Intrinsic Signals in the Unique Domain Target p56lckto the Plasma Membrane Independently of CD4

In T lymphocytes, the Src-family protein tyrosine kinase p56^lck (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell–specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles.

A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.

     

Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo

Background

Tau protein is the principal component of the neurofibrillary tangles found in Alzheimer's disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimer's disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease.

Results

In this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others.

Conclusions

Fyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.     

Activation of Lck is critically required for sphingosine-induced conformational activation of Bak and mitochondrial cell death

Despite extensive investigation, the molecular mechanism of anticancer activity of sphingolipid metabolites remains to be clarified. Here we demonstrate that sphingosine induces mitochondrial cell death via Lck-mediated conformational activation of Bak in Jurkat T cell lymphoma. Treatment of cells with sphingosine rapidly induced mitochondrial membrane potential loss, cytochrome c release from mitochondria, and apoptotic cell death. Sphingosine also induced conformational activation of Bak, but not Bax. siRNA targeting of Bak effectively attenuated sphingosine-induced mitochondrial cell death, indicating that Bak is involved in sphingosine-induced mitochondrial cell death. Sphingosine also induced activation of tyrosine kinase Lck. Inhibition of Lck by treatment of PP2, a Lck inhibitor or siRNA targeting of Lck suppressed sphingosine-induced conformational activation and oligomerization of Bak, mitochondrial membrane potential loss, and apoptotic cell death, implying that activation of Lck is critically required for sphingosine-induced conformational activation of Bak and mitochondrial cell death. The results elucidated in this study provide a novel cellular mechanism for the anticancer activity of sphingolipid metabolites.     

Autoimmune-associated PTPN22 R620W Variation Reduces Phosphorylation of Lymphoid Phosphatase on an Inhibitory Tyrosine Residue

A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid tyrosine phosphatase (LYP), which forms a complex with the kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T single nucleotide polymorphism results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent manner. T cell receptor-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue releases tonic inhibition of signaling by LYP. The R620W variation disrupts the interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which ultimately contributes to gain-of-function inhibition of T cell signaling.     

Conformational States Control Lck Switching between Free and Confined Diffusion Modes in T Cells

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.     

T cell receptor signal initiation induced by low-grade stimulation requires the cooperation of LAT in human T cells

Background

One of the earliest activation events following stimulation of the T cell receptor (TCR) is the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3-associated complex by the Src family kinase Lck. There is accumulating evidence that a large pool of Lck is constitutively active in T cells but how the TCR is connected to Lck and to the downstream signaling cascade remains elusive.

Methodology/Principal Findings

We have analyzed the phosphorylation state of Lck and Fyn and TCR signaling in human naïve CD4⁺ T cells and in the transformed T cell line, Hut-78. The latter has been shown to be similar to primary T cells in TCR-inducible phosphorylations and can be highly knocked down by RNA interference. In both T cell types, basal phosphorylation of Lck and Fyn on their activatory tyrosine was observed, although this was much less pronounced in Hut-78 cells. TCR stimulation led to the co-precipitation of Lck with the transmembrane adaptor protein LAT (linker for activation of T cells), Erk-mediated phosphorylation of Lck and no detectable dephosphorylation of Lck inhibitory tyrosine. Strikingly, upon LAT knockdown in Hut-78 cells, we found that LAT promoted TCR-induced phosphorylation of Lck and Fyn activatory tyrosines, TCRζ chain phosphorylation and Zap-70 activation. Notably, LAT regulated these events at low strength of TCR engagement.

Conclusions/Significance

Our results indicate for the first time that LAT promotes TCR signal initiation and suggest that this adaptor may contribute to maintain active Lck in proximity of their substrates.     

Cannabinoid Receptor Type 1- and 2-mediated Increase in Cyclic AMP Inhibits T Cell Receptor-triggered Signaling

The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G_i/o proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Δ9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the ζ-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.     

Impact of Exogenous Galectin-9 on Human T Cells: CONTRIBUTION OF THE T CELL RECEPTOR COMPLEX TO ANTIGEN-INDEPENDENT ACTIVATION BUT NOT TO APOPTOSIS INDUCTION*

Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca²⁺) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca²⁺ mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca²⁺ mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4⁺ T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca²⁺ mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4⁺ T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex.     

Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity.

The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45- T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45- YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45- YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation increased enzymatic activity. In contrast, the 394-Y-->F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45- YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45.     

The noncatalytic domains of Lck regulate its dephosphorylation by CD45

The Src-family tyrosine kinase, Lck, contains two key regulatory phosphotyrosine residues, tyrosine 394 (Tyr-394) and tyrosine 505 (Tyr-505), both of which can be dephosphorylated by CD45. Here, the interaction of CD45 with its substrate, Lck, was determined to be complex, involving multiple interactions with both the catalytic and noncatalytic regions of Lck. CD45 preferentially dephosphorylated Tyr-394 over Tyr-505 in Lck. This was not due to sequence specificity surrounding the phosphotyrosine, but was due to the noncatalytic domains of Lck. The interactions with the noncatalytic domains of Lck and CD45 enhanced the dephosphorylation of Tyr-394 whereas intramolecular interactions within Lck reduced, but did not abolish, the dephosphorylation of Tyr-505. This demonstrates that the noncatalytic domains of Lck regulate the dephosphorylation of both Tyr-394 and Tyr-505 by CD45.     

CD28 and lipid rafts coordinate recruitment of Lck to the immunological synapse of human T lymphocytes

In T lymphocytes, the Src family kinase Lck associates lipid rafts and accumulates at the immunological synapse (IS) during T cell stimulation by APCs. Using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS depends on CD4, whereas CD28 sustains Lck activation. However, in human resting T cells, CD28 is responsible for promoting recruitment of lipid rafts to the IS by an unknown mechanism. Thus, we performed a series of experiments to determine 1) whether Lck is recruited to the IS through lipid rafts; and 2) whether Lck recruitment to the IS of human resting T cells depends on CD4 or on CD28 engagement. We found that CD28, but not CD4, stimulation induced recruitment of Lck into detergent-resistant domains as well as its accumulation at the IS. We also found that Lck recruitment to the IS depends on the CD28 COOH-terminal PxxPP motif. Thus, the CD28-3A mutant, generated by substituting the prolines in positions 208, 211, and 212 with alanines, failed to induce Lck and lipid raft accumulation at the synapse. These results indicate that CD28 signaling orchestrates both Lck and lipid raft recruitment to the IS to amplify T cell activation.     

Mapping the binding site of full length HIV-1 Nef on human Lck SH3 by NMR spectroscopy

Summary The Nef protein of human immunodeficiency virus type 1 (HIV-1) is known to directly bind to the SH3 domain of human lymphocyte specific kinase (Lck) via a proline-rich region located in the amino terminal part of Nef. To address the question whether Nef binding to Lck SH3 involves residues outside the typical poly-proline peptide binding site and whether the Lck unique domain is involved in Nef–Lck interaction, we studied the direct interaction between both molecules using recombinant full-length HIV-1 Nef protein on one side and recombinantly expressed and uniformly ¹⁵N-isotope labeled Lck protein comprising unique and SH3 domains on the other side. Applying nuclear magnetic resonance spectroscopy we could show that only residues of Lck SH3, that are typically involved in binding poly-proline peptides, are affected by Nef binding. Further, for the first time we could rule out that residues of Lck unique domain are involved in binding to full length Nef protein. Thus, interactions of Lck unique domain to cellular partners e.g. CD4 or CD8, are not necessarily competitive with Lck binding to HIV-1 Nef.     

Trafficking of an acylated cytosolic protein: newly synthesized p56(lck) travels to the plasma membrane via the exocytic pathway

The Src-related tyrosine kinase p56(lck) (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.     

Superantigen stimulation reveals the contribution of Lck to negative regulation of T cell activation

The conventional paradigm of T cell activation through the TCR states that Lck plays a critical activating role in this signaling process. However, the T cell response to bacterial superantigens does not require Lck. In this study we report that not only is Lck dispensable for T cell activation by superantigens, but it actively inhibits this signaling pathway. Disruption of Lck function, either by repression of Lck gene expression or by selective pharmacologic inhibitors of Lck, led to increased IL-2 production in response to superantigen stimulation. This negative regulatory effect of Lck on superantigen-induced T cell responses required the kinase activity of Lck and correlated with early TCR signaling, but was independent of immunological synapse formation and TCR internalization. Our data demonstrate that the multistage role of Lck in T cell signaling includes the activation of a negative regulatory pathway of T cell activation.     

The Lck SH3 domain negatively regulates localization to lipid rafts through an interaction with c-Cbl.

Lck is a member of the Src family of protein-tyrosine kinases and is essential for T cell development and function. Lck is localized to the inner surface of the plasma membrane and partitions into lipid rafts via dual acylation on its N terminus. We have tested the role of Lck binding domains in regulating Lck localization to lipid rafts. A form of Lck containing a point mutation inactivating the SH3 domain (W97ALck) was preferentially localized to lipid rafts compared with wild type or SH2 domain-inactive (R154K) Lck when expressed in Lck-deficient J.CaM1 cells. W97ALck incorporated more of the radioiodinated version of palmitic acid, 16-[(125)I]iodohexadecanoic acid. Overexpression of c-Cbl, a ligand of the Lck SH3 domain, depleted Lck from lipid rafts in Jurkat cells. Additionally, Lck localization to lipid rafts was enhanced in c-Cbl-deficient T cells. The association of Lck with c-Cbl in vivo required a functional SH3 domain. These results suggest a model whereby the SH3 domain negatively regulates basal localization of Lck to lipid rafts via association with c-Cbl.     

Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains

p56lck (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membrane protein, and approximately half of the membrane-associated Lck is associated with a glycolipid-enriched membrane (GEM) fraction that is resistant to solubilization by Triton X-100 (TX-100). To compare the membrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibody to phosphotyrosine. Lck in the GEM fraction was found to be hyperphosphorylated on tyrosine, and this correlated with a lower kinase specific activity relative to the TX-100-soluble Lck. Peptide mapping and phosphatase diagests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr505. In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fraction. Cells lacking CD45 showed identical phosphorylation of Lck in GEM and TX-100-soluble membranes. We propose that the GEM fraction represents a specific membrane domain present in T-cells, and that the hyperphosphorylation of tyrosine and lower kinase activity of GEM-associated Lck is due to exclusion of CD45 from these domains. Lck associated with the GEM domains may therefore consitute a reservoir of enzyme that can be readily activated.     

Serine 6 of Lck tyrosine kinase: a critical site for Lck myristoylation, membrane localization, and function in T lymphocytes

Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.     

DHHC2 is a protein S-acyltransferase for Lck

Abstract

Lck is a non-receptor tyrosine kinase of the Src family that is essential for T cell activation. Dual N-terminal acylation of Lck with myristate (N-acylation) and palmitate (S-acylation) is essential for its membrane association and function. Reversible S-acylation of Lck is observed in vivo and may function as a control mechanism. Here we identify the DHHC family protein S-acyltransferase DHHC2 as an enzyme capable of palmitoylating of Lck in T cells. Reducing the DHHC2 level in Jurkat T cells using siRNA causes decreased Lck S-acylation and partial dislocation from membranes, and conversely overexpression of DHHC2 increases S-acylation of an Lck surrogate, LckN10-GFP. DHHC2 localizes primarily to the endoplasmic reticulum and Golgi apparatus suggesting that it is involved in S-acylation of newly-synthesized or recycling Lck involved in T cell signalling.     

Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip

The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.