T cell activation induces rapid tyrosine phosphorylation of a limited number of cellular substrates

Activation of murine T cells by antigen, antibodies binding the T cell antigen receptor, or stimulatory anti-Thy-1 antibodies results in rapid phosphorylation of the T cell receptor zeta chain on tyrosine residues. The T cell receptor is itself unlikely to be a tyrosine kinase; rather, it is probable that this receptor is coupled to a nonreceptor tyrosine kinase. To understand further this protein kinase pathway, additional targets of the tyrosine kinase have been sought by comparing anti-phosphotyrosine antibody immunoblots of cellular proteins from unactivated and activated T cell hybridomas. In addition to the T cell receptor zeta chain, two proteins of 53 and 62 kDa are phosphorylated on tyrosine residues after T cell activation. These phosphorylations require stimulatory anti-Thy-1 antibodies, antigen, or antireceptor antibody stimulation. The 53-kDa protein is preferentially phosphorylated by antigen or antireceptor antibody. Of interest is that variants of the murine T cell hybridoma lacking the T cell receptor zeta chain or lacking surface antigen receptor can nonetheless be stimulated by anti-Thy-1 antibodies to phosphorylate the 62-kDa substrate. In contrast to the tyrosine kinases of oncogenic viruses, the kinase coupled to the T cell antigen receptor appears to have a limited number of targets. These proteins are candidates for critical substrates in this protein tyrosine kinase pathway.     

Interleukin 3-specific tyrosine phosphorylation of a membrane glycoprotein of Mr 150,000 in multi-factor-dependent myeloid cell lines.

Tyrosine phosphorylation of cellular proteins induced by various hematopoietic growth factors such as interleukin 3 (IL3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL4) was studied in several multi-factor-dependent myeloid cell lines. Among the growth factors, IL3 specifically induced rapid tyrosine phosphorylation of a membrane glycoprotein of mol. wt 150 kd (gpp150) in the IL3-dependent cell lines, IC2 and DA-1. The IL3-induced tyrosine phosphorylation of gpp150 was detected within 30 s, reached a maximum at 3 min and decreased thereafter. The concentration of IL3 required for half-maximum stimulation of gpp150 tyrosine phosphorylation with 2.5 x 10(6)/ml cells was approximately 200 pM, which is the same as the dissociation constant for 125I-labeled IL3 binding. gpp150 was constitutively phosphorylated on tyrosine residue(s) in growth factor independent variants, IC2Tr and DA-1Tr, derived from IC2 and DA-1 respectively. Neither variant synthesized IL3. The present findings suggest that tyrosine phosphorylation of gpp150 is a critical event involved in both IL3-dependent and -independent growth.     

Stimulation of beta 1 integrin induces tyrosine phosphorylation of vascular endothelial growth factor receptor-3 and modulates cell migration.

Interactions between integrins and tyrosine kinase receptors can modulate a variety of cell functions. We observed a cooperative interaction between the beta(1) integrin and vascular endothelial growth factor receptor-3 (VEGFR-3 or Flt4) that appeared to be required for cell migration. By using VEGFR-3-transfected 293 cells (293/VEGFR-3) or primary dermal microvascular endothelial cells (DMEC), we found that stimulation with either soluble or immobilized extracellular matrix (ECM) proteins, collagen or fibronectin (FN), resulted in the increased tyrosine phosphorylation of VEGFR-3 in the absence of a cognate ligand. This increased tyrosine phosphorylation of VEGFR-3 was diminished by pretreatment with a blocking antibody against the beta(1) integrin. Cross-linking with anti-beta(1) integrin antibody induced a similar degree of tyrosine phosphorylation of VEGFR-3. Stimulation with collagen or FN induced an association between beta(1) integrin and VEGFR-3 in both 293/VEGFR-3 and primary DMEC cells. Collagen or FN-induced tyrosine phosphorylation of VEGFR-3 was inhibited by treatment with cytochalasin D, an inhibitor of actin polymerization. Collagen or FN was able to induce the migration of 293/VEGFR-3 or DMEC cells to a limited extent. However, migration was dramatically enhanced when a gradient of the cognate ligand, VEGF-D, was added. VEGF-D failed to induce cell migration in the absence of ECM proteins. Introducing a mutation at the kinase domain of VEGFR-3 or treatment with blocking antibody against either VEGFR-3 or beta(1) integrin inhibited cell migration induced by ECM and VEGF-D, indicating that signals from both beta(1) integrin and VEGFR-3 are required for this cell function.     

Activation of human T cells is associated with tyrosine phosphorylation of several cellular proteins

Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.     

Expression of interleukin 2 receptors on interleukin 3-dependent cell lines

Several mouse IL 3-dependent cell lines, IC2, LT4, FDC-P2, and PB-3C, derived from spleen or bone marrow cells were shown to express low affinity receptors for IL 2 (Kd; 0.5 to 8 X 10(-8) M). High affinity receptors for IL 2 were not detected on the IL 3-dependent cells within the experimental limitation of this study. The clones did not respond to IL 2 at all at the concentration as high as 25 micrograms/ml. The number of the receptors expressed on those clones was estimated to be 0.2 to 2 X 10(5)/cell, which is comparable with the number of those on IL 2-dependent T cell clones. Expression of IL 2 receptor was confirmed in mRNA levels for both IC2 and LT4 cells. A relatively low level expression of one (4.5 Kb) of four IL 2 receptor mRNA species was observed with those IL 3-dependent clones compared with IL 2-dependent T cells. It seems that these low affinity receptors may be expressed on IL 3-dependent cells that undergo differentiation or maturation in mast cell and some myeloid cell lineages.     

Stimulation of beta1 integrins on fibroblasts induces PDGF independent tyrosine phosphorylation of PDGF beta-receptors

We report that integrin-mediated signaling induces a rapid and transient tyrosine phosphorylation of platelet-derived growth factor (PDGF) beta-receptors in human diploid foreskin AG 1518 fibroblasts. A transient tyrosine phosphorylation of PDGF beta-receptors was evident one and two hours after cells had been plated on collagen type I and fibronectin, as well as on immobilized anti-integrin subunit IgG, but not on poly-L-lysine. In contrast EGF or PDGF alpha-receptors were not phosphorylated on tyrosine residues under these conditions. Tyrosine phosphorylation of PDGF beta-receptors induced by plating on collagen type I was inhibited by cytochalasin D and herbimycin A, unaffected by cycloheximide and enhanced by orthovanadate. Furthermore, a transient phosphorylation of PDGF beta-receptors occurred when AG 518 fibroblasts were cultured in three-dimensional collagen lattices or exposed to external strain exerted through centrifugation. The latter effect was evident already after two minutes. Clustering of cell surface beta1 integrins led to PDGF beta-receptor phosphorylation both in suspended and firmly attached AG 1518 fibroblasts. Plating of cells on collagen type I, fibronectin, and anti-beta1-integrin IgG resulted in the formation of PDGF beta-receptor aggregates as detected by immunofluorescence. Suramin or anti-PDGF-BB IgG had no effect on the plating-induced tyrosine phosphorylation of PDGF beta-receptors. PDGF-B chain mRNA, or protein, were not detected in AG 1518 fibroblasts. Our data suggest that a ligand-independent PDGF beta-receptor activation during cell adhesion and early phases of cell spreading is involved in integrin-mediated signaling in fibroblasts, and constitutes parts of a mechanism for cells to respond during the dynamic phases of externally applied tension as well as fibroblast-mediated tension during cell adhesion and collagen gel contraction.     

Human interleukin 1 is a cytocidal factor for several tumor cell lines

Highly purified interleukin 1 (IL 1) obtained from stimulated human monocytes appeared to be growth inhibitory and cytocidal for a human melanoma cell line, A375. Although IL 1 did not have an immediate cytolytic effect, with time in culture the growth of the target cells was irreversibly inhibited. The cells eventually lysed and decreased markedly in number; the IL 1 effect can therefore be said to be cytocidal. IL 1 activity could not be separated from the cytocidal activity by a variety of chromatography procedures by using conventional and high-performance liquid chromatography (HPLC). The A375 melanoma cell line was also sensitive to another human cytokine alpha-lymphotoxin (alpha-LT) derived from a human B cell line. IL 1 also appeared to be partially growth inhibitory and cytocidal for a LT-sensitive mouse fibroblast cell line, L929; but not for LT-resistant cells, including a subline of L929; a human epithelial carcinoma cell line, HeLa; a human osteosarcoma cell line, HOS; and a mouse SV40-transformed kidney cell line, TU5. However, the LT-sensitive mouse fibroblast cell line, L-M, was resistant to IL 1. Therefore, the cytocidal activity of IL 1 only partially overlapped the target cell selectivity of alpha-LT. Although natural IFN-alpha and recombinant IFN-beta were appreciably growth inhibitory for the A375 cell line, natural and recombinant IFN-alpha and recombinant IFN-beta and IFN-gamma exhibited little cytocidal activity. Purified IL 1 did not have any antiviral activity, and conversely, IFN and alpha-LT were not co-mitogenic for thymocytes. Furthermore, by ELISA and radioimmunoassays, antibodies against human alpha-LT, tumor necrosis factor, and IFN-gamma did not react with IL 1, indicating that IL 1 is antigenically distinct from these other cytokines. These in vitro results suggest that IL 1 may play a role in host defense against some tumors as a cytocidal factor.     

Protein tyrosine phosphorylation associated with activation of the interleukin 2 receptor.

The addition of interleukin 2 (IL2) to the IL2-dependent murine cytotoxic T cell line CTTL-2 induced increased tyrosine phosphorylation of a protein with a molecular weight of 80,000 and, to a lesser extent, proteins with molecular weights of 130,000, 100,000, and 69,000. To correlate the stimulation of tyrosine phosphorylation with increased tyrosine kinase activity, cell-free phosphorylation assays were performed. Phosphotyrosine-containing proteins were purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibodies. The level of tyrosine kinase activity was determined by incorporation of [gamma-32P]ATP into the exogenous substrate histone H2B. IL2 treatment of cells increased H2B phosphorylation 10-fold when compared with nonstimulated cells. Phosphorylation was first detected after 2.5 min of incubation with physiologically relevant (100 pM) IL2 doses. To examine if tyrosine kinase activity was resident within the IL2 receptor complex, cell-free phosphorylation assays were performed with ligand-receptor complexes following cross-linking with IL2 and purification by immunoabsorption with an anti-IL2 antibody. Tyrosine kinase activity was found specifically associated with the IL2 receptor complex. These results indicate that the IL2 receptor complex contains a tyrosine kinase activity that is induced by IL2 binding and suggest that components of the complex may be a substrate of this activity.     

Insulin induces suppressor of cytokine signaling-3 tyrosine phosphorylation through janus-activated kinase

Suppressor of cytokine signaling (SOCS) proteins were originally described as cytokine-induced molecules involved in negative feedback loops. We have shown that SOCS-3 is also a component of the insulin signaling network (). Indeed, insulin leads to SOCS-3 expression in 3T3-L1 adipocytes. Once produced, SOCS-3 binds to phosphorylated tyrosine 960 of the insulin receptor and inhibits insulin signaling. Now we show that in 3T3-L1 adipocytes and in transfected COS-7 cells insulin leads to SOCS-3 tyrosine phosphorylation. This phosphorylation takes place on Tyr(204) and is dependent upon a functional SOCS-3 SH2 domain. Purified insulin receptor directly phosphorylates SOCS-3. However, in intact cells, a mutant of the insulin receptor, IRY960F, unable to bind SOCS-3, was as efficient as the wild type insulin receptor to phosphorylate SOCS-3. Importantly, IRY960F is as potent as the wild type insulin receptor to activate janus-activated kinase (Jak) 1 and Jak2. Furthermore, expression of a dominant negative form of Jak2 inhibits insulin-induced SOCS-3 tyrosine phosphorylation. As transfected Jaks have been shown to cause SOCS-3 phosphorylation, we propose that insulin induces SOCS-3 phosphorylation through Jak activation. Our data indicate that SOCS-3 belongs to a class of tyrosine-phosphorylated insulin signaling molecules, the phosphorylation of which is not dependent upon a direct coupling with the insulin receptor but relies on the Jaks.     

Stimulation via CD3-Ti but not CD2 induces rapid tyrosine phosphorylation of a 68-kDa protein in the human Jurkat T cell line.

Tyrosine phosphorylation is an early biochemical event associated with surface receptor triggering in many cellular systems. In T lymphocytes, Ag receptor (CD3-Ti) stimulation results in tyrosine phosphorylation of the CD3 zeta subunit. The tyrosine kinase responsible for this modification after CD3-Ti triggering has not been identified. Here we reported that a 68-kDa T cell membrane-associated protein (pp68) in human Jurkat T cells is phosphorylated on tyrosine residues within 1 min after anti-CD3 mAb addition. This induced tyrosine phosphorylation is detected either by in vivo [32P]orthophosphate labeling of the Jurkat T cells or by in vitro [32P]ATP labeling after immunoprecipitation by antiphosphotyrosine antibody. In contrast, mAb stimulation via CD2 and CD4 structures does not induce phosphorylation of pp68. These data are among the first to provide evidence that CD3-Ti and CD2 activation pathways are distinct. Furthermore, they imply that pp68 is itself a tyrosine kinase and/or is a rapidly phosphorylated substrate of a tyrosine kinase.     

Trans-interaction of nephrin and Neph1/Neph3 induces cell adhesion that associates with decreased tyrosine phosphorylation of nephrin

Slit diaphragms are specialized junctions between glomerular epithelial cells (podocytes) that are crucial for glomerular ultrafiltration. The Ig superfamily members nephrin and Neph1 are essential components of the slit diaphragm, whereas the role of Neph1 homologue Neph3 in the slit diaphragm is unknown. In the present paper we show that Neph3 homodimerizes and heterodimerizes with nephrin and Neph1. We further investigated whether these interactions play a role in cell adhesion by using mouse L fibroblasts that lack endogenous cell-adhesion activity and found that Neph1 and Neph3 are able to induce cell adhesion alone, whereas nephrin needs to trans-interact with Neph1 or Neph3 in order to promote formation of cell-cell contacts. Tyrosine phosphorylation of nephrin was down-regulated after nephrin trans-interacted with either Neph1 or Neph3 leading to formation of cell-cell contacts. We further found that the expression of Neph3 was increased in nephrin-deficient mouse podocytes. The findings of the present paper show that nephrin and Neph1 or Neph3 trans-interactions promote cell-contact formation, suggesting that they may also function together in slit diaphragm assembly.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Phenotypic characteristics of cell lines requiring interleukin 3 for growth

A number of cell lines have been derived from bone marrow cultures in the presence of WEHI-3 conditioned media (CM) that continue to require WEHI-3 CM for growth in vitro. Because the WEHI-3 cell line has been shown to constitutively produce a lymphokine (IL 3) that induces the expression of 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of splenic lymphocytes from athymic mice, we examined whether these cell lines were dependent upon IL 3 for growth. The results demonstrate that the factor required for growth of these cell lines copurifies with IL 3 activity on G-100, DEAE cellulose, and CM cellulose column chromatography as well as in preparative isoelectric focusing and on hydrophobic supports in reverse phase high pressure liquid chromatography. The biologic activity of peak fractions in each case was similar in both types of assays. These results strongly suggest that the WEHI-3 CM-dependent cell lines are dependent on IL 3 for growth in vitro. All the cell lines have readily detectable levels of 20 alpha SDH but have differing cell surface phenotypes. The C3HSFFV line is devoid of conventional lymphoid cell surface markers with the exception of Lyt-1, whereas the FDC-P1 expresses Thy-1, Ly-5, and H-11. Other cell lines have intermediate phenotypes.     

Stimulation of tyrosine phosphorylation in lectin treated human lymphocytes

Large increases in tyrosine phosphorylation have been detected in subcellular matrixes isolated from lectin treated human lymphocytes. In lectin stimulated cells proteins of molecular weight 105, 75, 58 and 35 kDa contained phosphotyrosine (P-tyr) whereas non-stimulated cells had no 105 and low levels of P-tyr in proteins of 75, 58 and 35 kDa. In stimulated cells increased tyrosine kinase activity was also shown using gastrin as substrate. In both stimulated and non-stimulated cells the 58 kDa phosphoprotein was the most heavily labelled, after partial proteolysis of the 58 kDa different phosphopeptides were generated. A peptide with a sequence analogous to the autophosphorylated tyrosine site of pp60src inhibited tyrosine phosphorylation in stimulated cells. The lymphocyte system provides a useful tool to study normal tyrosine protein kinases and their role in cellular proliferation.     

Janus kinase 3 regulates interleukin 2-induced mucosal wound repair through tyrosine phosphorylation of villin

Janus kinase 3 (Jak3) is a non-receptor tyrosine kinase known to be expressed in hematopoietic cells. Studies of whole organ homogenates show that Jak3 is also expressed in the intestines of both human and mice. However, neither its expression nor its function has been defined in intestinal epithelial enterocytes. The present studies demonstrate that functional Jak3 is expressed in human intestinal enterocytes HT-29 Cl-19A and Caco-2 and plays an essential role in the intestinal epithelial wound repair process in response to interleukin 2 (IL-2). Exogenous IL-2 enhanced the wound repair of intestinal enterocytes in a dose-dependent manner. Activation by IL-2 led to rapid tyrosine phosphorylation and redistribution of Jak3. IL-2-stimulated redistribution of Jak3 was inhibited by the Jak3-specific inhibitor WHI-P131. IL-2 also induced Jak3-dependent redistribution of the actin cytoskeleton in migrating cells. In these cells Jak3 interacted with the intestinal and renal epithelial cell-specific cytoskeletal protein villin in an IL-2-dependent manner. Inhibition of Jak3 activation resulted in loss of tyrosine phosphorylation of villin and a significant decrease in wound repair of the intestinal epithelial cells. Previously, we had shown that tyrosine phosphorylation of villin is important for cytoskeletal remodeling and cell migration. The present study demonstrates a novel pathway in intestinal enterocytes in which IL-2 enhances intestinal wound repair through mechanisms involving Jak3 and its interactions with villin.     

Vanadate can replace interleukin 3 for transient growth of factor-dependent cells

A mouse interleukin 3 (IL-3)-dependent cell line, IC2, could survive and proliferate over a 48-h period in the absence of IL-3 when incubated with micromolar concentrations of sodium orthovanadate. The greatest response was obtained at 12.5 microM, as judged by stimulation of cell growth. Vanadate also stimulated synthesis of nucleotides and protein in IC2 cells. After 6 h of culture in the absence of IL-3, the intracellular ATP levels of IC2 cells fell dramatically; this fall was prevented by addition of vanadate to the culture. Studies with recombinant IL-3 revealed that vanadate potentiated the effect of submaximal doses of IL-3 on the growth of IC2 cells, but did not appear to act synergistically with IL-3 to give a maximal proliferative response of the cells. After 48 h of IL-3 replacement with vanadate, IC2 cells could respond to IL-3 with no loss of proliferative integrity. These results suggest that IL-3 dependence of IC2 cells is transiently substituted for by vanadate, and it may be a useful tool to understand the mechanism of action of IL-3.     

Identification in several human myeloid leukemias or cell lines of a DNA rearrangement next to the c-mos 3'-end

A characteristic DNA rearrangement, the loss of an EcoRI cleavage site next to the 3'-end of the human c-mos gene, has been found to be frequently present in DNA from transformed hematopoietic cells of the myeloid lineage but not in DNA from either normal or transformed cells of different tissue types. Three established cell lines, respectively a pro-monocytic line (CM-S) and two precursor granulocytic lines (My/K1 and My/K5), carry the same genome rearrangement, but not fibroblasts obtained from the marrow of the same patients. This DNA rearrangement is maintained in three different hybridomas derived by fusion of CM-S cells with normal human embryo hepatocytes.