Mutational Analysis of Lck in CD45-Negative T Cells: Dominant Role of Tyrosine 394 Phosphorylation in Kinase Activity

D’Oro, Ugo; Sakaguchi, Kazuyasu; Appella, Ettore; Ashwell, Jonathan D.

doi:10.1128/mcb.16.9.4996

Cited by 91 publications

(79 citation statements)

References 51 publications

(55 reference statements)

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“…This binding may help deliver activated Lck polypeptides to CD45. Given the capacity of CD45 to dephosphorylate the positive regulatory site of Lck (tyrosine 394) and cause its inactivation (11,12), this could be part of a negative feedback mechanism aimed at limiting TCR signaling. Such a concept was also suggested by the earlier findings that TCR stimulation resulted in an enhancement of the extent of association of CD45 with Lck and that such an increase was absent in T-cells lacking CD45-AP (30).…”

Section: Discussionmentioning

confidence: 99%

Interactions of CD45-associated Protein with the Antigen Receptor Signaling Machinery in T-lymphocytes

Veillette¹,

Soussou²,

Latour³

et al. 1999

Journal of Biological Chemistry

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CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56 lck , a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56 lck . The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56 lck could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56 lck . Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56 lck . Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.Activation of T-lymphocytes by antigen is initiated by protein tyrosine phosphorylation (1-4). Although the T-cell antigen receptor (TCR) 1 and the associated CD3 and subunits are devoid of intrinsic protein tyrosine kinase activity, they can recruit two classes of cytoplasmic protein tyrosine kinases to mediate this response, the Src family and the Syk/Zap-70 family. The Src-related enzymes Lck and FynT initiate TCR-mediated signals by phosphorylating a signaling motif in the cytoplasmic domain of CD3 and termed ITAM (for immunoreceptor tyrosine-based activation motif). Following this phosphorylation, the Syk/Zap-70 family kinases are activated through binding of their tandem Src homology 2 (SH2) domains to doubly phosphorylated ITAMs. Together with Src family kinases, Zap-70 and Syk are responsible for subsequent tyrosine phosphorylation of several signal transduction molecules including phospholipase C-␥1, Cbl, Vav, Slp-76, and Lat.CD45 is a 180 -220-kDa transmembrane protein tyrosine phosphatase expressed on all nucleated hemopoietic cells (5, 6). In T-cells it constitutes ϳ10% of all cell surface glycoproteins. Previous studies have shown that CD45 is necessary for T-cell activation because of its ability to promot...

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Section: Discussionmentioning

confidence: 99%

Interactions of CD45-associated Protein with the Antigen Receptor Signaling Machinery in T-lymphocytes

Veillette¹,

Soussou²,

Latour³

et al. 1999

Journal of Biological Chemistry

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“…Phosphorylation of the corresponding Tyr 416 in the prototypic SFK Src was shown to destabilize the interaction of the SH3 domain with the SH2-CD linker (35). Therefore, we introduced a Tyr 394 to Phe mutation in CLckY-2, which is known to strongly decrease kinase activity of wild-type Lck (36). The mutated CLckY-2-Y394F failed to reconstitute the signaling deficiency of J.CaM1.6 cells (Fig.…”

Section: Visualizing the Crosstalk Between Regulatory And Catalytic Dmentioning

confidence: 99%

Genetically Encoded Förster Resonance Energy Transfer Sensors for the Conformation of the Src Family Kinase Lck

Paster

Paar

Eckerstorfer

et al. 2009

The Journal of Immunology

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The current model for regulation of the Src family kinase member Lck postulates a strict correlation between structural condensation of the kinase backbone and catalytic activity. The key regulatory tyrosine 505, when phosphorylated, interacts with the Src homology 2 domain on the same molecule, effectively suppressing tyrosine kinase activity. Dephosphorylation of Tyr 505 upon TCR engagement is supposed to lead to unfolding of the kinase structure and enhanced kinase activity. Studies on the conformation-activity relationship of Lck in living cells have not been possible to date because of the lack of tools providing spatiotemporal resolution of conformational changes. We designed a biochemically active, conformation-sensitive Förster resonance energy transfer biosensor of human Lck using the complete kinase backbone. Live cell imaging in Jurkat cells demonstrated that our biosensor performed according to Src family kinase literature. A Tyr 505 to Phe mutation opened the structure of the Lck sensor, while changing the autophosphorylation site Tyr 394 to Phe condensed the molecule. The tightly packed structure of a high-affinity YEEI tail mutant showed that under steady-state conditions the bulk of Lck molecules exist in a mean conformational configuration. Although T cell activation commenced normally, we could not detect a change in the conformational status of our Lck biosensor during T cell activation. Together with biochemical data we conclude that during T cell activation, Lck is accessible to very subtle regulatory mechanisms without the need for acute changes in Tyr 505 and Tyr 394 phosphorylation and conformational alterations.

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“…Mutational analysis suggests that the phosphorylation of Y394 is dominant to that of Y505 in determining the kinase activity of Lck (D'Oro et al 1996), and therefore the deactivation event probably involves dephosphorylation of the Y394 by a phophatase, which is likely to be either CD45 (D'Oro et al 1996) or SHP-1 (Chiang & Sefton 2001). This implies that there will be a micro-environment enriched in activated Lck molecules in the vicinity of an engaged TCR.…”

Section: Introductionmentioning

confidence: 99%

Feedback control of T–cell receptor activation

Chan

Stark

George

2004

Proc. R. Soc. Lond. B

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The specificity and sensitivity of T-cell recognition is vital to the immune response. Ligand engagement with the T-cell receptor (TCR) results in the activation of a complex sequence of signalling events, both on the cell membrane and intracellularly. Feedback is an integral part of these signalling pathways, yet is often ignored in standard accounts of T-cell signalling. Here we show, using a mathematical model, that these feedback loops can explain the ability of the TCR to discriminate between ligands with high specificity and sensitivity, as well as provide a mechanism for sustained signalling. The model also explains the recent counter-intuitive observation that endogenous 'null' ligands can significantly enhance T-cell signalling. Finally, the model may provide an archetype for receptor switching based on kinase-phosphatase switches, and thus be of interest to the wider signalling community.

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Mutational Analysis of Lck in CD45-Negative T Cells: Dominant Role of Tyrosine 394 Phosphorylation in Kinase Activity

Cited by 91 publications

References 51 publications

Interactions of CD45-associated Protein with the Antigen Receptor Signaling Machinery in T-lymphocytes

Interactions of CD45-associated Protein with the Antigen Receptor Signaling Machinery in T-lymphocytes

Genetically Encoded Förster Resonance Energy Transfer Sensors for the Conformation of the Src Family Kinase Lck

Feedback control of T–cell receptor activation

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