Superantigen-induced T cell:B cell conjugation is mediated by LFA-1 and requires signaling through Lck, but not ZAP-70.

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.

Differential T-cell antigen receptor signaling mediated by the Src family kinases Lck and Fyn.

Src family tyrosine kinases play a key role in T-cell antigen receptor (TCR) signaling. They are responsible for the initial tyrosine phosphorylation of the receptor, leading to the recruitment of the ZAP-70 tyrosine kinase, as well as the subsequent phosphorylation and activation of ZAP-70. Molecular and genetic evidence indicates that both the Fyn and Lck members of the Src family can participate in TCR signal transduction; however, it is unclear to what extent they utilize the same signal transduction pathways and activate the same downstream events. We have addressed this issue by examining the ability of Fyn to mediate TCR signal transduction in an Lck-deficient T-cell line (JCaM1). Fyn was able to induce tyrosine phosphorylation of the TCR and recruitment of the ZAP-70 kinase, but the pattern of TCR phosphorylation was altered and activation of ZAP-70 was defective. Despite this, the SLP-76 adapter protein was inducibly tyrosine phosphorylated, and both the Ras-mitogen-activated protein kinase and the phosphatidylinositol 4, 5-biphosphate signaling pathways were activated. TCR stimulation of JCaM1/Fyn cells induced the expression of the CD69 activation marker and inhibited cell growth, but NFAT activation and the production of interleukin-2 were markedly reduced. These results indicate that Fyn mediates an alternative form of TCR signaling which is independent of ZAP-70 activation and generates a distinct cellular phenotype. Furthermore, these findings imply that the outcome of TCR signal transduction may be determined by which Src family kinase is used to initiate signaling.

Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.

The lck SH3 domain is required for activation of the mitogen-activated protein kinase pathway but not the initiation of T-cell antigen receptor signaling.

Initiation of T-cell antigen receptor (TCR) signaling is dependent upon the activity of protein tyrosine kinases. The Src family kinase Lck is required for the initial events in TCR signaling, such as the phosphorylation of the TCR complex and the activation of ZAP-70, but little is known of its role in downstream signaling. Expression of a mutated form of Lck lacking SH3 domain function (LckW97A) in the Lck-deficient T-cell line JCaM1 revealed a requirement for Lck beyond the initiation of TCR signaling. In cells expressing LckW97A, stimulation of the TCR failed to activate the mitogen-activated protein kinase (MAPK) pathway, despite normal TCR zeta chain phosphorylation, ZAP-70 recruitment, and ZAP-70 activation. Activation of extracellular signal-regulated kinase (ERK) and MAPK kinase (MEK), as well as the induction of CD69 expression, was greatly impaired in JCaM1/LckW97A cells. In contrast, the phosphorylation of phospholipase Cgamma1 (PLCgamma1) and corresponding elevations in intracellular calcium concentration ([Ca2+]i) were intact. Thus, cells expressing LckW97A exhibit a selective defect in the activation of the MAPK pathway. These results demonstrate that Lck has a role in the activation of signaling pathways beyond the initiation of TCR signaling and suggest that the MAPK pathway may be selectively controlled by regulating the function of Lck.

Mercurial-induced alterations in neuronal divalent cation homeostasis.

Mercurials such as Hg2+ and methylmercury (MeHg) are environmental contaminants. Both are neurotoxic upon chronic and acute exposure, however, these toxic manifestations are distinct. The mechanisms underlying this cytotoxicity remain unknown, but may be related to a disruption in divalent cation homeostasis because both disrupt Ca(2+)-dependent processes in several model systems. These effects include a block in nerve-evoked neurotransmitter release as well as an increase in spontaneous transmitter release. This suggests that mercurials simultaneously decrease Ca2+ influx following nerve stimulation, and increase intracellular Ca2+ concentration ([Ca2+]i) in the nerve terminal. Although these effects appear to be at odds, they can be justified mechanistically. Both Hg2+ and MeHg block voltage-activated Ca2+ channels in the nerve terminal. The mechanism of block by these mercurials is different, since Hg2+ and MeHg are competitive and noncompetitive inhibitors of Ca2+ influx, respectively. The functional consequence in both instances remains decreased Ca2+ influx into the nerve terminal following the invasion of an action potential leading to decreased nerve-evoked release of neurotransmitter. The effects of mercurials on voltage-activated Ca2+ channels are distinct from those which mediate the increases in spontaneous transmitter release. Reducing extracellular Ca2+ concentration ([Ca2+]e) decreased, but did not prevent, the mercurial-induced increases in spontaneous transmitter release, suggesting that both intra- and extracellular sources of Ca2+ contribute to mercurial-induced elevations in [Ca2+]i in a nerve terminals. The effects of MeHg on divalent cation homeostasis have been studied using isolated nerve terminals from the rat brain (synaptosomes) and cells in culture (NG108-15 and isolated cerebellar granule cells) loaded with the Ca(2+)-selective fluorescent indicator fura-2. In synaptosomes, MeHg caused an Ca(2+)e-independent elevation in intrasynaptosomal Zn2+ concentration ([Zn2+]i) as well as an Ca(2+)e-dependent elevation in [Ca2+]i. The elevations in [Zn2+]i and [Ca2+]i were mediated by release of Zn2+ from soluble synaptosomal proteins and increased plasma membrane permeability, respectively. In NG108-15 cells, the effects of MeHg on divalent cation concentrations were more complex. First, MeHg mobilized Ca2+ from an intracellular store sensitive to inositol-1,4,5-tris-phosphate (IP3) which was independent of IP3 generation. Second, MeHg increased the intracellular concentration of an endogenous polyvalent cation, possibly Zn2+. Finally, MeHg caused an increase in the plasma membrane permeability to Ca2+ which was attenuated by high concentrations of the voltage-activated Ca2+ channel blocker nifedipine or by the voltage-activated Na+ channel blocker tetrodotoxin (TTX). While these studies demonstrate mercurials interfere with divalent cation regulation in neuronal systems, the consequences of these effects are not yet known.

Methylmercury-induced elevations in intrasynaptosomal zinc concentrations: an 19F-NMR study.

Methylmercury (MeHg) increases the concentration of intracellular Ca2+ ([Ca2+]i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca2+]i was strictly dependent on extracellular Ca2+ (Ca2+e); similarly, in NG108-15 cells, a component of the elevations in [Ca2+]i was Ca2+e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca2+e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn2+. Using 19F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'- tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn2+ concentration ([Zn2+]i). In buffer containing 200 microM EGTA to prevent the Ca2+e-dependent elevations in [Ca2+]i, the [Zn2+]i was 1.37 +/- 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn2+]i was 1.88 +/- 0.53 nM. Treatment of synaptosomes for 40 min with 125 microM MeHg yielded [Zn2+]i of 2.69 +/- 0.55 nM, whereas 250 microM MeHg significantly elevated [Zn2+]i to 3.99 +/- 0.68 nM. No Zn2+ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine (TPEN, 100 microM) following 250 microM MeHg exposure. [Ca2+]i in buffer containing 200 microM EGTA was 338 +/- 26 nM and was 370 +/- 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca2+]i was 498 +/- 28 or 492 +/- 53 nM during a 40-min exposure to 125 or 250 microM MeHg, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Methylmercury alters intrasynaptosomal concentrations of endogenous polyvalent cations.

The effects of the neurotoxic organomercurial methylmercury (MeHg) on intrasynaptosomal polyvalent cation concentrations were examined using fura-2. In the presence of extracellular Ca2+ (Ca2+e), MeHg caused a concentration-dependent, biphasic elevation in the ratio of fluorescence intensity at the emission wavelength of 505 nm following excitation at 340 and 380 nm (340/380 nm ratio). The first phase was independent of Ca2+e and complete within 5 sec. The second phase was dependent upon Ca2+e and was not complete within 6 min. MeHg increased the synaptosomal membrane permeability to Mn2+, suggesting that the second phase was due to influx of Ca2+e. Ruthenium red (20 microM), mitochondrial depolarization (10 mM NaN3 plus 4 micrograms/ml oligomycin), thapsigargin (1 microM), or caffeine (40 mM) did not elevate [Ca2+]i or alter the response of the synaptosomes to MeHg. Upon closer inspection, we noticed that MeHg simultaneously increased the fluorescence intensity at the excitation wavelengths of 340 and 380 nm and at the Ca(2+)-insensitive excitation wavelength of 360 nm. Pretreatment of synaptosomes with the cell-permeant heavy metal chelator TPEN (50 microM) blocked the MeHg-induced elevations in the 360-nm intensity and the 340/380 nm ratio. TPEN given after MeHg reversed the elevations in the 360-nm intensity. The cell-impermeant heavy metal chelator DTPA (150 microM) had no effect. We conclude that MeHg disrupts polyvalent cation homeostasis by at least two mechanisms. The first involves release of endogenous non-Ca2+ polyvalent cations, while the second is due to increased Ca2+ permeability of the plasma membrane.