazinolysis. Approximately 6 mol of sugar chains was released from 1 mol of CD45. These sugar chains were converted to radioactive oligosaccharides by reduction with NaB3H4 and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. Binding of the sialylated oligosaccharides to an SNA-agarose column as well as methylation analysis revealed that the oligosaccharides have only alpha-2,6-linked sialic acid residues. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that CD45 contains mainly bi-, tri-, and tetraantennary complex-type sugar chains. About 46% of the tetraantennary complex-type sugar chains had the poly(N-acetyllactosamine) groups and 18% of the 2,4-branched triantennary complex-type sugar chains had the fucosyl N-acetyllactosamine group. A portion of the bi- and 2,4-branched triantennary complex-type sugar chains were bisected. In addition to these sugar chains, a small amount of high mannose-type and hybrid-type sugar chains were detected.

Reduced tyrosine phosphorylation in polyamine-starved cells.

Onset of cell proliferation is associated with enhanced turnover of the polyamines putrescine, spermidine, and spermine, particularly evident in the massive increase in the activity of the rate-limiting enzyme in their production, ornithine decarboxylase (ODC). The physiological functions of these polyamines, however, have remained unclear. Here we report that treatment of LSTRA cells for 2-18 h with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreased the amount of phosphotyrosine in several cellular substrates including the T cell protein tyrosine kinase p56lck. No reductions in the amount of p56lck, overall synthesis of protein and DNA, or cell viability were observed until much later. DFMO did not affect the catalytic activity of p56lck in vitro and the activity of p56lck immunoprecipitated from DFMO-treated cells was unaltered. Addition of putrescine, the reaction product of ODC, completely reversed the effect of DFMO on tyrosine phosphorylation. Finally, we provide evidence that polyamines reduce the activity of cellular protein tyrosine phosphatases toward endogenous substrates. Our results suggest that polyamines may influence the extent of tyrosine phosphorylation during cell proliferation and malignant transformation, perhaps by modulating the rate of dephosphorylation of specific target proteins.

Phenylarsine oxide augments tyrosine phosphorylation in hematopoietic cells.

Tyrosine phosphorylation and dephosphorylation are implicated in the regulation of cell growth and differentiation. A diverse identification of key regulatory proteins by their content of phosphotyrosine has been hampered by the very low level of tyrosine phosphorylation. This is presumably caused by the relative preponderance of phosphotyrosine phosphatase activity in many cells. We report that treatment of hematopoietic cells with phenylarsine oxide (PAO), a membrane-permeable phosphotyrosine phosphatase inhibitor, induced a dramatic accumulation of phosphotyrosine in a number of cellular proteins. No changes in serine or threonine phosphorylation were detected. The PAO-induced accumulation of phosphotyrosine occurred well before any signs of toxicity or irreversible damage to the cells were seen. Addition of dithiothreitol reversed the effect of PAO. Our data demonstrate that phosphotyrosine phosphatase activity has a major impact on the level of phosphotyrosine in cellular proteins, even in cells with high protein tyrosine kinase activity. Cells with constitutively elevated tyrosine kinase activity are easily detected following treatment with PAO and substrates with an otherwise too low phosphotyrosine content or too rapid phosphate turnover can be studied. This effect of PAO allows determinations of tyrosine phosphorylation-dependent complex formation between proteins.

The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity.

Protein tyrosine kinases participate in the transduction and modulation of signals that regulate proliferation and differentiation of cells. Excessive or deregulated protein tyrosine kinase activity can cause malignant transformation. The catalytic activity of the T cell protein tyrosine kinase p56lck is normally suppressed by phosphorylation of a carboxyl-terminal tyrosine, Tyr-505, by another cellular protein tyrosine kinase. Here we characterize a human cytosolic 50 kDa protein tyrosine kinase, p50csk, which specifically phosphorylates Tyr-505 of p56lck and a synthetic peptide containing this site. Phosphorylation of Tyr-505 suppressed the catalytic activity of p56lck. We suggest that p50csk negatively regulates p56lck, and perhaps other cellular src family kinases.

Regulation of the p59fyn protein tyrosine kinase by the CD45 phosphotyrosine phosphatase.

Triggering of the T cell antigen receptor/CD3 (TcR/CD3) complex leads to rapid tyrosine phosphorylation of regulatory proteins that participate in initiating T cell activation and proliferation. This signal transduction event requires the presence of the TcR/CD3-associated protein tyrosine kinase p59fyn. There is also evidence that the CD45 phosphotyrosine phosphatase is involved in TcR/CD3 signalling. We show here by capping experiments using double indirect immunofluorescence techniques that the receptor phosphotyrosine phosphatase CD45 and the intracellular protein tyrosine kinase p59fyn specifically co-distribute in functional T lymphocytes. Furthermore, we provide evidence that isolated p59fyn is a substrate for CD45 as indicated by the rapid dephosphorylation of the regulatory Tyr531 of p59fyn by CD45. This dephosphorylation is accompanied by a severalfold increase in the catalytic activity of p59fyn as measured by its autophosphorylation and phosphorylation of an exogenous substrate. We also demonstrate that CD45-mediated dephosphorylation and activation of p59fyn apparently occurs at a slow basal rate in resting T cells. This represents the first identification of a physiologic regulator of p59fyn and implies a mechanism for the role of CD45 in TcR/CD3 signal transduction.

Phosphorylation of the beta-subunit of CD11/CD18 integrins by protein kinase C correlates with leukocyte adhesion.

Adhesion of activated leukocytes to cells is of critical functional importance. The adhesion is known to be mediated mainly by the CD11/CD18 integrins, also known as leukocytic cell adhesion molecules, or Leu-CAM. We have now studied the phosphorylation of Leu-CAM by protein kinase C and the correlation of phosphorylation with the generation of the adhesive phenotype among human peripheral blood mononuclear leukocytes during cell activation. We here show that a good correlation exists between the phosphorylation of the beta subunit of Leu-CAM (CD18), and the extent of cell-to-cell adhesion. The phosphorylated CD18 subunit was associated with both CD11a and CD11b. Purified protein kinase C was able to phosphorylate the beta subunit of isolated Leu-CAM in vitro. The phosphorylation occurred mainly on serine residues.

The pivotal role of the Leu-CAM and ICAM molecules in human leukocyte adhesion.

Cellular adhesion is of fundamental importance in leukocyte physiology. It is a complex, strictly regulated process, which involves the participation of several cell surface glycoproteins. Among the most important are the Leu-CAMs or the CD11/CD18 integrin receptors, and their adhesion ligands ICAM-1 (CD54) and ICAM-2. In this review we summarize some recent work on various aspects of these molecules.

Renin substrate in human amniotic fluid.

Purified human amniotic fluid renin substrate (RS) was compared to purified plasma RS. RS in plasma and amniotic fluid were similar in molecular weight, isoelectric point and immunological properties. Immunoreactivity of radio-iodinated amniotic fluid RS was lower than that of plasma RS. Measured by direct radioimmunoassay, RS-levels were only 10-22% of those obtained with indirect assay in 22 amniotic fluid samples. This difference suggests that amniotic fluid RS is less immunoreactive than plasma RS, possibly due to biochemical alteration or complex formation. No such difference in immunoreactivity was noticed in RS of decidual and placental cytosolic fraction.