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. 2012 Jun 12:3:155.
doi: 10.3389/fimmu.2012.00155. eCollection 2012.

Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement

Dominik Filipp  1 , Ondrej Ballek, Jasper Manning
Affiliations

Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement

Dominik Filipp et al. Front Immunol. .

Abstract

In spite of a comprehensive understanding of the schematics of T cell receptor (TCR) signaling, the mechanisms regulating compartmentalization of signaling molecules, their transient interactions, and rearrangement of membrane structures initiated upon TCR engagement remain an outstanding problem. These gaps in our knowledge are exemplified by recent data demonstrating that TCR triggering is largely dependent on a preactivated pool of Lck concentrated in T cells in a specific type of membrane microdomains. Our current model posits that in resting T cells all critical components of TCR triggering machinery including TCR/CD3, Lck, Fyn, CD45, PAG, and LAT are associated with distinct types of lipid-based microdomains which represent the smallest structural and functional units of membrane confinement able to negatively control enzymatic activities and substrate availability that is required for the initiation of TCR signaling. In addition, the microdomains based segregation spatially limits the interaction of components of TCR triggering machinery prior to the onset of TCR signaling and allows their rapid communication and signal amplification after TCR engagement, via the process of their coalescence. Microdomains mediated compartmentalization thus represents an essential membrane organizing principle in resting T cells. The integration of these structural and functional aspects of signaling into a unified model of TCR triggering will require a deeper understanding of membrane biology, novel interdisciplinary approaches and the generation of specific reagents. We believe that the fully integrated model of TCR signaling must be based on membrane structural network which provides a proper environment for regulatory processes controlling TCR triggering.

Keywords: Fyn; Lck; TCR triggering; compartmentalization; heavy and light DRMs; membrane microdomains; spatio-temporal regulation.

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Figures

Figure 1
Figure 1
A traditional model postulates that Lck kinase activity is regulated by reversible phosphorylation of two tyrosines residues: positive regulatory tyrosine Y394 in kinase domain (K.D.) and the negative regulatory tyrosine Y505 in the C-terminal end. When Y505 is phosphorylated (depicted here as P in red circle) by C-terminal src kinase (Csk), it forms an intramolecular bond with its own SH2 domain, locking the kinase domain in a substrate inaccessible, closed inactive conformation (top left structure). CD45 can dephosphorylate pY505, leaving the Lck structure in an open, primed conformation displaying a relatively low kinase activity (top middle structure). To achieve full kinase activity, the phosphorylation of Y394 (depicted here as P in green circle) must be achieved through trans/autophosphorylation (top right structure); its dephosphorylation is performed by CD45 or SHIP-1 or alternatively by PTPα in case of Fyn. It has been recently demonstrated that Lck can also persist in a double phosphorylated state (DPho), when both regulatory tyrosines (Y505 and Y394) are phosphorylated concurrently and the kinase retains its activity (bottom structure). DPho state is likely achieved in a two-step process. The first step involves the interaction of high affinity ligand(s) with Lck SH2 and/or SH3 domain which disrupts the closed conformation and is followed by CD4-Lck aggregation-mediated trans/auto phosphorylation of pY394. Alternatively, it is generated by the action of Csk on pY394 Lck. Whether CD45 converts these isoforms to a dephosphorylated, primed conformation or to an active pY394 state in vivo, is unclear (dashed arrows). Red arrows denote negative regulation; green arrows denote positive regulation.
Figure 2
Figure 2
The proposed model of TCR triggering mechanism. (A) Several types of membrane microdomains segregate distinct pools of functionally related molecules. Two SFK kinases Lck and Fyn reside in different types of microdomains where their kinase activities in resting T cells are controlled via distinct homeostatic mechanisms. The spatial confinement of Fyn within the light DRMs which are conducive to self-phosphorylation is subjected to a negative feedback loop mechanism controlled by PAG-recruited Csk. Kinase active Fyn phosphorylates PAG on Y371 that allows the membrane recruitment of Csk which in turn phosphorylates the negative regulatory Y528 of Fyn, thus dampening its activity. The dephosphorylation of pY528 as well as pY417 Fyn is mediated by either PTPα or by CD45. The pool of pY394 Lck associated with CD4 resides in heavy DRMs, where it colocalizes with TCR/CD3 and CD45. In resting T cells, CD3ζ is hypophosphorylated and precomplexed with the inactive ZAP-70 kinase. A distinct pool of heavy DRM-associated kinase inactive CD4-Lck complexes that are largely depleted of CD45 has been also detected. The identity of adaptor proteins able to recruit Csk to Lck is unknown. The adaptor protein LAT likely occupies another type of light DRMs. The activation of T cells is accompanied by a cascade of events depicted in the figure by numbers 1–6 in orange circles. pMHC-TCR engagement (1) promotes the clustering of CD4-Lck resulting in the activation of Lck (2). In the situation where only a few pMHC complexes are engaged, the preactivated pool of pY394Lck should be sufficient to initiate the signaling cascade; (3) activated Lck proceeds to phosphorylate ITAMs of CD3 chains which are concomitantly released from sequestration by the inner leaflet of PM upon TCR engagement, by a mechanism that has not been elucidated; ZAP-70 kinase recruited to pY-ITAMs is activated by Lck (4); CD45 and other phosphatases that possess a bulky ectodomain are moved laterally from contact zones by a size-exclusion mechanism and likely by virtue of their association with the cytoskeleton which coordinate their membrane redistribution (not depicted; 5); the free C-terminal end of activated Lck is able to interact with elements of the microtubular cytoskeletal network which aid its enrichment in light DRMs likely via the coalescence of heavy and light DRMs (6). (B) The amplification of the TCR signal in light DRMs seems to be critical for the engagement of downstream signaling components. A fraction of TCR/CD3/ZAP-70active and kinase active pY394 Lck translocates to light DRM and amplifies the signals in two independent ways: (i) colocalization with Fyn disrupts its negative regulatory homeostatic mechanism allowing the proximity-mediated transphosphorylation of the Fyn kinase domain, its activation and subsequent phosphorylation of Fyn targets Pyk2 and ADAP; and (ii) activated ZAP-70 phosphorylates its main targets LAT and SLP76 adaptors, thus allowing the engagement of downstream targets such as NF-AT, Ras-Raf signaling, and cytoskeleton rearrangement.
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