The Role of SPECs, Small Cdc42-binding Proteins, in F-actin Accumulation at the Immunological Synapse

Kathryn H Ching,Adam E Kisailus,Peter D Burbelo

doi:10.1074/jbc.m500128200

Kathryn H Ching, Adam E Kisailus + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m500128200

Copy DOI

Abstract

SPEC1 and SPEC2 are structurally similar Cdc42-binding proteins of 79 and 84 amino acid residues, respectively. We investigated the role of SPEC2 in T cell function due to its high mRNA expression in lymphocytes. Western blot analysis revealed abundant SPEC2 protein in lymphocytes, which in glutathione S-transferase-capture experiments specifically interacted with only GTP-bound Cdc42. Immunofluorescence experiments revealed that the SPEC2 protein was diffusely localized in the cytoplasm and at the cell membrane in unstimulated Jurkat T cells and Raji B cells. Recruitment of SPEC2 within Jurkat T cells to the antigen-presenting cell interface occurred following incubation with staphylococcal enterotoxin E superantigen-loaded B cells and colocalized there with F-actin and Cdc42. T cell receptor (TCR) activation studies using anti-CD3 antibody-coated polystyrene beads showed that SPEC2 was recruited to the site of bead contact, which was not observed with anti-major histocompatibility complex antibody-coated beads. Accumulation of SPEC2 following TCR engagement occurred as early as 5 min, before obvious F-actin accumulation. Biochemical studies with Jurkat T cells demonstrated that N-terminal cysteine residues in SPEC2 were palmitoylated. Overexpression studies of the related SPEC1 showed that it also was recruited to the activated TCR. Mutational analysis revealed that localization of SPEC1 to the TCR required two N-terminal cysteine residues. Furthermore, a SPEC1 Cdc42 Rac-interacting binding mutant, containing an intact N terminus but defective in Cdc42 binding, completely blocked F-actin accumulation at the activated TCR. Taken together these results suggest that SPECs may play important roles in Cdc42-mediated F-actin accumulation at the immunological synapse.

Highlights

Cytoskeletal rearrangements play an important role in T cell activation following engagement of the T cell receptor (TCR)1
SPEC2 Is Expressed in Immune Tissue and Cells—Previous gene array mRNA expression analysis of human cells and tissues2 revealed that both SPEC1 and SPEC2 mRNA were ubiquitously expressed, but that the highest levels of human SPEC1 and SPEC2 mRNA expression occurred in T lymphocytes, dendritic, and whole blood cells [27]
6% of the conjugates expressing this mutant showed significant F-actin accumulation at the activated receptor (Fig. 6, lower right panel). These results suggest that SPEC1, and likely SPEC2, do not require interaction with Cdc42 for recruitment to the activated TCR. These results suggest the dual cysteine residues conserved between SPEC1 and SPEC2 are involved in targeting to the activated TCR, and that this region in both molecules may play an important role in regulating F-actin accumulation at the immunological synapse

Summary

Introduction

Cytoskeletal rearrangements play an important role in T cell activation following engagement of the T cell receptor (TCR)1. SPEC2 Colocalizes with Cdc42 and F-actin at the Immunological Synapse—Staining for endogenous SPEC2 using the anti-SPEC2 antibody in Jurkat T cells and Raji B cells adherent to poly-L-lysine-coated coverslips revealed that the SPEC2 protein was diffusely localized in the cytoplasm and weakly at the plasma membrane in Jurkat T cells (see unstimulated Jurkat T cells in Fig. 3B below and data not shown).

Results

Conclusion