The actin cytoskeleton plays a major
role as an integral component of lymphocyte activation .
TR-mediated stimulation leads to the organization of supramolecular activation clusters (SMACs) or "organized contacts" at the interfaces of physical contact between T cells and antigen presenting cells (APCs).
The central area of a SMAC contains the TR, CD3, the LCK (p56lck) and FYN (p59fyn) kinases, and protein kinase C (PKC) θ, while the peripheral regions are enriched in the adhesion molecule LFA1 and the cytoskeletal protein talin. Based on measurements of physical dimensions and molecular interactions, it has also been proposed that the coreceptor CD4 and CD8, the costimulatory molecule CD28, and the adhesion molecule CD2 may cosegregate with the central SMAC domain, whereas the protein tyrosine phosphatase receptor type C (PTPRC, CD45) and adhesion molecule sialophorin (SPN, CD43) might be excluded from the central contact zone.
The forces driving the formation of SMACs require actin polymerization in the T cell but not in the APC. Engagement of CD2 initiales a process of protein segregation, receptor clustering, and polarization of the T cell cytoskeleton. The CD2-associated protein (CD2AP), a SH3 domain-containing adaptor molecule, can mediate this cytoskeletal polarization. Since the binding of CD2AP to CD2 depends on T cell activation, it has been suggested that TR activation mobilizes CD2AP, which engages CD2, resulting in receptor clustering, the building of intracellular scaffolds, and T cell polarization. However, mice lacking CD2 have no apparent defects in T cell activation and lymphocyte development.
VAV1 links TR stimulation to activation of the Rho-family kinases (Rac1), CDC42, and ARHA (RhoA). VAV1-deficient T cells and thymocytes exhibit defects in antigen receptor- induced actin polymerization and recruitment of actin to the CD3ζ chain. Cap formation is also impaired by a deficiency of the CDC42-associated Wiscott Aldrich syndrome protein (WAS). VAV1, Rac1 and WAS link antigen receptor engagement to cytoskeletal reoganization, receptor clustering, and cap formation.
The GTPases Rac1, CDC42, and RhoA function as molecular switches in cells and orchestrate receptor- mediated cellular responses such as cytoskeletal changes and DNA synthesis. Target molecules for Rac1, CDC42, and ARHA (RhoA) include phosphatidylinositol 4-phosphate 5-kinase (PIP5K1A), WAS, myosin light chain (MLC) phosphatase, and the p21/CDC42/Rac1-actived kinase (PAK1). PIP5K1A phosphorylates PIP, resulting in the production and local accumulation of phosphatidylinositol 4, 5-bisphosphate (PIP2). High concentrations of PIP2 can dissociate actin-binding proteins such as profilin and gelsolin and promote interactions between actin and the cytoskeletal proteins vinculin and talin, which could be one mechanism for local actin polymerization and anchoring of the actin cystoskeleton to the cell membrane. In B cells, stimulation of the CD19 coreceptor leads to the recruitment of VAV1, which then regulates PIP5K1A activation. Thus, regulation of actin polymerization and cap formation by VAV1 might be achieved in part by the RhoA-or Rac1-mediated activation of PIP5K1A. However, VAV1 also directly associates with talin and vinculin. VAV1 might scaffold a signalling pathway promoting actin polymerization and anchor actin nucleation molecules required for the formation of caps and SMACs.
In the absence of VAV1, early thymocyte development at the pre-TR stage, positive and negative thymocyte selection, peptide/MHC- and antigen receptor"mediated thymocyte apoptosis, antigen receptor"induced cell cycle progression, and the activation of immune response genes such as the IL-2 gene are impaired. Similarly, deletion of the WAS gene severely impairs T cell proliferation and cytokine production. Moreover, inhibition of actin polymerization by Cytochalasin D mimics defects in VAV1- or WAS-deficient T cells or T cell lines overexpressing dominant-negative Rac1. Cytochalasin D inhibits IL-2 production, proliferation, TR capping, and peptide/MHC-mediated thymocyte apoptosis but does not impair known signalling pathways with the exception of Ca2+ mobilization. Thus, VAV1, Rac1 and WAS-regulated cytoskeletal reorganization and receptor clustering are required for T cell maturation and the induction of physiological T cell responses.
Activation of PKCθ can bypass the functional defects in T cells deficient for VAV1, Rac1 or WAS function. Of the several PKC isoforms, VAV1 associates only with the Ca2+-independent PKCθ molecule. PKCθ is highly expressed in the hematopoietic system, particulary in T cells, and cooperates with calcineurin to induce transcription of the T cell growth factor IL-2. This observation is reminiscent of the PKCθ coordinated transactivation of the IL-2 gene by calcineurin and VAV1/Rac1. In addition, PKC θ translocates to the central areas of the SMACs, whereas PKCα, β1, δ, ∈ and ζ remain in other regions. Thus, PKCθ is a candidate for an effector kinase that links cap and SMAC formation to downstream signalling pathways.
Overexpression of VAV1 activates NF-AT-dependent
transcription, suggesting that NF-AT is a nuclear terminus of cap-
and actin-dependent signalling.
Receptor clustering could favor sustained signalling in three ways:
T cells display ∼30,000-40,000 TR on their surface,
but only 50-100 peptide/MHC complexes on APCs are required for T cell
activation. Moreover, it appears that survival of naïve peripheral CD4
and CD8 T cells depends on continuous recognition of self-peptide/MHC
complexes by the TR. Why are these T cells not constantly activated?
Ligand-induced formation of caps and SMACs may introduce an additional
level of regulation of lymphocyte effector functions. Thus, TR-mediated
MAPK, SAPK/JNK, or NF-KB induction in the absence of caps and
SMACs might regulate cell survival. However, formation of higher order clusters appears necessary
to induce immune responses such as proliferation and expression of regulatory cytokines .
The widespread use of dimerization domains, localization domains, and protein-protein interaction domains as well as scaffolding proteins may reflect the importance of effective molarity as a regulatory mechanism in intracellular signalling.