Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain

Sheng, Ren; Jung, Da-Jung; Silkov, Antonina; Kim, Hyun‐Jin; Singaram, Indira; Wang, Zhi Gang; Yao, Xin; Kim, Eui; Park, Mi Jeong; Thiagarajan-Rosenkranz, Pallavi; Smrt, Sean T.; Honig, Barry; Baek, Kwanghee; Ryu, Sung Ho; Lorieau, Justin L.; Kim, You-Me; Cho, Wonhwa

doi:10.1074/jbc.m116.720284

Cited by 44 publications

(59 citation statements)

References 57 publications

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“…Recent studies revealing that the activation of TCR upon the dynamic 2D binding with pMHC in physiological conditions is likely to occur through mechanisms that are quite different from those observed when the receptor is stimulated by antibody crosslinking 54 , 55 . On the other hand, the differential effects of MCID expression on the pMHC- and soluble anti-CD3-elicited TCR activation suggests that these effects are not due to TCR-unrelated alterations such as a higher Lck activity 56 or positive feedback effects from down-stream signaling events such as a stronger calcium influx 10 . Additionally, in our opinion, the effects of the phosphatase on TCR activation were not due to diminished cell contacts, owing to results from a previous study that showed that the reduction of PI(4,5)P2 levels in T cells promotes rather than inhibits the contacts between T cells and APCs 57 , which could be a result of decreased T cell membrane rigidity 58 .…”

Section: Discussionmentioning

confidence: 99%

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation

Chouaki-Benmansour

Ruminski

Sartre

et al. 2018

Sci Rep

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Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.

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Section: Discussionmentioning

confidence: 99%

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation

Chouaki-Benmansour

Ruminski

Sartre

et al. 2018

Sci Rep

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“…In addition, the SH2 domain of Lck can also interact with lipid within the plasma membrane upon TCR activation. This binding might be crucial for a lateral diffusion of Lck to interact with the triggered TCR–CD3 complex . These data indicate that localization of Lck to TCR–CD3s that are phosphorylated on few tyrosines facilitates the phosphorylation of the other ITAM tyrosines within CD3.…”

Section: Lckmentioning

confidence: 80%

“…An indirect interaction might be mediated by RhoH, a haematopoietic‐specific Rho GTPase . The direct interaction might be mediated by the SH2 domain of Lck and phosphorylated ITAMs . In addition, the SH2 domain of Lck can also interact with lipid within the plasma membrane upon TCR activation.…”

Section: Lckmentioning

confidence: 99%

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Selected signalling proteins recruited to the T‐cell receptor–CD3 complex

2017

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SummaryThe T-cell receptor (TCR)-CD3 complex, expressed on T cells, determines the outcome of a T-cell response. It consists of the TCR-ab heterodimer and the non-covalently associated signalling dimers of CD3ec, CD3ed and CD3ff. TCR-ab binds specifically to a cognate peptide antigen bound to an MHC molecule, whereas the CD3 subunits transmit the signal into the cytosol to activate signalling events. Recruitment of proteins to specialized localizations is one mechanism to regulate activation and termination of signalling. In the last 25 years a large number of signalling molecules recruited to the TCR-CD3 complex upon antigen binding to TCR-ab have been described. Here, we review knowledge about five of those interaction partners: Lck, ZAP-70, Nck, WASP and Numb. Some of these proteins have been targeted in the development of immunomodulatory drugs aiming to treat patients with autoimmune diseases and organ transplants.

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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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Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain

Cited by 44 publications

References 57 publications

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation

Selected signalling proteins recruited to the T‐cell receptor–CD3 complex

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

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