Evidence of redox-linked signaling for producing a giant signal complex.
Previously we showed that a thiol-reactive heavy metal, HgCl2, crosslinked multiple cell surface receptors through a ligand-independent pathway, which produced massive aggregates of phosphotyrosine (PTYR)-containing proteins beneath plasma membrane [Nakashima et al. (1994): J Immunol 152: 1064-1071]. In this study we characterized these unique aggregates at the molecular level. The lysates in Brij 96 of thymocytes treated with HgCl2 were separated into the supernatant and pellet fractions by simple centrifugation. Selected PTYR-containing proteins and p56lck 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-PAGE under nonreducing condition. This suggested that PTYR-containing proteins and p56lck themselves do not form dimer or polymer directly by thiol-mediated bond. The pellet fraction was further found to include some other signal delivery elements, such as GTPase activating protein, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase. Finally, all of these signal elements and selected PTYR-containing proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox-linked pathway of formation of a giant signal complex.
Katano
Y
Y
Department of Immunology, Nagoya University School of Medicine, Japan.
Pu
M Y
MY
Akhand
A A
AA
Hamaguchi
M
M
Koga
Y
Y
Isobe
K
K
Fukuda
Y
Y
Hayakawa
T
T
Nakashima
I
I
D000818:Animals; D001798:Blood Proteins / Q000032:analysis*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis; D002498:Centrifugation; D003432:Cross-Linking Reagents; D020690:GTPase-Activating Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes / Q000737:chemistry* / Q000201:enzymology; D008627:Mercuric Chloride / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis; D019000:Phosphotyrosine; D010938:Plant Oils; D011092:Polyethylene Glycols / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D011506:Proteins; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D001798:Blood Proteins; D003432:Cross-Linking Reagents; D020690:GTPase-Activating Proteins; D010938:Plant Oils; D011506:Proteins; D019000:Phosphotyrosine; D011092:Polyethylene Glycols; D014443:Tyrosine; D008627:Mercuric Chloride; C033084:polyethylene glycol oleyl ether; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017871:Calcium-Calmodulin-Dependent Protein Kinases