αII-spectrin in T cells is involved in the regulation of cell-cell contact leading to immunological synapse formation?

T-lymphocyte activation after antigen presentation to the T-Cell Receptor (TCR) is a critical step in the development of proper immune responses to infection and inflammation. This dynamic process involves reorganization of the actin cytoskeleton and signaling molecules at the cell membrane, leading to the formation of the Immunological Synapse (IS). The mechanisms regulating the formation of the IS are not completely understood. Nonerythroid spectrin is a membrane skeletal protein involved in the regulation of many cellular processes, including cell adhesion, signaling and actin cytoskeleton remodeling. However, the role of spectrin in IS formation has not been explored. We used molecular, imaging and cellular approaches to show that nonerythroid αII-spectrin redistributes to the IS during T-cell activation. The redistribution of spectrin coincides with the relocation of CD45 and LFA-1, two components essential for IS formation and stability. We assessed the role of spectrin by shRNA-mediated depletion from Jurkat T cells and show that spectrin-depleted cells exhibit decreased adhesion and are defective in forming lamellipodia and filopodia. Importantly, IS formation is impaired in spectrin-depleted cells. Thus, spectrin may be engaged in regulation of distinct events necessary for the establishment and maturity of the IS: besides the involvement of spectrin in the control of CD45 and LFA-1 surface display, spectrin acts in the establishment of cell-cell contact and adhesion processes during the formation of the IS.

PKC-θ is a negative regulator of TRAIL-induced and FADD-mediated apoptotic spectrin aggregation.

INTRODUCTION: During studies on chemotherapy-induced apoptosis in lymphoid cells, we noted that aggregation of spectrin occurred early in apoptosis, i.e. before activation of initiator caspase(s) and prior to exposure of phosphatidylserine (PS). We also found that protein kinase C theta (PKC-θ) co-localized with spectrin in these aggregates. Our previously published studies indicated that in formation of early apoptotic spectrin aggregates, either PKC-θ or other apoptosis-related proteins are involved. Taking into consideration above data, we decided to test the effect of PKC-θ and Fas-associated death domain protein (FADD) on spectrin aggregation in these cells during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. MATERIAL AND METHODS: For PKC-θ gene (PRKCQ) or FADD gene expression silencing in Jurkat T cells we used lentiviral particles containing shRNA and scrambled shRNA, respectively. Spectrin aggregates were detected by Western blotting after Triton-X 100 extraction in pellet and soluble fractions or by confocal imaging. RESULTS: TRAIL-induced apoptosis results in spectrin aggregation and leads to translocation and aggregation of PKC-θ. We found that phorbol-myristate acetate, a PKC activator and translocation inducer, has only a small effect on spectrin aggregation. To further confirm this, we have also shown that knock down ofPRKCQin Jurkat T cells accelerates the formation of TRAIL-induced spectrin aggregates. Transient overexpression of theß-spectrin C-terminal fragment, containing multiple S/T phosphorylation sites, potential substrate sites for PKC-θ, accelerated the formation of spectrin aggregates. Silencing of downstream TRAIL receptor effector gene,FADD, delayed aggregation of spectrin, but did not reduce PKC-θ localization to the plasma membrane. CONCLUSIONS: In summary, our results show for the first time involvement of spectrin aggregation in TRAIL receptor-FADD apoptotic pathway and indicate that TRAIL-induced spectrin aggregate formation is mediated by FADD and negatively regulated by PKC-θ.

Release of an ~55kDa fragment containing the actin-binding domain of ß-spectrin by caspase-8 during FND-induced apoptosis depends on the presence of protein 4.1.

Analyses of the status of the membrane spectrin-based skeleton during fludarabine/mitoxantrone/dexamethasone-induced (FND-induced) apoptosis revealed proteolytic degradation of ß-spectrin, with the prevalent appearance of a specific fragment with a molecular weight of ~55kDa, containing the actin-binding domain (ABD). Appearance of this fragment was dependent on induction of apoptosis. In silico proteolysis of spectrin identified caspase-8 as a candidate protease responsible for the generation of this ~55kDa ABD-containing fragment. Analyses of spectrin and procaspase-8 localization during early apoptosis indicated temporary (<30-120min) submembranous colocalization of both proteins. Proteolytic release of the N-terminal ~55kDa fragment of purified spectrin by recombinant caspase-8 does not occur in normal cells, but does occur in isolated membrane, such as red blood cell ghosts, or in vitro in the presence of apoptotic cell extracts. Surprisingly, proteolysis of purified spectrin by recombinant caspase-8 resulted in the generation of the ~55kDa fragment only in the presence of purified protein 4.1. This suggests that only the appropriate spatial arrangement of the spectrin-based membrane skeleton or the appropriate conformational state of spectrin, which are both known to be induced by 4.1, can sensitize ß-spectrin to cleavage by caspase-8 at the N-terminal ABD-containing region.

The emerging role of protein kinase Cθ in cytoskeletal signaling.

Cytoskeletal rearrangements often occur as the result of transduction of signals from the extracellular environment. Efficient awakening of this powerful machinery requires multiple activation and deactivation steps, which usually involve phosphorylation or dephosphorylation of different signaling units by kinases and phosphatases, respectively. In this review, we discuss the signaling characteristics of one of the nPKC isoforms, PKCθ, focusing on PKCθ-mediated signal transduction to cytoskeletal elements, which results in cellular rearrangements critical for cell type-specific responses to stimuli. PKCθ is the major PKC isoform present in hematopoietic and skeletal muscle cells. PKCθ plays roles in T cell signaling through the IS, survival responses in adult T cells, and T cell FasL-mediated apoptosis, all of which involve cytoskeletal rearrangements and relocation of this enzyme. PKCθ has been linked to the regulation of cell migration, lymphoid cell motility, and insulin signaling and resistance in skeletal muscle cells. Additional roles were suggested for PKCθ in mitosis and cell-cycle regulation. Comprehensive understanding of cytoskeletal regulation and the cellular "modus operandi" of PKCθ holds promise for improving current therapeutic applications aimed at autoimmune diseases.