EVIDENCE OF REDOX-LINKED SIGNALING FOR PRODUCING A GIANT SIGNAL COMPLEX

Previously we showed that a thiol-reactive heavy metal, HgCl2, crossli nked multiple cell surface receptors through a ligand-independent path way, which produced massive aggregates of phosphotyrosine (PTYR)-conta ining proteins beneath plasma membrane [Nakashima et al. (1994):J Immu nol 152:1064-1071]. In this study we characterized these unique aggreg ates at the molecular level. The lysates in Brij 96 of thymocytes trea ted with HgCl2 were separated into the supernatant and pellet fraction s by simple centrifugation. Selected PTYR-containing proteins and p56( lck) appeared in the pellet fraction as quickly as 5 s after exposure to HgCl2, and were further increased in amount by 5 min. Although the mechanism of triggering these events was redox-linked, the majority of proteins in the Brij 96-insoluble aggregates were dissociated in SDS- PACE under nonreducing condition. This suggested that PTYR-containing proteins and p56(lck) themselves do not form dimer or polymer directly by thiol-mediated bond. The pellet fraction was further found to incl ude some other signal delivery elements, such as GTPase activating pro tein, phosphatidylinositol 3 kinase, and mitogen-activated protein kin ase. Finally, all of these signal elements and selected PTYR-containin g proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox-linked p athway of formation of a giant signal complex. (C) 1995 Wiley-Liss, In c.