Ionic CD3−Lck interaction regulates the initiation of T-cell receptor signaling

Li, Lunyi; Guo, Xingdong; Shi, Xiaoshan; Li, Changting; Wu, Wei; Yan, Chun; Wang, Haopeng; Li, Hua; Xu, Chenqi

doi:10.1073/pnas.1701990114

Cited by 80 publications

(97 citation statements)

References 63 publications

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“…Lck is anchored to the plasma membrane through its SH4 domain via post-translational acylation on three specific sites: a myristoylated Gly2 8 and palmitoylated Cys3 and Cys5. The latter two are crucial for membrane binding and biological activity, enabling Lck diffusion in the inner leaflet of the plasma membrane and its recruitment to the immunological synapse 9 The unique domain interacts with the CD3ε subunit in the TCR-CD3 complex 10 as well as the co-receptors CD4 and CD8 11 via zinc-mediated bonds. However, Lck does not require the co-receptors for recruitment to the immunological synapse or for TCR triggering 12 , suggesting that freely diffusing Lck is sufficient for T cell activation.…”

mentioning

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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mentioning

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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“…The requirement of the CD3ε BRS for Lck recruitment was confirmed through biolayer interferometry and NMR experiments . The electrostatic recruitment of Lck to CD3ε was shown to occur through the Lck Unique domain (Lck‐UD), which bears an acidic‐rich motif enabling the electrostatic association with the CD3ε and CD3ζ BRS through ionic interactions.…”

Section: Electrostatic Interaction‐driven Immune Receptor Signalingmentioning

confidence: 87%

“…A similar approach was used to confirm CD3ζ CT PM binding through electrostatic interactions with anionic lipids although the contribution of each CD3ζ BRS motifs was not assessed . As expected, the cytoplasmic tails of CD3δ and CD3γ do not associate electrostatically to the PM due to their lack of BRS …”

Section: Electrostatic Interaction‐driven Lipid Bindingmentioning

confidence: 99%

Electrostatic interactions: From immune receptor assembly to signaling

Connolly

Gagnon

2019

Immunological Reviews

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Our ability to mount a long-lasting and protective immune response relies on a variety of immune receptors that enable the recognition of ongoing infections, which triggers the adaptation of a myriad of immune cells. The organization of several immune receptors, such as the T cells receptor and several natural killer cell receptors, utilizes different modules for ligand recognition and signaling. These receptors require specific recognition mechanisms between the different modules in order to ensure proper assembly and function. Once assembled, immune receptors must remain inactive in the absence of ligand to prevent the onset of unwanted immune response.Indeed, several mechanisms exist to prevent aberrant immune receptor signaling in the absence of ligand to avert the initiation of uncontrolled autoimmunity. However, once a ligand is recognized, immune receptors must rapidly and specifically engage kinases to initiate highly regulated signaling cascades that lead to the initiation of transcriptional programs that dictate the immune response. Over the last decade, compelling evidence have been presented which suggest that electrostatic interactions are critical for many aspects of immune receptor functions. In the work that follows, we present an overview of the literature that have provided evidence that illustrate how electrostatic interactions regulate immune receptor assembly, inactive state, triggering, and signaling.

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“…The CD28 Y206/Y209 sites are surrounded by multiple proline residues, and this proline-rich binding domain is likely responsible for LCK recruitment when CD28 Y206 and Y209 are unphosphorylated (36). A similar proline-rich region is also thought to help recruit LCK to CD3ε and CD2 (35,37). Perhaps this proline-rich Y206/Y209 site on CD28 is able to bind and recruit LCK more readily than other sites but, given its lower affinity for the catalytic pocket of LCK, it can be outcompeted by other sites on the same protein.…”

Section: Discussionmentioning

confidence: 99%

“…This is true for all CD3ζ ITAM tyrosine sites except A1 (Figure 1B). The peptide containing site A1 also contains a tyrosine at position 64, which is not part of an ITAM and is not predicted to play a significant role in CD3ζ phosphorylation based on known LCK binding motifs and computational predictions (35)(36)(37)(38). Therefore, we added a Y64F mutation in the CD3ζ recombinant protein and validated that it does not influence the overall phosphorylation kinetics within this system (Supplemental Figure S2).…”

Section: E Half Max Timementioning

confidence: 94%

Computational model of chimeric antigen receptors explains site-specific phosphorylation kinetics

Rohrs

Zheng

Graham

et al. 2018

Preprint

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Chimeric antigen receptors (CARs) have recently been approved for the treatment of hematological malignancies, but our lack of understanding of the basic mechanisms that activate these proteins has made it difficult to optimize and control CAR-based therapies. In this study, we use phospho-proteomic mass spectrometry and mechanistic computational modeling to quantify the in vitro kinetics of individual tyrosine phosphorylation on a variety of CARs. We show that the ten tyrosine sites on the CD28-CD3ζ CAR are phosphorylated by LCK independently and with different kinetics. The addition of CD28 at the N-terminal of CD3ζ increases the overall rate of CD3ζ phosphorylation. Our computational model explains experimental observations of CD3ζ immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation and provides new mechanistic hypotheses for how these activating domains can be more optimally phosphorylated. Specifically, the model highlights the importance of protein binding to single ITAM tyrosine sites to enhance the amount of doubly phosphorylated ITAMs. This quantitative modeling framework can be used to better understand CAR signaling and T cell activation.

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Ionic CD3−Lck interaction regulates the initiation of T-cell receptor signaling

Cited by 80 publications

References 63 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

Electrostatic interactions: From immune receptor assembly to signaling

Computational model of chimeric antigen receptors explains site-specific phosphorylation kinetics

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