A weak Lck tail bite is necessary for Lck function in T cell antigen receptor signaling

Src family kinases are suppressed by a "tail bite" mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH) 2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)(505) affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.     

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.     

Fatty acyl chain-dependent but charge-independent association of the SH4 domain of Lck with lipid membranes

The SH4 domain of Src family of nonreceptor protein tyrosine kinases represents the extreme N-terminal 1–16 amino acid region which mediates membrane association of these proteins and facilitates their functions. The SH4 domains among Src members lack well-defined sequence consensus and vary in the net charge. However, they readily anchor to the cytoplasmic face of the plasma membrane upon fatty acid acylation. Here, we report the membrane association of differentially acylated SH4 domain of Lck kinase, which has net negative charge at physiological pH. Our results suggest that despite the net negative charge, the SH4 domain of Lck associates with membranes upon fatty acid acylation. While myristoylation at the N-terminus is sufficient for providing membrane anchorage, multiple acylation determines orientation of the peptide chain with respect to the lipid bilayer. Hence, fatty acylation serves more than just a lipid anchor. It has an important role in regulating the spatial orientation of the peptide domain with respect to the lipid bilayer, which could be important for the interaction of the other domains of these kinases with their partners.     

The SH3 domain of Lck modulates T-cell receptor-dependent activation of extracellular signal-regulated kinase through activation of Raf-1

Engagement of the T-cell antigen receptor (TCR) results in the proximal activation of the Src family tyrosine kinase Lck. The activation of Lck leads to the downstream activation of the Ras/Raf/MEK/ERK signaling pathway (where ERK is extracellular signal-related kinase). Under conditions of weak, but not strong, stimulation through the TCR, a version of Lck that contains a single point mutation in the SH3 (Src homology 3) domain (W97ALck) fails to support the activation of ERK, despite initiating signaling through the TCR, as demonstrated by the robust activation of ZAP-70, PLC-γ, and Ras. We determined that the signaling lesion in W97ALck-expressing cells lies at the level of Raf-1 activation and is dependent on the presence of tyrosines 340/341 in the Raf-1 sequence. These data demonstrate a second function for Lck in TCR-mediated signaling to ERK. Additionally, we found that a significant fraction of Lck is localized to the Golgi apparatus and that, compared with wild-type Lck, W97ALck displays aberrant Golgi membrane localization. Our results support a model where under conditions of weak stimulation through the TCR, in addition to activated Ras, Golgi apparatus-localized Lck is needed for the full activation of Raf-1.     

T cell-specific adapter protein inhibits T cell activation by modulating Lck activity

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-gamma1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3zeta-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.     

Genetic Evidence of a Role for Lck in T-Cell Receptor Function Independent or Downstream of ZAP-70/Syk Protein Tyrosine Kinases

T-cell antigen receptor (TCR) engagement results in sequential activation of the Src protein tyrosine kinases (PTKs) Lck and Fyn and the Syk PTKs, ZAP-70 and Syk. While the Src PTKs mediate the phosphorylation of TCR-associated signaling subunits and the phosphorylation and activation of the Syk PTKs, the lack of a constitutively active Syk PTK has prohibited the analysis of Lck function downstream of these initiating signaling events. We describe here the generation of an activated Syk family PTK by substituting the kinase domain of Syk for the homologous region in ZAP-70 (designated as KS for kinase swap). Expression of the KS chimera resulted in its autophosphorylation, the phosphorylation of cellular proteins, the upregulation of T-cell activation markers, and the induction of interleukin-2 gene synthesis in a TCR-independent fashion. The KS chimera and downstream ZAP-70 or Syk substrates, such as SLP-76, were still phosphorylated when expressed in Lck-deficient JCaM1.6 T cells. However, expression of the KS chimera in JCaM1.6 cells failed to rescue downstream signaling events, demonstrating a functional role for Lck beyond the activation of the ZAP-70 and Syk PTKs. These results indicate that downstream TCR signaling pathways may be differentially regulated by ZAP-70 and Lck PTKs and provide a mechanism by which effector functions may be selectively activated in response to TCR stimulation.     

Modulation of Lck function through multisite docking to T cell-specific adapter protein

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr(280), Tyr(290), and Tyr(305) were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)(280) and Tyr(P)(290) phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)(505) phosphopeptide, whereas the TSAd Tyr(P)(305) peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20-30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is approximately 1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.     

Dual fatty acylation of p59(Fyn) is required for association with the T cell receptor zeta chain through phosphotyrosine-Src homology domain-2 interactions

The first 10 residues within the Src homology domain (SH)-4 domain of the Src family kinase Fyn are required for binding to the immune receptor tyrosine-based activation motif (ITAM) of T cell receptor (TCR) subunits. Recently, mutation of glycine 2, cysteine 3, and lysines 7 and 9 was shown to block binding of Fyn to TCR zeta chain ITAMs, prompting the designation of these residues as an ITAM recognition motif (Gauen, L.K.T., M.E. Linder, and A.S. Shaw. 1996. J. Cell Biol. 133:1007-1015). Here we show that these residues do not mediate direct interactions with TCR ITAMs, but rather are required for efficient myristoylation and palmitoylation of Fyn. Specifically, coexpression of a K7,9A-Fyn mutant with N-myristoyltransferase restored myristoylation, membrane binding, and association with the cytoplasmic tail of TCR zeta fused to CD8. Conversely, treatment of cells with 2-hydroxymyristate, a myristoylation inhibitor, blocked association of wild-type Fyn with zeta. The Fyn NH2 terminus was necessary but not sufficient for interaction with zeta and both Fyn kinase and SH2 domains were required, directing phosphorylation of zeta ITAM tyrosines and binding to zeta ITAM phosphotyrosines. Fyn/zeta interaction was sensitive to octylglucoside and filipin, agents that disrupt membrane rafts. Moreover, a plasma membrane bound, farnesylated Fyn construct, G2A,C3S-FynKRas, was not enriched in the detergent insoluble fraction and did not associate with zeta. We conclude that the Fyn SH4 domain provides the signals for fatty acylation and specific plasma membrane localization, stabilizing the interactions between the Fyn SH2 domain and phosphotyrosines in TCR zeta chain ITAMs.     

Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.     

Subdomain X of the kinase domain of Lck binds CD45 and facilitates dephosphorylation

CD45 is a transmembrane, two-domain protein-tyrosine phosphatase expressed exclusively in nucleated hematopoietic cells. The Src family kinase, Lck, is a major CD45 substrate in T cells and CD45 dephosphorylation of Lck is important for both T cell development and activation. However, how the substrate specificity of phosphatases such as CD45 is achieved is not well understood. Analysis of the interaction between the cytoplasmic domain of CD45 and its substrate, Lck, revealed that the active, membrane-proximal phosphatase domain of CD45 (CD45-D1) bound to the phosphorylated Lck kinase domain, the SH2 domain, and the unique N-terminal region of Lck. The second, inactive phosphatase domain (CD45-D2) bound only to the kinase domain of Lck. CD45-D2 was unable to bind phosphotyrosine, and its interaction with the kinase domain of Lck was independent of tyrosine phosphorylation. The binding of CD45-D2 was localized to subdomain X (SD10) of Lck. CD45-D2 bound similarly to Src family kinases but bound Csk to a lesser extent and did not bind significantly to the less related kinase, Erk1. CD45 dephosphorylated Lck and Src at similar rates but dephosphorylated Csk and Erk1 at lower rates. Replacement of Erk1 SD10 with that of Lck resulted in the binding of CD45-D2 and the conversion of Erk1 to a more efficient CD45 substrate. This demonstrates a role for CD45-D2 in binding substrate and identifies the SD10 region in Lck as a novel site involved in substrate recognition.     

Regulation of the Src family kinase Lck by Hsp90 and ubiquitination

Regulation of the Src-related tyrosine kinase Lck is crucial to the outcome of T-cell receptor (TCR) stimulation. It was previously shown that the stability of the constitutively active mutant LckY505F is controlled by Hsp90 (M. J. Bijlmakers and M. Marsh, Mol. Biol. Cell. 11:1585-1595, 2000). Here we establish that following TCR stimulation, endogenous activated Lck in T cells is also degraded in the presence of the Hsp90 inhibitor geldanamycin. Using Lck constructs expressed in COS-7 cells, we show that the presence of activating Lck mutations results not only in the enhanced dependence on Hsp90 but also in enhanced ubiquitination of Lck. Although both processes were induced by mutations Y505F and W97A that release the SH2 and SH3 inhibitory intramolecular interactions, respectively, neither process required Lck kinase activity or activation-dependent phosphorylation at serines 42 and 59 or tyrosine 394. By binding to the ATP-binding site, the Src family inhibitor PP2 reduced ubiquitination and overcame the need for Hsp90 monitoring of active Lck. We conclude that the levels of active Lck are influenced by two opposing processes, targeting for degradation by ubiquitination and rescue from degradation by Hsp90 monitoring. Based on the PP2 result, we propose that activation-induced conformational changes of the Lck kinase domain instigate both regulatory processes.     

The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization.

Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T-cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP-70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM.     

DHHC2 is a protein S-acyltransferase for Lck

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.     

Binding, domain orientation, and dynamics of the Lck SH3-SH2 domain pair and comparison with other Src-family kinases

The catalytic activity of Src-family kinases is regulated by association with its SH3 and SH2 domains. Activation requires displacement of intermolecular contacts by SH3/SH2 binding ligands resulting in dissociation of the SH3 and SH2 domains from the kinase domain. To understand the contribution of the SH3-SH2 domain pair to this regulatory process, the binding of peptides derived from physiologically relevant SH2 and SH3 interaction partners was studied for Lck and its relative Fyn by NMR spectroscopy. In contrast to Fyn, activating ligands do not induce communication between SH2 and SH3 domains in Lck. This can be attributed to the particular properties of the Lck SH3-SH2 linker which is shown to be extremely flexible thus effectively decoupling the behavior of the SH3 and SH2 domains. Measurements on the SH32 tandem from Lck further revealed a relative domain orientation that is distinctly different from that found in the Lck SH32 crystal structure and in other Src kinases. These data suggest that flexibility between SH2 and SH3 domains contributes to the adaptation of Src-family kinases to specific environments and distinct functions.     

Palmitoylation of either Cys-3 or Cys-5 is required for the biological activity of the Lck tyrosine protein kinase.

Palmitoylation can regulate both the affinity for membranes and the biological activity of proteins. To study the importance of the palmitoylation of the Src-like tyrosine protein kinase p56lck in the function of the protein, Cys-3, Cys-5, or both were mutated to serine, and the mutant proteins were expressed stably in fibroblasts and T cells. Both Cys-3 and Cys-5 were apparent sites of palmitoylation in Lck expressed in fibroblasts, as only the simultaneous mutation of both Cys-3 and Cys-5 caused a large reduction in the incorporation of [3H]palmitic acid. The double mutant S3/5Lck was no longer membrane bound when examined by either immunofluorescence or cell fractionation. This indicated that palmitoylation was required for association of Lck with the plasma membrane. Since the S3/5Lck protein was myristoylated, myristoylation of Lck is not sufficient for membrane binding. When Cys-3, Cys-5, or both Cys-3 and Cys-5 were changed to serine in activated F505Lck, palmitoylation of either Cys-3 or Cys-5 was found to be necessary and sufficient for the transformation of fibroblasts and for the induction of spontaneous, antigen-independent interleukin-2 production in the T-helper cell line DO-11.10. Nonpalmitoylated F505Lck exhibited little activity in vivo, where it did not induce elevated levels of tyrosine phosphorylation, and in vitro, where it was unable to phosphorylate angiotensin in an in vitro kinase assay. These findings suggest that F505Lck must be anchored stably to membranes to become activated. Because palmitoylation is dynamic, it may be involved in regulating the cellular localization of p56(lck), and consequently its activity, by altering the proximity of p56(lck) to its activators and/or targets.     

Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids

The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.     

Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck.

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.     

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.