Biochemical characterization of distinct regions of SPEC molecules and their role in phagocytosis.

Cdc42 signaling pathways play important roles in immune cell polarization and cytoskeletal changes. Although the small Cdc42-binding proteins SPEC1 and SPEC2 play a role in F-actin accumulation in activated T lymphocytes, little is known about their precise activities in other cell types. Here, we mapped the Cdc42-binding activity of SPEC1 to the CRIB sequence and a downstream alpha helical region.

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.

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity.