Anergic Th1 cells express altered levels of the protein tyrosine kinases p56lck and p59fyn.

Tolerance in T lymphocytes can result from clonal anergy, or paralysis, of Ag-specific T cells. To investigate the molecular mechanisms responsible for anergy, a system in which tolerance can be induced in vitro was employed. Anergy, as defined by long-lived nonresponsiveness to normal antigenic stimulation for IL-2 production, was produced in cloned murine CD4+ Th1 cells.

Cross-linking of surface IgM or IgD causes differential biological effects in spite of overlap in tyrosine (de)phosphorylation profile.

Although displaying similar amounts of surface IgM and IgD, ECH 408-1 cells only succumb to apoptosis after cross-linking of IgM (not IgD), suggesting that different signaling pathways couple to both receptors. Immunoprecipitation studies revealed the presence of several proteins selectively associated with IgM and IgD, thus ruling out that the lack of inhibitory signaling mediated by IgD might be due to membrane expression in the absence of associated proteins belonging to the B cell receptor complex. 32P metabolic labeling and immunoprecipitation studies demonstrated that IgM and IgD are associated with phosphoproteins of 32-33 kDa in an isotype-specific fashion.

Synthetic fragments of the CD4 receptor cytoplasmic domain and large polycations alter the activities of the pp56lck tyrosine protein kinase.

In CD4+ T cells, the src-like tyrosine kinase pp56lck is associated with the CD4 receptor and cross-linking of CD4 results in the activation of this enzyme. The mechanism responsible for this activation is not known, although there is evidence that the activities of the src family of enzymes are regulated by tyrosine phosphorylation. Here we report that pp56lck-catalyzed angiotensin II phosphorylations are activated 20-fold in vitro by synthetic peptides reproducing portions of the murine CD4 cytoplasmic domain.

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils.

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Stimulation of the neutrophils with chemotactic factor fMet-Leu-Phe (10 nM) caused rapid increases of tyrosine phosphorylation of several proteins with apparent molecular masses of (Group A) 54-58 kDa and 100-125 kDa and (Group B) 36-41 kDa. Stimulation of Group A proteins was observed by fMet-Leu-Phe (10 nM, maximum at 20 s) and A23187 (1 microM, 1 min).

Stimulation of the antigen and interleukin-2 receptors on T lymphocytes activates distinct tyrosine protein kinases.

The T cell antigen receptor complex (TCR) and the interleukin 2 (IL-2) receptor are responsible for signal transduction that results in T lymphocyte activation and proliferation. Stimulation of either the TCR or the IL-2 receptor induces an increase in tyrosine phosphorylation of several cellular proteins indicating that signal transduction by both of these receptors involves the activation of a tyrosine protein kinase. Although the tyrosine protein kinases activated by these receptors have not yet been characterized the receptors themselves are known not to contain a tyrosine protein kinase domain.

An activating combination of CD2 antibodies stimulates tyrosine phosphorylation in a T lymphocyte cell line.

The activating combination of CD2 antibodies Leu-5b plus 9.1 stimulates tyrosine phosphorylation in the human T cell line Jurkat. The tyrosine phosphorylation has the same molecular weight pattern as that seen when cells are stimulated on the CD3 receptor with OKT3 antibodies.

The 75,000-dalton interleukin-2 receptor transmits a signal for the activation of a tyrosine protein kinase.

The high-affinity receptor for interleukin-2 (IL-2) is composed of two distinct subunits with molecular weights of 55,000 and 75,000 (p55 and p75). While the presence of the high-affinity receptor requires the simultaneous expression of p55 and p75, these subunits can also be expressed independently, resulting in IL-2 receptors with low and intermediate affinities, respectively. IL-2 can induce proliferation in cells expressing either the intermediate affinity p75 receptor or the p55.

Tyrosine phosphorylation in human neutrophil.

Protein tyrosine phosphorylation in human neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. The addition of the human hormone granulocyte-macrophage colony stimulating factor to human neutrophils caused an increase in the tyrosine phosphorylation levels of several proteins. The increases in at least two of these proteins having molecular masses of 40 kDa (p40) and 54 kDa (p54) were rapid and were inhibited in pertussis toxin treated cells.

Pertussis toxin activates protein kinase C and a tyrosine protein kinase in the human T cell line Jurkat.

Pertussis toxin activates T lymphocytes by a mechanism that is independent of its ADP-ribosylation activity. The toxin stimulates increases in diacylglycerol and intracellular calcium apparently by interacting with a cell surface receptor. Consistent with the production of these second messengers we have found that pertussis toxin activates protein kinase C in the Jurkat cell line.

The insulin-like effect of vanadate on lipolysis in rat adipocytes is not accompanied by an insulin-like effect on tyrosine phosphorylation.

Tyrosine phosphorylation of the insulin receptor and other intracellular proteins in rat adipocytes was examined using an immunoblot technique with antiphosphotyrosine antibody. Insulin at 10(-7) M increased the tyrosine phosphorylation of the 95K subunit of the insulin receptor (15-fold) and proteins of 180K (7-fold) and 60K (23-fold). Increases in insulin-dependent phosphorylation of the three proteins were detectable at 10(-10) M insulin and attained steady state within 30 sec of insulin (10(-7) M) addition.

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor.

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity.

Activation of a tyrosine protein kinase is an early event in the stimulation of T lymphocytes by interleukin-2.

The ability of the T lymphocyte growth factor interleukin 2 (IL-2) to activate a tyrosine protein kinase in vivo was assessed by using antibodies to phosphotyrosine in conjunction with immunoblots. Treatment of the murine IL-2-dependent cytotoxic T cell line CTLL-2 with IL-2 resulted in an increase in tyrosine phosphorylation of several proteins of molecular weights ranging from 38,000 to 120,000. The tyrosine phosphorylation in the various proteins increased in a concomitant fashion and reached a maximum level within 15 min.

Chemotactic factor induced tyrosine phosphorylation of membrane associated proteins in rabbit peritoneal neutrophils.

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Two tyrosine phosphorylated proteins were found with apparent molecular weights of 62,000 (p62) and 125,000. Both were enriched in the membrane fraction.

Demonstration that LSTRA cells have an elevated level of proteins phosphorylated on tyrosine residues.

The LSTRA cell line has been shown to have an exceptionally high level of a tyrosine protein kinase (pp56lck). We now report that LSTRA cells also have a much higher level of proteins phosphorylated on tyrosine residues in comparison to several other cell lines with normal levels of pp56lck. The level of phosphotyrosine-containing proteins in LSTRA cells was comparable to that seen in K562 cells, a cell line known to have a constitutively active tyrosine protein kinase.

Sites of in vivo phosphorylation of the T cell tyrosine protein kinase in LSTRA cells and their alteration by tumor-promoting phorbol esters.

The LSTRA cell line contains an elevated level of a tyrosine protein kinase of apparent molecular weight of 56,000 (pp56Tcell). Analysis of the tryptic fragments of this protein labeled in vivo with 32P shows that it contains four sites of tyrosine phosphorylation and one site of serine phosphorylation. Two of the sites of in vivo tyrosine phosphorylation are also labeled in vitro when membranes are incubated with [gamma-32P]ATP.

Tumor promoters cause changes in the state of phosphorylation and apparent molecular weight of a tyrosine protein kinase in T lymphocytes.

The T cell lymphoma LSTRA contains an elevated level of a tyrosine protein kinase of molecular weight of 56,000 (pp56Tcell) that is present in normal T lymphocytes. Treatment of 32P-labeled LSTRA cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), followed by immunoprecipitation of pp56Tcell, revealed that PMA causes complex changes in the state of phosphorylation of pp56Tcell, and the appearance of several new forms of pp56Tcell with higher apparent molecular weights on sodium dodecyl sulfate-polyacrylamide gels. The 32P-labeled pp56Tcell from untreated LSTRA cells contains phosphotyrosine and phosphoserine in a ratio of 2:1.

Covalently bound myristate in a lymphoma tyrosine protein kinase.

The murine lymphoma cell line LSTRA expresses high levels of a membrane-associated tyrosine protein kinase, which we now show to be acylated by [3H]myristate in vivo. This [3H]myristate-labeled tyrosine protein kinase is immunoprecipitated from detergent extracts of postnuclear particulate fractions with an antibody directed against its single site of tyrosine phosphorylation. This site has an amino acid sequence also found in the transforming proteins of the Rous sarcoma and Y73 viruses.

Identification of the tyrosine protein kinase from LSTRA cells by use of site-specific antibodies.

The lymphoma cell line LSTRA contains an elevated level of tyrosine protein kinase activity. It has been suggested that this elevated level of activity is due to the presence of a phosphoprotein with a molecular weight of 56,000 (pp56, formerly referred to as a 58,000-dalton protein). This paper describes the preparation of antibodies against pp56 through the use of a synthetic peptide that contains the sequence around the site of tyrosine phosphorylation in pp56, which is identical to the phosphorylation site in pp60src.

A comparison of the insulin- and epidermal growth factor-stimulated protein kinases from human placenta.

The characteristics of the insulin- and epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinases in a wheat germ lectin-Sepharose-purified preparation of solubilized placenta membranes were compared. The specific activity of the insulin-stimulated kinase in this preparation was 72 nmol/min/mg whereas the specific activity of the EGF-stimulated kinase was 312 nmol/min/mg using a synthetic peptide as the phosphorylatable substrate. The two enzymes showed similar divalent metal ion requirements and nucleotide specificities.

The use of synthetic peptides for defining the specificity of tyrosine protein kinases.

The tyrosine protein kinases are a large family of enzymes that may be involved in regulating cell growth and differentiation. An important property of these enzymes is their substrate specificity. Defining the specificity of these enzymes will contribute to a greater understanding of their biological functions.

Epidermal growth factor stimulates tyrosine phosphorylation of the myosin regulatory light chain from smooth muscle.

Epidermal growth factor (EGF) stimulates the phosphorylation by A431 membranes of tyrosine residues in the myosin regulatory light chain (LC20) from chicken gizzard smooth muscle. In the presence of EGF, the Km of the EGF receptor kinase for LC20 is 73 microM and the V max is 17 nmol/min/mg. Two moles of phosphate are incorporated into tyrosine per mol of LC20.

Antibodies to a defined region of pp60src neutralize the tyrosine-specific kinase activity.

Site-specific antibodies to pp60src, the transforming protein of Rous sarcoma virus (RSV), have been prepared by immunizing rabbits with a chemically synthesized pentadecapeptide corresponding to residues 498-512 (Cys-Trp-Arg-Lys-Asp-Pro-Glu-Glu-Arg-Pro-Thr-Phe-Lys-Tyr-Leu) as deduced from the nucleotide sequence of the Prague C src gene. Antibodies specific for the synthetic peptide were purified from immune sera by affinity chromatography on peptide-bound Sepharose and characterized by a number of immunocytochemical techniques. Immunoprecipitation and Western blot analyses of normal and RSV-transformed cell lines revealed that this peptide antibody identified the authentic viral src gene product.