The role of SPECs, small Cdc42-binding proteins, in F-actin accumulation at the immunological synapse.
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.
Ching
Kathryn H
KH
Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University Medical Center, Washington, DC 20057, USA.
Kisailus
Adam E
AE
Burbelo
Peter D
PD
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D005455:Fluorescent Antibody Technique; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*
D000199:Actins; D020764:cdc42 GTP-Binding Protein