Tyrosine Kinase Activity Of P56lck Research Articles

Inhibitory effects of atractylone on mast cell-mediated allergic reactions

This study investigated a salutary effect of atractylone (Atr) which is an active constituent of Pyeongwee-San (KMP6) on mast cell-mediated allergic reactions. Our previous report indicated that KMP6 regulated allergic reactions. Thus, this study sought to determine the potential of Atr in vitro models, compound 48/80-stimulated rat peritoneal mast cells (RPMCs), phorbol 12-myristate 13-acetate (PMA) plus A23187-stimulated human mast cell line (HMC-1) cells, and stem cell factor (SCF)-stimulated RPMCs as well as in vivo models, IgE-mediated passive cutaneous anaphylaxis (PCA), compound 48/80-induced systemic anaphylaxis, and compound 48/80-induced ear swelling. The results showed that Atr inhibited compound 48/80-induced RPMCs degranulation, intracellular calcium level, tryptase release, and histamine release. Atr inhibited the up-regulation of p56lck tyrosine kinase activity by compound 48/80. And Atr reduced tryptase and histamine releases from PMA plus A23187-stimulated HMC-1 cells. In addition, Atr decreased histidine decarboxylase activity and expression in the activated HMC-1 cells. Atr inhibited SCF-induced morphological alteration and filamentous actin formation in RPMCs. Atr improved IgE-induced PCA reaction by decreasing the levels of histamine, IgE, interleukin (IL)-4, IL-5, IL-6, vascular endothelial growth factor, and IL-13 in the serum of PCA-induced mice. Furthermore, Atr mitigated compound 48/80-induced systemic anaphylaxis and ear swelling. Taken together, these results of this study indicate that Atr regulates the degranulation of mast cell, proving its potential in the treatment of mast cell-mediated allergic reactions.

Damnacanthal inhibits the NF-κB/RIP-2/caspase-1 signal pathway by inhibiting p56lck tyrosine kinase

Damnacanthal is a major constituent of Morinda citrifolia L. (noni) and exhibits anti-cancer and anti-inflammatory activities. However, the effects of damnacanthal on allergic diseases have not been determined. In this study, we investigated the effect of damnacanthal on mast cell-mediated allergic inflammatory responses. Damnacanthal significantly and dose-dependently inhibited compound 48/80-induced systemic anaphylactic shock, histamine release and intracellular calcium levels. In particular, IgE-mediated passive cutaneous anaphylaxis was significantly inhibited by the oral administration of damnacanthal. In addition, we report for the first time that p56lck tyrosine kinase was expressed in phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated mast cells. Furthermore, damnacanthal inhibited the up-regulation of p56lck tyrosine kinase activity by PMACI and repressed PMACI-induced histidine decarboxylase expression and activity. Damnacanthal also inhibited PMACI-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α expressions by suppressing nuclear factor-kappa B (NF-κB) activation and suppressed the activation of caspase-1 and the expression of receptor interacting protein-2. This study shows damnacanthal inhibits the NF-κB/receptor-interacting protein-2/caspase-1 signal pathway by inhibiting p56lck tyrosine kinase and suggests that damnacanthal has potential for the treatment of mast cell-mediated allergic disorders.

4-thiophenoxy-N-(3,4,5-trialkoxyphenyl)pyrimidine-2-amines as potent and selective inhibitors of the T-cell tyrosine kinase p56lck.

We have developed a series of 4-thiophenoxy-N-(3,4,5-trialkoxyphenyl) pyrimidine-2-amines as potent and selective inhibitors of p56lck tyrosine kinase activity. In particular, the most potent inhibitor shows cellular activity in T-cell receptor (TCR) stimulated models of cytokine release, which suggests an immunomodulatory role for this class of inhibitor.

Molecular mechanisms of L-selectin-induced co-localization rafts and shedding

Leukocyte recruitment to lymph nodes or inflammatory sites is regulated by adhesion and activation. L-selectin (CD62L) is expressed on leukocytes and mediates tethering and rolling of leukocytes on endothelial cells. Upon stimulation L-selectin is down-regulated by proteolytic cleavage but the molecular mechanisms regulating this shedding step are poorly defined. To study intracellular mechanisms, we induced shedding of L-selectin by cross-linking with an immobilized L-selectin antibody (Dreg56) in Jurkat cells. The loss of surface expression was quantitated by flow cytometry and the increase of soluble L-selectin was determined by Western blot analysis. We find that Jurkat and p56 lck-deficient JCaM1.6 cells released L-selectin to similar extent (18±4% and 17±3%, respectively) and revealed comparable inhibition with the src-tyrosine kinase inhibitor PP2. Glutathione (GSH), an inhibitor of the neutral sphingomyelinase, PD98059, a MAP-kinase (MAP-K) inhibitor and metalloprotease inhibitors (MPI) (TAPI, Ro 31-9790, and BB-3103) reduced significantly L-selectin-induced shedding by 60–80%. In Jurkat cells, L-selectin was present in Triton X-100 insoluble membrane rafts and was constitutively tyr-phosphorylated. Dreg56 cross-linking enhanced phosphorylation and recruitment of L-selectin into rafts which was significantly decreased by pretreatment of cells with PD98059. We conclude, that the metalloproteinase-mediated cleavage of L-selectin from cell surface is triggered by intracellular signaling pathways that are independent of p56 lck tyrosine kinase activity, but require other tyrosine kinases and the neutral sphingomyelinase. The cleavage of L-selectin might involve membrane rafts as signaling platform.

Damnacanthal is a highly potent, selective inhibitor of p56lck tyrosine kinase activity.

Damnacanthal, an anthraquinone isolated from a plant extract, was found to be a potent, selective inhibitor of p56lck tyrosine kinase activity. The structure, potency, and selectivity of damnacanthal were confirmed by independent synthesis and testing. Damnacanthal exhibited an IC50 of 17 nM for inhibition of p56lck autophosphorylation and an IC50 of 620 nM for phosphorylation of an exogenous peptide by p56lck. Damnacanthal had > 100-fold selectivity for p56lck over the serine/threonine kinases, protein kinase A and protein kinase C, and > 40-fold selectivity for p56lck over four receptor tyrosine kinases. It also demonstrated modest (7-20-fold), but highly statistically significant, selectivity for p56lck over the homologous enzymes p60src and p59fyn. Mechanistic studies demonstrated that damnacanthal was competitive with the peptide binding site, but mixed noncompetitive with the ATP site. Although damnacanthal contains a potentially reactive aldehyde moiety, equilibrium dialysis experiments demonstrated that significant amine formation between damnacanthal and amines occurred only at high concentrations of reactants. However, damnacanthal appeared to bind nonspecifically to membrane lipids and was not active in whole cell tyrosine kinase assays. Damnacanthal is the most potent, selective inhibitor of p56lck tyrosine kinase activity described to date and may represent the starting point for the identification of novel, selective inhibitors of p56lck which are active in whole cell as well as in cell-free systems.

CD45 phosphatase in Jurkat cells is necessary for response to applied ELF magnetic fields

Oscillations of free intracellular calcium [Ca 2+] i were seen in individual Jurkat cells as response to a 50 Hz, 0.15 mT magnetic field (MF). In contrast, a CD45-deficient Jurkat cell line was unable to respond to MF stimulation. The phosphatase activity of CD45 has been implicated to regulate p56 lck tyrosine kinase activity by removing an inhibitory phosphate. By using Jurkat cells that expressed a chimeric molecule, comprising the cytoplasmic phosphatase domain of CD45, the MF induced calcium response was restored. This showed the necessity for an intact signal transduction pathway leading to a calcium increase as a result of stimulation of cells by MF. Thus, our data suggest that the target for the applied MF are molecules involved in early events in the signalling pathway from the T cell antigen receptor.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.

Tyr394 and Tyr505 are autophosphorylated in recombinant Lck protein-tyrosine kinase expressed in Escherichia coli.

The activity of the Src family protein-tyrosine kinase p56lck is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505. Tyr394 is autophosphorylated after p56lck activation, whereas phosphorylation of Tyr505 is believed to be due to p50csk which negatively modulates p56lck activity. To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S-transferase expression system to express wild-type Lck, the mutants [Y394F]Lck and [Y505F]Lck, a kinase-deficient p56lck with a mutation of the ATP-binding site [K273E]Lck and a double mutant [Y394F, Y505F]Lck. We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild-type Lck both in vivo and in vitro. Wild-type Lck, [Y505F]Lck and [Y394F]Lck were phosphorylated on tyrosine. Both the kinase-deficient mutant[K273E]Lck and the double mutant [Y394F, Y505F]Lck did not react with monoclonal anti-phosphotyrosine antibody [anti-Y(P) mAb], thus providing evidence that (a) the bacterial strains used lacked intrinsic protein-tyrosine kinase activities, and therefore tyrosine phosphorylations of wild-type Lck, [Y505F]Lck and [Y394F]Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56lck can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505. Phosphopeptide mapping analysis confirmed that p56lck can undergo autophosphorylation on these two tyrosine residues. We propose that autophosphorylation at Tyr505 of p56lck may represent an accessory mechanism for the down-regulation of the tyrosine kinase activity of p56lck.

Molecular Analysis of the T-Cell Acute Lymphoblastic Leukemia-Associated t(1;7)(p34;q34) That Fuses LCK and TCRB

Abstract Previously we had characterized the t(1;7)(p34;q34) translocation from HSB-2. This translocation fused the beta T-cell receptor gene (TCRB) constant region and transcriptional enhancer with the type I transcription unit of the LCK gene on the derivative 1 [der(1)] chromosome. The type II promoter was translocated to the der(7) chromosome. Regarding the mechanism of the t(1;7) in HSB-2, we identified an alternating purine-pyrimidine tract (G-T)17 at the 1p34/LCK breakpoint. Additionally, sequence analysis of both breakpoint junctions provided data that implicate the V(D)J recombinase in formation of the t(1;7). A heptamer-nonamer recognition sequence with a 12-bp spacer was found in the immediate vicinity of the 1p34/LCK breakpoint and, thus, chromosomal breakage at 1p34 may be explained as resulting from recombinase activity. Because phosphorylation of Tyr-505 in vivo regulates the tyrosine kinase activity of p56lck we amplified a region from LCK exon 12 that contains the codon for Tyr-505 and showed no mutation of this codon in HSB-2 DNA and, therefore, p56lck in HSB-2 is not activated by mutation of Tyr-505. We have analyzed LCK gene expression in HSB-2 and SUP-T12 cell lines. RNase protection analysis identified almost exclusively type I transcripts in HSB-2. An independent t(1;7) in SUP-T12 also resulted in the juxtaposition of LCK to TCRB. The breakpoint in SUP-T12 occurred 2 kb 5′ of the type II promoter, leaving an intact LCK gene on the der(1) chromosome. RNase protection analysis identified both type I and type II LCK transcripts in a 3:1 ratio in SUP-T12. Factors other than proximity to the TCRB enhancer must affect promoter utilization in this cell line.

Molecular analysis of the T-cell acute lymphoblastic leukemia- associated t(1;7)(p34;q34) that fuses LCK and TCRB

Previously we had characterized the t(1;7)(p34;q34) translocation from HSB-2. This translocation fused the beta T-cell receptor gene (TCRB) constant region and transcriptional enhancer with the type I transcription unit of the LCK gene on the derivative 1 [der(1)] chromosome. The type II promoter was translocated to the der(7) chromosome. Regarding the mechanism of the t(1;7) in HSB-2, we identified an alternating purine-pyrimidine tract (G-T)17 at the 1p34/LCK breakpoint. Additionally, sequence analysis of both breakpoint junctions provided data that implicate the V(D)J recombinase in formation of the t(1;7). A heptamer-nonamer recognition sequence with a 12-bp spacer was found in the immediate vicinity of the 1p34/LCK breakpoint and, thus, chromosomal breakage at 1p34 may be explained as resulting from recombinase activity. Because phosphorylation of Tyr-505 in vivo regulates the tyrosine kinase activity of p56lck we amplified a region from LCK exon 12 that contains the codon for Tyr-505 and showed no mutation of this codon in HSB-2 DNA and, therefore, p56lck in HSB-2 is not activated by mutation of Tyr-505. We have analyzed LCK gene expression in HSB-2 and SUP-T12 cell lines. RNase protection analysis identified almost exclusively type I transcripts in HSB-2. An independent t(1;7) in SUP-T12 also resulted in the juxtaposition of LCK to TCRB. The breakpoint in SUP-T12 occurred 2 kb 5' of the type II promoter, leaving an intact LCK gene on the der(1) chromosome. RNase protection analysis identified both type I and type II LCK transcripts in a 3:1 ratio in SUP-T12. Factors other than proximity to the TCRB enhancer must affect promoter utilization in this cell line.

Inhibition of Interleukin 2 (IL-2)-stimulated tyrosine kinase activity by leflunomide

We have used antigen-specific T-cell clones plus antigen to examine the biochemical effects of leflunomide on T cells during an immune response. Leflunomide inhibited both antigen and IL-2-stimulated proliferation of these clones, with an ED50 of 8–10 μM for either stimuli. Conversely, antigen-stimulated cytokine production and up-regulation of the IL-2 receptor (CD25) were relatively insensitive to inhibition by leflunomide. IL-2-receptor-mediated signaling, however, was inhibited by this drug. Using32P-orthophosphate metabolic labeling and anti-phosphotyrosine immunoprecipitation, we were able to show that leflunomide inhibited IL-2-stimulated tyrosine kinase activity. Furthermore, we demonstrated that leflunomide directly inhibited the IL-2 stimulated tyrosine kinase activity of p56lck. These data suggest that inhibition of IL-2-stimulated p56lck activity could play a role in leflunomide-mediated immunosuppression.

Differential CD4-dependent regulation of naive and memory CD4+ T cell adhesion is not related to differences in expression and function of CD4 and p56lck.

We have previously reported that antigen-independent adhesion of CD45RO+ memory CD4+ T cells to B cells is negatively regulated by CD4-MHC class II interaction, whereas that of CD45RA+ naive CD4+ T cells is not. We have now found that both cross-linking of CD4 ligands [anti-CD4 mAbs, HIV gp160 (env) protein and a 12mer peptide encompassing the 35-46 sequence of the HLA-DR beta 1 domain] on CD4+ naive T cells and activation-induced conversion of naive CD4+ T cells to memory T cells leads to CD4-dependent down-regulation of adhesion. To further elucidate CD4-dependent differential regulation of naive and memory T cell adhesion to B cells, we investigated the expression and function of CD4 and p56lck, a tyrosine kinase associated with the cytoplasmic domain of CD4. p56lck tyrosine kinase activity was equally enhanced by anti-CD4 mAbs and gp160 (120) in the two subsets. Furthermore, cell-surface CD4 down-modulation by phorbol myristate acetate or anti-CD4 mAbs was similar in the two subsets, which express the same amounts of both cell-surface CD4 and CD4-associated p56lck. Finally, the pattern of tyrosine phosphorylation of cellular proteins induced by gp120 (160) was similar in the two subsets. Taken together, these results indicate that the different sensitivity of naive and memory CD4+ T cells to CD4-dependent regulation of adhesion is not accounted for by differences in the tyrosine kinase activity of p56lck; it probably, therefore, involves a step downstream of p56lck or another pathway differentially used in naive and memory CD4+ T cells.

P59fyn tyrosine kinase regulates p56lck tyrosine kinase activity and early TCR-mediated signaling.

To study the role of p59fyn in T cell activation, we used antisense RNA to inhibit p59fyn expression in a T cell clone. Transfectants with reduced levels of p59fyn were functionally impaired in their responses to antigen, Con A+recombinant IL-1 and cross-linking with anti-TCR mAb. Induction of tyrosine phosphorylation on most intracellular substrates was greatly reduced. We also noted that the lck kinase activity was greatly reduced even though the amount of lck protein was equivalent to that present in parental D10 cells. Our results suggest that the protein tyrosine kinase p59fyn is critical in TCR-mediated signaling and also suggests that p59fyn may regulate p56lck tyrosine kinase activity.

Both T cell receptor (TcR)-CD3 complex and CD2 increase the tyrosine kinase activity of p56lck. CD2 can mediate TcR-CD3-independent and CD45-dependent activation of p56lck.

T cell activation by triggering the T cell receptor (TcR)-CD3 complex leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins. To date, there has been no direct evidence on the identity of the tyrosine kinase activity implicated in this signaling pathway. In this study, we demonstrate that activation of human T cells with anti-CD3 monoclonal antibody increases tyrosine kinase activity of p56lck. This extends our previous findings which demonstrated the involvement of p56lck kinase activity in the CD2 signal transduction pathway. The results from peripheral blood lymphocytes and Jurkat cell line showed in both cases an early and transient change in the specific activity of p56lck, followed by a shift to a higher apparent molecular mass. Therefore, to test directly the role of TcR-CD3 in CD2-induced activation of p56lck, we utilized mutant variants of the Jurkat cell line lacking in cell surface TcR-CD3. We found that cell surface expression of TcR-CD3 is not required for the activation of p56lck via CD2 but is necessary for the appearance of the reduced-electrophoretic-mobility form of p56lck observed after CD2 triggering. By isolating CD45- mutants from Jurkat cells, we observed that surface expression of the tyrosine phosphatase CD45 is required in order to increase p56lck activity following CD2 stimulation, while CD4-induced activation of the kinase remained unchanged. These data provide evidence for a specific functional linkage between CD2 and p56lck, in which CD45 may play an essential role.

Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: Implications for mechanisms of IL-2-signal transduction

Recently, the SRC-like non-receptor protein tyrosine kinase p56-LCK has been shown to physically associate with the interleukin-2 receptor (IL-2-R) complex and to undergo rapid elevations in its tyrosine kinase activity upon stimulation of T lymphocytes with IL-2. The functional significance of p56-LCK kinase activation for IL-2-mediated lymphocyte responses, however, has never been directly assessed. Using gene transfer approaches, we have achieved markedly elevated levels of p56-LCK kinase activity in the IL-2-dependent cytolytic T-cell line CTLL-2 and the helper line HT-2. CTLL-2 and HT-2 cells that were stably transfected with expression plasmids encoding either the normal human p56-LCK or a constitutively active version of the mouse p56-LCK kinase (LCK[Y505]) contained striking elevations in the levels of tyrosine phosphorylation on several proteins (34–36, 50–60, 62–68, 77–78, 104–110 kDa), as determined by immunoblot analysis using anti-phosphotyrosine antibodies. CTLL-2 and HT-2 LCK- and LCK(Y505F)-transfected cells remained dependent on IL-2 for their growth and survival in culture despite the findings that (i) IL-2 specifically stimulated elevations in the activity of the endogenous p56-LCK in untransfected CTLL-2 cells without affecting the activities of the other SRC-like kinases in these cells (p59-FYN, p62-YES) and that (ii) IL-2-mediated regulation of p56-LCK correlated with IL-2-driven proliferation of these T cells. Specifically, no elevation in the proliferation (DNA synthesis) or growth of these T cells was found at any of the concentrations of IL-2 examined (0.01–25 U/ml), relative to untransfected and control transfected cells. Furthermore, when cultured in the absence of IL-2, transfected T cells whose relative levels of p56-LCK activity were elevated by ≈ 20–50-fold died with the same kinetics as control cells and underwent apoptosis, as defined by uptake of trypan blue dye and DNA fragmentation assays, respectively. Taken together, these data indicate that while IL-2 can up-regulate the enzymatic activity of p56-LCK, elevated levels of p56-LCK tyrosine kinase activity are insufficient to stimulate IL-2-mediated pathways required for T-cell growth and survival. These findings thus imply the existence of other signal-transducing molecules, besides p56-LCK, that physically participate in IL-2R complexes and that are necessary for initiation of the biochemical events ultimately responsible for IL-2's pleiotropic actions on lymphocytes.

The lymphocyte-specific tyrosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2.

Incubation of the human T cells, Jurkat, with two sets of activating anti-CD2 mAb (T11(2) + T11(3), D66 + T11(1)) induced delocalization of p56lck and CD2 receptors from the plasma membrane and increased the tyrosine kinase activity of p56lck. The anti-CD2 mAb combination (T11(2) + T11(3)) that produced the most rapid increase in p56lck kinase activity also induced the most rapid delocalization of the kinase. In stimulated cells, both p56lck and CD2 receptors are detected in cytoplasmic vesicles. The internalization of p56lck in endocytic vesicles was established by confocal microscopy. By double staining it was shown that only part of the p56lck colocalized with the internalized CD2 receptor suggesting distinct sorting processes. Internalization of p56lck appeared to be specific of CD2 stimulation as: 1) in Jurkat cells triggered with an anti-CD3 mAb, p56lck was not internalized whereas CD3 receptors were completely endocytosed; 2) when cells were stimulated via CD4, the kinase and CD4 receptors remained associated with the plasma membrane. In addition, internalization of p56lck upon stimulation of CD2 receptors was not modified in CD2+/CD3-Jurkat cells indicating that CD3 is not involved in this process. The identification of different subcellular localizations of p56lck in resting and stimulated T cells should represent an important step in the definition of its functional activity.

Coassociation of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lymphocytes.

In the present report, we demonstrated that modulation of CD26 from T cell surface induced by antiCD26 (1F7) led to enhanced phosphorylation of CD3 zeta tyrosine residues and increased CD4 associated p56lck tyrosine kinase activity. We further showed that CD26 was comodulated on the T cell surface with CD45, a known membrane-linked protein tyrosine phosphatase and that anti-CD26 was capable of precipitating CD45 from T cell lysates. These findings strongly suggest that CD26 may be closely associated with the CD45 protein tyrosine phosphatase on T cell surface and further support the notion that the interaction of CD26 with CD45 results in enhanced tyrosine kinase activity, zeta chain phosphorylation, and T cell activation.

The tyrosine kinase activity of p56lck is increased in human T cells activated via CD2

An early biochemical event associated with T cell activation is tyrosine phosphorylation. We have previously shown that p56lck, a lymphocyte-specific protein tyrosine kinase, is hyperphosphorylated on serine and tyrosine residues 15 minutes after activation via CD2 with a concomitant shift to a higher molecular mass. We now demonstrate that the tyrosine kinase activity of p56lck is increased within seconds following CD2 triggering. This activity decreases thereafter correlating with the appearance of changes in phosphorylation previously described. These results suggest that p56lck may play an important role in the CD2 activation pathway.

Effect of human immunodeficiency virus gp120 glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human T lymphocytes

The human immunodeficiency virus binds to CD4+ T lymphocytes through the interaction of its envelope glycoprotein (gp120) with the CD4 molecule. The src-related protein tyrosine kinase p56lck is physically associated with CD4 and is co-immunoprecipitated by CD4 monoclonal antibody (mAb). Activators of protein kinase C (PKC) cause the dissociation of p56lck from CD4. Here we report that gp120 mAb immunoprecipitated the p56lck.CD4.gp120 complex after short term treatment (20 min) of human T lymphocytes with gp120. The p56lck that was associated with the CD4.gp120 complex was dissociated by activators of PKC. This effect was abolished by pretreatment of cells with PKC inhibitors. Thus the p56lck.CD4.gp120 immune complex immunoprecipitated by gp120 mAb behaves in a similar manner, with respect to PKC activation or inhibition, to the p56lck.CD4 complex immunoprecipitated by CD4 mAb. Short term treatment of cells with gp120, followed by gp120 mAb, resulted in an increase in the tyrosine kinase activity of p56lck associated with CD4. However, the amount of enzyme associated with CD4 remained unchanged. Long term treatment (20 h) of human T lymphocytes with gp120 resulted in the down-regulation of cell surface CD4 molecules. A parallel decrease in CD4-associated gp120 was also observed. In addition, gp120 caused the dissociation of p56lck and CD4. However, the dissociation of the p56lck from CD4 occurred at much faster rate than the down-regulation of surface CD4 molecules. Such mechanisms may account for the down-regulation of cell surface CD4 molecules and the depletion of functional CD4+ T lymphocytes which are characteristic of human immunodeficiency virus infections and acquired immune deficiency syndrome pathogenesis.

Cross-Linking of T-Cell Surface Molecules CD4 and CD8 Stimulates Phosphorylation of the lck Tyrosine Protein Kinase at the Autophosphorylation Site

p56lck, a lymphocyte-specific tyrosine protein kinase, binds to the cytoplasmic tails of the T-cell surface molecules CD4 and CD8. Cross-linking of CD4 expressed on the surface of murine thymocytes, splenocytes, and CD4+ T-cell lines induced tyrosine phosphorylation of p56lck dramatically. Cross-linking of CD8 stimulated tyrosine phosphorylation of p56lck strongly in murine L3 and GA4 cells, slightly in splenocytes, but not detectably in thymocytes. Differing effects of cross-linking on in vitro tyrosine kinase activity of p56lck were observed. An increase in the in vitro kinase activity of p56lck, when assayed with [Val5]-angiotensin II as an exogenous substrate, was found to accompany cross-linking of CD4 in three cell lines. No stimulation of the in vitro kinase activity, however, was observed after cross-linking of CD8 in L3 cells. The phosphorylation of p56lck at Tyr-394, the autophosphorylation site, was stimulated by cross-linking in all cell lines examined. Tyr-394 was the predominant site of increased tyrosine phosphorylation in two leukemic cell lines. In the other two cell lines, the phosphorylation of both Tyr-394 and an inhibitory site, Tyr-505, was found to increase. In contrast to cross-linking with antibodies, no striking increase in the tyrosine phosphorylation of p56lck was stimulated by antigenic stimulation. Therefore, the effect of antibody-induced aggregation of CD4 and CD8 on the tyrosine phosphorylation of p56lck differs, at least quantitatively, from what occurs during antigen-induced T-cell activation.