Phospholipase C-gamma 1 association with CD3 structure in T cells.

Recently, we and others have reported tyrosine phosphorylation of phospholipase C-gamma 1 (PLC gamma 1) enzyme after CD3 activation of T cells, and have proposed that PLC gamma 1 mediates signal transduction through the T cell receptor (TCR/CD3). Here, using immunoblotting and immune complex PLC assays, we show that CD3 stimulation of Jurkat cells induces the association of PLC gamma 1 enzyme with CD3 complex. PLC activity is also found to co-precipitate with the CD3 zeta chain from activated cells. In addition, in vitro PLC assays show that CD3 activation leads to about 10-fold stimulation of PLC gamma 1 activity. These results, along with the observation that Jurkat cells preferentially express PLC gamma 1, indicate that PLC gamma 1 participates in CD3 signaling.

Two variants of DRw52, DR3, and DQw2 specificities distinguish two different DR3-bearing extended haplotypes.

HLA-DR beta and DQ beta MHC antigens present on B-lymphoblastoid cell lines homozygous for either [HLA-B8,DR3,SC01] or [HLA-B18,DR3,F1C30] were studied by two-dimensional gel electrophoresis. Comparison of neuraminidase-treated DR proteins from these cells showed that DR3-bearing cells express two DR beta genes. However, one DR beta chain (beta a) is nonpolymorphic, whereas the other beta chain (beta b) is polymorphic. Two variants of DR beta (DR3 or DRw52) and two of DQ beta (DQw2) were found, with all the examples of each extended haplotype carrying only one of these sets of variants. The variants thus absolutely distinguished the two DR3-bearing extended haplotypes. These results support the hypothesis of extended haplotypes at the protein level, and demonstrate the fixity of alleles on them in the HLA-B-D/DR region.

Defective signal transduction via T-cell receptor-CD3 structure in T cells from rheumatoid arthritis patients.

T cells from patients with active RA are known to produce low levels of IL-2 and proliferate poorly in response to various mitogenic stimuli. The present work shows that cross-linking of CD3 antigen on patients' T-cell surface induces two- to threefold lower Ca2+ response than in T cells from age-matched controls. Immunofluorescence studies indicate that the attenuated signal may be due to the suppressed expression of CD3 and/or CD45 molecules on patients' T cells. In the majority of the patients, the level of CD45 expression is reduced by 60%-70% as compared with that in the control T cells. Therefore, the poor mitogenic response of patient cells is apparently due to a defect in early stages of signal transduction through the T-cell receptor (TCR-CD3).

Tyrosine-specific protein kinases of normal tissues.

Tyrosine-specific protein kinases from normal tissue have been studied using synthetic peptides as substrate. Spleen had much higher activity of the enzyme in the particulate fraction than any other normal tissue (except purified T lymphocytes). The tyrosine protein kinase from the particulate fraction of rat spleen was partially purified and characterized. The kinase could phosphorylate src-related as well as unrelated peptides and casein at tyrosine residues. The enzyme in the membrane seemed to have somewhat different substrate specificity than the solubilized, partially purified enzyme. Serum containing antibody to pp60v-src did not precipitate the kinase; however, the protein kinase could phosphorylate the heavy chain of IgG from TBR serum (but not from normal serum). The possible relationship of the tyrosine-specific protein kinase of spleen with pp60c-src and other tyrosine-specific protein kinases is discussed.

Specificity of signal transduction through CD16, TCR-CD3 and BCR receptor chains containing the tyrosine-associated activation motif.

FcR gamma III (CD16) is a member of a family of receptor proteins that bind to Fc regions of IgG molecules. Ligation of CD16 transduces signal and alters tyrosine phosphorylation of proteins in natural killer, B and T cells. However, the identity of protein tyrosine kinases (PTKs) involved in CD16 signaling and the mode of their interaction with the receptor are not completely understood. Here, we show that the transmembrane form of CD16 transfected in a CD3- Jurkat cell line associates with p56lck and ZAP-70 PTKs. Using chimeric proteins consisting of the extracellular domain of CD16 and the cytoplasmic portions of the zeta chain, we find that these PTKs interact with the 17-residue activation motif present in the zeta chain. Substitution of tyrosine residues in the motif with phenylalanine blocks the interaction of PTKs and abrogates signal transduction. Comparative studies demonstrate that similar to zeta, MB1 protein of the IgM receptor also binds to p56lck and ZAP-70 in T cells and induces strong activation responses, whereas CD3 gamma, delta and epsilon chains do not interact with ZAP-70 and transduce weak signals. Together, these results demonstrate that: (i) PTKs bound to CD16 mediate signal transduction, (ii) ZAP-70 plays a crucial role in signaling and (iii) apart from tyrosine residues in the activation motif, other structural constraints also regulate the interaction of PTKs with the receptor molecule.

Receptor-like role of HLA-class I antigens: regulation of T cell activation.

Class I major histocompatibility antigens are known to restrict the cytotoxic activity of T lymphocytes. However, experiments using monoclonal antibodies against class I antigens showed that these antigens also play some role in the regulation of T cell activation. Three monoclonal antibodies, namely W6/32 (anti-class I HLA-A, B, C, antigens), 4E (anti-class I HLA-B antigens), and BBM.1 (anti-beta 2-microglobulin) significantly suppressed the phytohemagglutinin-induced T cell proliferation. The inhibitory effect of anti-class I antibody was found to depend on the presence of monocyte/macrophage-type adherent cells. In the presence of antibody, adherent cells released a factor that suppressed T cell proliferation. These results suggest that HLA class I antigens on Mo1+ monocyte/macrophage cells function like ligand-receptor molecules, and regulate the secretion of suppressor factor(s).

Tyrosine protein kinase activity during embryogenesis.

The activity of tyrosine-specific protein kinase was estimated during early embryonic development of the sea urchin, Strongylocentrotus purpuratus, using the synthetic peptide Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly as a probe. The peptide was not phosphorylated by the purified cyclic AMP-dependent protein kinase, phosphorylase kinase, glycogen synthase kinase 3, or casein kinase 2 but was phosphorylated by the purified epidermal growth factor-receptor kinase from A-431 cells. The sea urchin tyrosine protein kinase activity was determined in a particulate fraction (17,000 x g pellet) and in a membrane fraction obtained after discontinuous sucrose gradient centrifugation; these fractions contained higher specific activities of the kinase than the cytosolic or nuclear fractions. Unfertilized eggs had very low tyrosine protein kinase activity (0.22 pmol of peptide phosphorylated per mg of protein/min) in the particulate fraction, but the activity increased 2.5-fold by 1 h after fertilization and almost 20-fold by the gastrula stage. The enzyme activity of the membrane fraction increased similarly during this time period. The tyrosine protein kinase had an apparent Km of 8.9 mM for the peptide, and showed one-half-maximal velocity at about 35 microM MgATP. The phosphorylation of membrane fractions in vitro at different stages of embryonic development resulted in nine endogenous protein-staining bands (Mr = 26,000 to greater than 200,000; 10% Na dodecyl SO4 gels) which contained phosphotyrosine; some of the bands incorporated 32P differentially as a function of development. In the unfertilized egg membrane fraction, none of the protein-staining bands were shown to contain detectable 32P-tyrosine. Thus, fertilization results in increases in tyrosine-specific protein kinase(s) activity which continues to increase during early embryonic development; it is suggested that these protein kinases have a functional role during early development and differentiation.

Spatio-temporal distribution of gap junctions in zebra fish embryo.

The distribution of gap junctions in zebra fish embryo is a dynamic process. They appear during cleavage stages and are uniformly distributed between deep cells in early blastula. By mid-blastula stage their density increases all over the embryo. At this stage lightly stained L cells and densely stained D cells (Dasgupta and Singh 1981; Wilhelm Roux's Arch Dev Biol 190:358) develop some differences in their surface properties due to which the frequencies of gap junctions between homologous (L-L and D-D) cells are two- to threefold higher than between heterologous (L-D) cells. Before the initiation of epipoly the gap junction occurrence increases in the envelopinglayer (EVL) and then decreases. The possible functional significance of the organization of these junctions is discussed.

CD4-independent signal transduction through the T-cell receptor (TCR/CD3).

The membrane-bound CD4 glycoprotein has been proposed to act like a co-receptor along with the T-cell antigen receptor (TCR/CD3) during ligand recognition and cell activation. Due to its association with the protein tyrosine kinase (PTK) p56lck, CD4 is believed to transduce a signal and support CD3 activation of T cells. In this study we have shown that CD3 ligation on murine T-cell hybridomas induces tyrosine phosphorylation of proteins, including phospholipase C-gamma 1 (PLC gamma 1), both in the presence as well as in the absence of CD4-linked p56lck. Furthermore, using HPB clones deficient in CD3/PTK association, it has been found that the presence of CD4/p56lck does not overcome the defect in signalling. Not even co-aggregation of CD4 with CD3 triggers tyrosine phosphorylation of proteins in these cells. Together, the present results indicate that CD3-linked PTK(s) plays a primary role in the induction of signalling through TCR/CD3, and the presence of CD4/p56lck is neither necessary nor sufficient to elicit these events. In the light of these results a possible role for CD4 in antigen presentation has been proposed.

Tyrosine protein kinase activity of rat spleen and other tissues.

Using a synthetic peptide (Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly) as a substrate, various normal tissues from the rat were probed for tyrosine protein kinase activity. Spleen was shown to contain much higher tyrosine protein kinase activity than other rat tissues (lung, brain, testes, liver, kidney, heart, and thymus, in decreasing order of specific activity). Most of the tyrosine protein kinase activity of the various rat tissues (greater than 80%) was present in the particulate fraction, and Nonidet P-40, a nonionic detergent, could activate the particulate form of the enzyme 2-20-fold in many of the tissues. Epidermal growth factor (1 microgram/ml), cyclic AMP, cyclic GMP, or Ca2+ did not increase spleen tyrosine protein kinase activity. Half-maximal enzyme activity was observed at 60-80 microM MgATP and at 2.2 mM peptide, and both Mg2+ (10 mM) and Mn2+ (0.5-1.0 mM) were effective divalent metal ions for the expression of activity. When the particulate fraction of spleen was incubated with [gamma-32P]ATP followed by polyacrylamide gel electrophoresis in the presence of Na dodecyl SO4, a number of alkali-stable bands were identified by autoradiography. Two major bands at Mr = 53,000 and 56,000 were shown to contain phosphotyrosine. Two similar alkali-stable bands containing phosphotyrosine but with lower amounts of 32P labeling were also observed in the particulate fractions of various other tissues (lung, brain, kidney, and testes). The particulate form of tyrosine protein kinase of rat spleen could be solubilized by using high concentrations of Nonidet P-40 (5%) at an alkaline pH (pH 9.0). Partial purification and subsequent filtration on Sephacryl S-200 yielded a peak of tyrosine protein kinase activity with an apparent molecular weight of 55,000. The two major phosphorylated bands of Mr = 53,000 and 56,000 co-migrated with the peak of enzyme activity. The solubilized and partially purified enzyme preparation phosphorylated only tyrosine residues when either endogenous proteins or casein were used as substrates. These results suggest that relatively high activities of tyrosine protein kinase exist in a normal tissue (rat spleen). Major endogenous substrates of the enzyme(s) appear to be represented by two proteins of Mr = 53,000 and 56,000; one or both of these substrates may be the tyrosine protein kinase itself.

Involvement of major histocompatibility complex class I antigens in T cell activation.

During the last few years ample evidence has been collected indicating a regulatory role for major histocompatibility complex class I antigens (Ag) in T cell activation. However, due to differential effects (stimulatory and inhibitory) of anti-class I antibodies (Ab) observed under different conditions, no coherent scheme of the mechanism of action of these Ag has emerged. Here, we present evidence that the mode of action of anti-class I Ab depends upon the presence or absence of monocytes/macrophages (M phi) in the culture. The Ab inhibit Ag presentation by binding to M phi. Coating of tetanus toxin -pulsed M phi with anti-class I Ab is sufficient to suppress T cell activation. On the contrary, these Ab enhance lectin- as well as phorbol ester-induced T cell activation in the absence of M phi. Cross-linking of class I Ag on T cell surface mobilizes cytoplasmic Ca2+, and also enhances the CD3-induced Ca2+ flux inside the cells indicating a functional relationship between CD3 and class I Ag. Though surface modulation and immunoprecipitation experiments do not indicate any physical association between these two types of molecules on the T cell surface, capping studies show that cross-linking of class I Ag induces an association of these Ag with CD3. Binding of anti-CD3 Ab enhances the strength of association between CD3 and class I Ag, and the former co-caps completely with the latter. Based on these observations we propose that during antigen presentation M phi (or Ag-presenting cells) and T cells, besides interacting via peptide--class II Ag/CD3--T cell receptor complex formation, also interact through class I Ag. The latter interaction may stabilize the contact formation between T cells and Ag-presenting cell and support T cell activation.

Receptor-like nature of class I HLA: endocytosis via coated pits.

The present investigations show that class I HLA are internalized by macrophage/monocyte type cells. Anti-class I antibody-binding assays show that about 30% of class I Ag present on cell surface are endocytosed within 1 h. Electronmicroscopic investigations reveal that, like other well established receptor molecules, internalization of HLA is mediated by coated pits and coated vesicles. The endocytosed Ag are transferred from endosomes to trans-Golgi reticulum and trans-Golgi cisternae, suggesting recycling of these Ag back to the cell surface. In the presence of phorbol ester tetradecanoyl phorbol acetate, there is a modest increase in the rate of internalization. These results are consistent with the hypothesis that class I Ag on monocytes/macrophages behave like receptor molecules. Malignant transformation of monocytic cells apparently causes the loss of this property of class I Ag.

Protein kinase A-mediated phosphorylation of the T-cell surface antigen CD27.

CD27 is a T-cell surface antigen expressed on the majority of peripheral T cells and belongs to a newly defined receptor family including the low-affinity nerve growth factor receptor, tumour necrosis factor (TNF) receptors, the B-cell activation antigen CD40, and the Fas antigen. Although the function of CD27 has not been defined, several experimental observations support the notion that this molecule plays an important role in the process of T-cell activation. In this paper, we have demonstrated that a rapid hyperphosphorylation of CD27 is induced by a cyclic AMP-inducing agent, forskolin, and a membrane-permeable cAMP analogue, 8-bromo-cAMP, as well as phorbol 12-myristate 13-acetate (PMA). In addition, increased phosphorylation of CD27 in T-cell activation either via CD2 or CD3 pathways was strongly suppressed by a cyclic nucleotide-dependent kinase inhibitor, H-8, but only slightly by a protein kinase C inhibitor, staurosporine. These results suggest that protein kinase A might be a key kinase responsible for CD27 phosphorylation in the process of T-cell activation. CD27 is the first T-cell surface antigen demonstrated to be phosphorylated by the cyclic AMP-protein kinase A-mediated pathway.

The role of class I histocompatibility antigens in the regulation of T-cell activation.

Class I major histocompatibility antigens in humans (HLA antigens) were found to participate in the regulation of T-cell activation and proliferation induced by phytohemagglutinin. W6/32, a monomorphic antibody directed against class I HLA-A,B,C antigens, significantly inhibited the phytohemagglutinin-induced cell proliferation of peripheral blood lymphocytes. Almost complete suppression of cell activation was achieved on a subfraction of peripheral blood lymphocytes enriched in Mo1+ monocyte/macrophage cells. This inhibition of cell proliferation takes place at an early stage of activation and was found to be adherent cell dependent. Removal of monocyte/macrophage type cells from peripheral blood lymphocytes completely abrogated the inhibitory influence of anti-HLA-class I antibody, and, upon adding them back, suppression reappeared. Indirect immunofluorescence demonstrated that the expression of receptors for interleukin 2 and transferrin was impaired in the presence of antibody. Although the amount of interleukin 2 synthesized by these cells was also reduced, the addition of exogenous purified interleukin 2 did not restore cell proliferation. Mitogenesis induced by the Ca2+ ionophore A23187 was similarly suppressed, but mitogenesis induced by the phorbol diester phorbol myristate acetate, which activates cells by directly stimulating protein kinase C, was not suppressed. These results are consistent with a hypothesis that HLA class I antigens regulate an early event(s) of the Ca2+-dependent pathway of activation of T lymphocytes and that this event(s) apparently occurs before protein kinase C stimulation.

PLC gamma 1, a possible mediator of T cell receptor function.

Stimulation of T cell antigen receptor (TCR/CD3) following the recognition of peptide-major histocompatibility antigen complex induces phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, the phospholipase C (PLC) enzyme mediating this process has not been identified. We report that PLC gamma 1 protein is expressed in human T cells. It is a phosphoprotein, and the activation of cyclic AMP-dependent protein kinase (PKA) or of protein kinase C (PKC) with forskolin or phorbol ester, respectively, increases the level of phosphorylation. CD3 stimulation of T cells induces tyrosine phosphorylation of PLC gamma 1 and causes 8-10-fold higher yield of PLC activity with anti-phosphotyrosine antibody (APTyr Ab) from activated cells than from non-activated cells. Genistein, an inhibitor of protein tyrosine kinase, decreases this yield of AP-Tyr Ab-bound PLC activity from activated cells and lowers the level of Ca2+ mobilization. Furthermore, phorbol ester and forskolin treatment of cells before CD3 stimulation reduces the level of tyrosine phosphorylation of PLC gamma 1 and the PLC activity associated with APTyr Ab. These results suggest that CD3 stimulation activates PIP2 hydrolysis by inducing tyrosine phosphorylation of PLC gamma 1, which is regulated negatively by PKC and PKA.

MHC class I antigens regulate CD3-induced tyrosine phosphorylation of proteins in T cells.

The MHC class I antigens enhance the T cell response to various mitogenic stimuli. Class I antigens co-cap and associate with the CD3 structure on these cells. The present work shows that co-aggregation of these MHC antigens with CD3 induces a sustained elevated Ca2+ response in T cells. The duration required for a 50% decline in peak response is five to 10 times longer when class I antigens are cross-linked with CD3 as compared to that when cells are stimulated through CD3 alone. Further analysis reveals that class I antigens prolong the duration of CD3-induced tyrosine phosphorylation of several proteins. No protein tyrosine kinase activity is found associated with these MHC antigens which could explain their influence on CD3 activation. Similarly, class I antigens do not depend on the membrane-bound protein tyrosine phosphatase CD45, since they elevate the degree of CD3-induced Ca2+ response in a CD45-deficient Jurkat cell line. On the contrary, in a CD45- HPB cell line defective in CD3 signaling, co-aggregation of class I antigens with CD3 does not induce Ca2+ flux. Therefore, the effect of class I antigens on CD3 function depends on the ability of CD3 to transduce a signal. Furthermore, cytofluorometric studies show that cross-linking of class I antigens with CD3 inhibits the internalization of the latter. Thus, class I antigens seem to prolong the duration of signal transduction through CD3 by retarding its down-regulation.

The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.

The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface of helper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4+ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55-60 kDa, which is expressed specifically in T cells. The PTK is the human analogue of the murine pp56LSTRA (pp56lck) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO4/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0-5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PTK at 55-60 kDa. The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.

The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.

Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex.