A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45

Courtney, Adam H.; Amacher, J.F.; Kadlecek, Theresa A.; Mollenauer, Marianne; Au-Yeung, Byron B; Kuriyan, John; Weiss, Arthur

doi:10.1016/j.molcel.2017.06.024

Cited by 56 publications

(78 citation statements)

References 59 publications

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“…While it is relatively well documented that the conformational states control enzymatic activity, how membrane-bound Lck finds and phosphorylates its substrates is not well understood. For example, the link between phosphorylation state and activity is well established 39 , as well as some interactions of Lck with other proteins 40-43 and lipids 44 Most studies so far have focused on whether or not T cell stimulation results in an ‘activation’ of Lck itself, i.e., whether there is an overall increase of Lck molecules in the open conformation and whether a stable pool of open Lck already exists in resting T cells. However, it is also possible that in the dynamic environment of the inner leaflet of the plasma membrane, Lck switches between open and closed states, as many other types of enzymes do 45-47 .…”

Section: Discussionmentioning

confidence: 99%

Conformational states control Lck switching between free and confined diffusion modes in T cells

Hilzenrat

Pandžić²,

Yang

et al. 2018

Preprint

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17T cell receptor (TCR) phosphorylation by Lck is an essential step in T cell activation. It is 18 known the conformational states of Lck control enzymatic activity; however, the underlying 19 principles of how Lck finds its substrate in the plasma membrane remain elusive. Here, 20 single-particle tracking is paired with photoactivatable localization microscopy (sptPALM) to 21 observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules 22 switched between free and confined diffusion in resting and stimulated T cells. 23 Conformational state, but not partitioning into membrane domains, caused Lck confinement 24 as open conformation Lck was more confined than closed. Further confinement of kinase-25 dead versions of Lck suggests that Lck interacts with open active Lck to cause confinement, 26 irrespectively of kinase activity. Our data supports a model that confined diffusion of open 27Lck results in high local phosphorylation rates and closed Lck diffuses freely to enable wide-28 range scanning of the plasma membrane. 29T cell signaling is a tightly controlled process involving both simultaneous and sequential 30 spatiotemporal events, involving membrane remodeling and redistribution of key signaling 31 proteins 1,2 . Engagement of the T cell receptor (TCR) with an antigenic pMHC on the surface 32 of an antigen-presenting cell (APC) leads to the formation of immunological synapses 3 and 33 initiates downstream signaling events that lead to T cell activation 4 . The Src family kinase 34 Lck plays a crucial role in the signaling cascade. TCR engagement results in the membrane 35 release 5 and phosphorylation of the immunoreceptor tyrosine-based motifs (ITAMs) located 36 in the cytoplasmic tails of the CD3ζ chain by Lck 6 . Phosphorylated sites on the TCR-CD3 37 complex become docking sites for the zeta chain-associated protein kinase 70 (ZAP70), that 38 is further phosphorylated by Lck 7 before recruiting other proteins in the signaling cascade 39 that are necessary for complete T cell activation. 40The role of Lck in T cell activation as a signaling regulator is of particular interest due to its 41 dynamic characteristics. Lck is a 56 kDa protein comprised of a Src homology (SH) 4 domain 42 at the N-terminus, followed by a unique domain, an SH3 domain, an SH2 domain, a kinase 43 domain and short C-terminal tail. Lck is anchored to the plasma membrane through its SH4 44 domain via post-translational acylation on three specific sites: a myristoylated Gly2 8 and 45 palmitoylated Cys3 and Cys5. The latter two are crucial for membrane binding and biological 46 activity, enabling Lck diffusion in the inner leaflet of the plasma membrane and its 47 recruitment to the immunological synapse 9 . The unique domain interacts with the CD3ε 48 subunit in the TCR-CD3 complex 10 as well as the co-receptors CD4 and CD8 11 via zinc-49 mediated bonds. However, Lck does not require the co-receptors for recruitment to the 50 immunological synapse or for TCR triggering 12 , suggesting that freely diffusing Lck is...

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Section: Discussionmentioning

confidence: 99%

Conformational states control Lck switching between free and confined diffusion modes in T cells

Hilzenrat

Pandžić²,

Yang

et al. 2018

Preprint

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“…Second, such 10-to 20-nm extension of a TCR-pMHC complex under force reduces its size difference from CD45, thus possibly preventing CD45 exclusions from TCRs during TCR initial triggering according to the kinetic segregation model (Davis and van der Merwe, 2006). This might be beneficial for TCR initial triggering because CD45 has been reported to be essential for early TCR proximal signaling by regulating Lck activation (Courtney et al, 2017;Dustin and Depoil, 2011). This hypothesis is actually supported by a recent finding (Cai et al, 2017) that no profound CD45 exclusion is observed on TCR pre-enriched microvillar tips of T cells during the very early stage of TCRs searching for cognate antigens on APCs, whereas, at the later stage of IS formation, CD45 is excluded from TCRs in the center of supramolecular activation clusters (Cai et al, 2017;Chang et al, 2016;Choudhuri et al, 2005).…”

Section: Force-induced Mhc Conformational Changes Facilitate Tcr Antimentioning

confidence: 99%

Mechano-regulation of Peptide-MHC Class I Conformations Determines TCR Antigen Recognition

et al. 2019

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“…1B), thereby preventing exclusion of CD45 from the region of T cell-APC contacts (36). This may allow CD45 to dephosphorylate the C-terminal tail of Lck, which is required to convert this critical kinase to its active conformation (37). In contrast, CD45 exclusion favors the phosphorylated state of CD3 ITAMs, which promotes T-cell activation (13).…”

Section: Jbc Reviews: Structural Basis Of T-cell Receptor Activationmentioning

confidence: 99%

The structural basis of T-cell receptor (TCR) activation: An enduring enigma

2019

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T cells are critical for protective immune responses to pathogens and tumors. The T-cell receptor (TCR)–CD3 complex is composed of a diverse αβ TCR heterodimer noncovalently associated with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T-cell signaling pathways, the mechanism whereby TCR engagement by pMHC is first communicated to the CD3 signaling apparatus, a process termed early T-cell activation, remains largely a mystery. In this review, we examine the molecular basis for TCR activation in light of the recently determined cryoEM structure of a complete TCR–CD3 complex. This structure provides an unprecedented opportunity to assess various signaling models that have been proposed for the TCR. We review evidence from single-molecule and structural studies for force-induced conformational changes in the TCR–CD3 complex, for dynamically-driven TCR allostery, and for pMHC-induced structural changes in the transmembrane and cytoplasmic regions of CD3 subunits. We identify major knowledge gaps that must be filled in order to arrive at a comprehensive model of TCR activation that explains, at the molecular level, how pMHC-specific information is transmitted across the T-cell membrane to initiate intracellular signaling. An in-depth understanding of this process will accelerate the rational design of immunotherapeutic agents targeting the TCR–CD3 complex.

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A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45

Cited by 56 publications

References 59 publications

Conformational states control Lck switching between free and confined diffusion modes in T cells

Conformational states control Lck switching between free and confined diffusion modes in T cells

Mechano-regulation of Peptide-MHC Class I Conformations Determines TCR Antigen Recognition

The structural basis of T-cell receptor (TCR) activation: An enduring enigma

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