phosphothreonine and oxophenylarsine

Several authors previously showed that the interaction between 14-3-3 proteins and plasma membrane H(+)-ATPase leads to an activated complex in which the enzyme is endowed with more favorable kinetic parameters and a more physiological pH optimum. In this paper we report immunological studies with antibodies covering a different specific region of the protein, including the N- and the C-terminal ends. The results showed that, beside a free and a complexed form, a third form of H(+)-ATPase in the cell must exist with low activity and no more activation due to the loss of a part of the C-terminal regulatory domain. A model in which 14-3-3 proteins activate H(+)-ATPase by protecting it from a specific proteolytic attack is presented and its generalization is discussed.

Topics: 14-3-3 Proteins; Amino Acid Sequence; Arsenicals; Blotting, Western; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Ethylmaleimide; Glycosides; Glycosylation; Hydrogen-Ion Concentration; Immunoblotting; Models, Biological; Molecular Sequence Data; Phosphorylation; Phosphothreonine; Phosphotyrosine; Plant Proteins; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Proton-Translocating ATPases; Sequence Homology, Amino Acid; Tyrosine 3-Monooxygenase

Dithiothreitol (DTT) and other dithiol antioxidants with closely spaced thiol pairs strongly activate leukocyte function antigen-1 (LFA-1, alphaLbeta2 integrin) to bind intercellular adhesion molecule-1 (ICAM-1). Because direct biochemical modification of LFA-1 by DTT is not apparently involved, we investigated the possible role of a reduction-oxidation (redox)-sensitive adhesion-regulatory pathway. Phenylarsine oxide (PAO), an oxidant selectively reactive with closely spaced pairs of thiol groups, inhibited LFA-1-dependent adhesion of human natural killer and HSB2 T leukemia cells to murine cells expressing human ICAM-1. PAO also induced disappearance of a conformation-sensitive LFA-1 epitope recognized by KIM127 antibodies and promoted an increase in total apparent cytoskeleton-linked LFA-1 in which a novel cytochalasin D-resistant linkage was involved. Exposure of PAO-pretreated cells to DTT caused a decline in LFA-1/cytoskeleton linkages in conjunction with rapid restoration of KIM127 epitope expression and LFA-1 adhesive function. Implicating an intracellular site of action were findings that (1) an epitope-tagged PAO probe bound predominantly to intracellular proteins but not detectably to immunoprecipitation-purified LFA-1 chains, and (2) membrane permeant but not impermeant dithiol antioxidants reversed PAO adhesion-inhibitory effects. These results support the concept of a reversible redox-sensitive linkage between LFA-1 and cytoskeleton by which oxidants and antioxidants may exert profound opposing effects on LFA-1 conformation and adhesive function.

Topics: Adenosine Triphosphate; Antibodies, Monoclonal; Arsenicals; Cell Adhesion; Cells, Cultured; Cytoskeleton; Humans; Integrins; Killer Cells, Natural; Lymphocyte Function-Associated Antigen-1; Oxidants; Oxidation-Reduction; Phosphoserine; Phosphothreonine; Signal Transduction; Sulfhydryl Compounds; Temperature; Tetradecanoylphorbol Acetate